阅读说明：本技术 双功能抗pd-1/il-7分子 (Bifunctional anti-PD-1/IL-7 molecules ) 是由 N·普瓦里耶 C·马里 A·莫雷洛 J·杜兰德 于 2019-12-17 设计创作，主要内容包括：本发明涉及包含抗PD-1抗体和IL-7的双功能分子及其用途。(The present invention relates to bifunctional molecules comprising an anti-PD-1 antibody and IL-7 and uses thereof.)

1. A bifunctional molecule comprising:

(a) an anti-human PD-1 antibody or antigen-binding fragment thereof, comprising:

(i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2 and HCDR3, and

(ii) a light chain variable domain (VL) comprising LCDR1, LCDR2, and LCDR3, and

(b) human interleukin 7(IL-7) or a fragment or variant thereof,

wherein said antibody or fragment thereof is covalently linked to said human IL-7 or fragment or variant thereof as a fusion protein, preferably via a peptide linker.

2. The bifunctional molecule of claim 1, wherein the N-terminal end of the human IL-7 or fragment thereof is linked to the C-terminal end of the heavy and/or light chain or both of the anti-human PD-1 antibody or antigen-binding fragment thereof.

3. The bifunctional molecule of any one of claims 1-2, wherein the antibody or antigen-binding fragment thereof is a chimeric, humanized, or human antibody.

4. The bifunctional molecule of claims 1-3, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises:

(i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2 and HCDR3, and

(ii) a light chain variable domain (VL) comprising LCDR1, LCDR2 and LCDR3,

wherein:

-the heavy chain CDR1(HCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 1;

-the heavy chain CDR2(HCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 2;

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 3, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N and E;

-the light chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 12, wherein X is G or T;

-the light chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO:15,

-the light chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16.

5. The bifunctional molecule of claims 1-4, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises or consists of: (a) a VH comprising or consisting of the amino acid sequence of SEQ ID NO 17, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N and E; (b) VL comprising or consisting of the amino acid sequence of SEQ ID NO. 26, wherein X is G or T.

6. The bifunctional molecule of claims 1-5, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises or consists of: (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:24, and (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28.

7. The bifunctional molecule of claims 1-3, wherein the anti-PD 1 antibody is selected from pembrolizumab, nivolumab, pidilizumab, cimiralizumab, PDR001, and monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F 4.

8. The bifunctional molecule of any one of claims 1-7, wherein the IL-7, or a variant thereof, comprises or consists of an amino acid sequence having at least 75% identity to wild-type human IL-7 (wth-IL-7).

9. Bifunctional molecule according to any of claims 1-8, wherein the IL-7 comprises or consists of the amino acid sequence shown in SEQ ID No. 51.

10. The bifunctional molecule of any one of claims 1-8, wherein the IL-7 is an IL-7 variant, wherein the IL-7 variant exhibits a sequence identical to a sequence comprising SEQ ID NO:51 or the amino acid sequence set forth in SEQ ID NO:51 (wth-IL-7) is at least 75% identical, wherein the variant comprises at least one amino acid mutation that i) reduces the affinity of the IL-7 variant for IL-7R compared to the affinity of wth-IL-7 for the IL-7 receptor (IL-7R), and ii) the pharmacokinetics of the bifunctional molecule comprising the IL-7 variant is improved compared to the bifunctional molecule comprising IL-7.

11. Bifunctional molecule according to claim 10, wherein the at least one mutation is an amino acid substitution or a group of amino acid substitutions selected from: (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F or W142Y, (iii) D74E, D74Q or D74N, (iv) Q11E, Y12F, M17L, Q22E and/or K81R; or any combination thereof.

12. The bifunctional molecule of claim 10, wherein the IL-7 variant comprises an amino acid substitution set selected from: C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, and C47S-C92S and C34S-C129S.

13. The bifunctional molecule of claim 10, wherein the IL-7 variant comprises an amino acid substitution selected from: W142H, W142F and W142Y.

14. The bifunctional molecule of claim 10, wherein the IL-7 variant comprises an amino acid substitution selected from: D74E, D74Q, and D74N.

15. Bifunctional molecule according to any of claims 1-8 and 10, wherein the IL-7 variant comprises or consists of the amino acid sequence shown in SEQ ID NOs 53-66.

16. Bifunctional molecule according to any of claims 1-8 and 10, wherein the IL-7 variant comprises or consists of the amino acid sequence shown in SEQ ID NO 54, 56 or 63.

17. The bifunctional molecule of any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant domain (preferably IgGl or IgG4 heavy chain constant domain).

18. The bifunctional molecule of any one of claims 1-17, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgGl heavy chain constant domain, optionally with a substitution or a combination of substitutions selected from: T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A + M252Y/S254T/T256E; K322A and K444A, preferably selected from the following: N297A, optionally in combination with M252Y/S254T/T256E, and L234A/L235A.

19. The bifunctional molecule of any one of claims 1-17, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgG4 heavy chain constant domain, optionally with a substitution or combination of substitutions selected from: S228P; L234A/L235A, S228P + M252Y/S254T/T256E.17 and K444A.

20. Bifunctional molecule according to any of claims 1-19, wherein the antibody or fragment thereof is linked to IL-7 or a variant thereof by a linker sequence, preferably selected from (GGGGS) ₃、(GGGGS)₄、(GGGGS)₂GGGGS, GGGS, GGG, GGS and (GGGS)₃Even more preferably (GGGGS)₃。

21. The bifunctional molecule of any one of claims 12-16, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgGl heavy chain constant domain, optionally with a substitution or a combination of substitutions selected from: T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A + M252Y/S254T/T256E; K322A and K444A, preferably selected from the following: N297A, optionally in combination with M252Y/S254T/T256E, and L234A/L235A; and the antibody or fragment thereof is linked via a linker (GGGGS)₃Linked to an IL-7 variant.

22. An isolated nucleic acid molecule or set of isolated nucleic acid molecules encoding the bifunctional molecule of any one of claims 1-21.

23. A vector comprising the nucleic acid or set of nucleic acid molecules of claim 22.

24. A host cell comprising the vector of claim 23 or the nucleic acid or set of nucleic acid molecules of claim 22.

25. A method for producing a bifunctional molecule according to any of claims 1-21, comprising the step of culturing a host cell according to claim 24 and optionally the step of isolating said bifunctional molecule.

26. A pharmaceutical composition comprising the bifunctional molecule of any one of claims 1-21, the nucleic acid or group of nucleic acid molecules of claim 22, the vector of claim 23, or the host cell of claim 24, and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition according to claim 26, wherein it further comprises an additional therapeutic agent, preferably selected from the following: alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferative agents, antivirals, aurora kinase inhibitors, apoptosis-promoting agents (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T-cell cement) antibodies, antibody drug conjugates, biological response modifiers, Bruton's Tyrosine Kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, Heat Shock Protein (HSP) -90 inhibitors, Histone Deacetylase (HDAC) inhibitors, hormonal therapies, immunological drugs, inhibitors of apoptosis protein Inhibitors (IAPs), intercalating antibiotics, anti-viral agents, aurora kinase inhibitors, apoptosis-promoting agents (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T-cell cement) antibodies, antibody drug conjugates, biological response modifiers, and anti-inflammatory drugs, Kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target protein of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate) -ribose polymerase (PARP) inhibitors, platinum-based chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid/retinoid plant alkaloids, small inhibitory ribonucleic acids (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines and the like, epitopes or neo-epitopes from tumor antigens, and combinations of one or more of these agents.

28. Pharmaceutical composition according to claim 26 or 27, the bifunctional molecule of any one of claims 1-21, the nucleic acid or group of nucleic acid molecules according to claim 22, or the vector according to claim 23, or the host cell according to claim 24 for use as a medicament.

29. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell according to claim 28 for use in the treatment of a disease selected from cancer.

30. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell of claim 29, wherein the cancer is selected from the following: a hematologic malignancy or solid tumor that expresses PD-1 and/or PD-L1, such as a cancer selected from: lymphohematopoietic tumors, angioimmunoblastic T-cell lymphomas, myelodysplastic syndromes and acute myeloid leukemia, cancers induced by viruses or associated with immunodeficiency, such as cancers selected from: kaposi's sarcoma (e.g., associated with kaposi's sarcoma herpes virus); cervical cancer, anal cancer, penile cancer and vulvar squamous cell carcinoma and oropharyngeal cancer (e.g., associated with human papilloma virus); b-cell non-hodgkin's lymphoma (NHL) including diffuse large B-cell lymphoma, burkitt's lymphoma, plasmablast lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classical hodgkin's lymphoma, and lymphoproliferative disorders (e.g., associated with epstein-barr virus (EBV) and/or kaposi's sarcoma herpes virus); hepatocellular carcinoma (e.g., associated with hepatitis b and/or hepatitis c virus); merkel cell carcinoma (e.g., associated with merkel cell polyoma virus (MPV)); and cancers associated with Human Immunodeficiency Virus (HIV) infection; and a cancer selected from: metastatic or non-metastatic, melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, hodgkin's lymphoma, head and neck cancer, urothelial cancer, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic merkel cell cancer, gastric or esophageal cancer, and cervical cancer.

31. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell of any one of claims 28-30 for use in combination with radiotherapy or an additional therapeutic agent, preferably selected from an alkylating agent, an angiogenesis inhibitor, an antibody, an antimetabolite agent, an antimitotic agent, an antiproliferative agent, an antiviral agent, an aurora kinase inhibitor, an apoptosis promoter (e.g., a Bcl-2 family inhibitor), a death receptor pathway activator, a Bcr-Abl kinase inhibitor, a BiTE (bispecific T-cell cement) antibody, an antibody drug conjugate, a biological response modifier, a Bruton's Tyrosine Kinase (BTK) inhibitor, a cyclin-dependent kinase inhibitor, a cell cycle inhibitor, a cyclooxygenase-2 inhibitor, DVD, an leukemia virus oncogene homolog (ErbB2) receptor inhibitor, a cancer cell line, a cancer cell line, or a cancer cell line, a cell line, Growth factor inhibitors, Heat Shock Protein (HSP) -90 inhibitors, Histone Deacetylase (HDAC) inhibitors, hormone therapy, immunological drugs, inhibitors of Inhibitor of Apoptosis Protein (IAP), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, inhibitors of mammalian target proteins of rapamycin, microRNA, inhibitors of mitogen-activated extracellular signal-regulated kinase, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), inhibitors of poly (ADP) (adenosine diphosphate) -ribose polymerase (PARP), platinum-based chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid/retinoid plant alkaloids, small inhibitory ribonucleic acid (siRNA), immune drugs, apoptosis protein Inhibitors (IAP), anti-tumor drugs, anti-inflammatory drugs (NSAIDs), anti-tumor drugs, anti-cancer drugs, anti-inflammatory drugs, anti-cancer drugs, topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines and the like, epitopes or neo-epitopes from tumor antigens, and combinations of one or more of these agents.

32. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell according to claim 28, for use in the treatment of infectious diseases, preferably chronic infectious diseases, even more preferably chronic viral infections.

33. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell of claim 32, wherein the infectious disease is caused by a virus selected from the group consisting of: HIV, hepatitis virus, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus and arbo encephalitis virus.

Technical Field

The present invention relates to the field of immunotherapy. The present invention provides a bifunctional molecule comprising an anti-PD-1 antibody or an antibody-binding fragment thereof.

Background

Methods of targeting T-cell inhibition checkpoints to relieve inhibition using therapeutic antibodies are an area of intense research (for review see pardol, Nat Rev Cancer, 2012; 12: 253-264). Immune checkpoints targeting adaptive immunity have shown great therapeutic efficacy against a variety of cancers, but they have been shown in only a limited proportion of patients. Combining immune checkpoint therapy with other immunotherapeutic strategies has shown great efficiency in preclinical models, but remains a clinical challenge.

Immune cell activation is controlled by the integration of balanced costimulatory and cosuppressive signals. T Cell Receptor (TCR) -mediated T cell activation is regulated by both costimulatory and cosuppressive signals. The antigen-independent second signal modifies the first signal, which is provided by the interaction of the antigen peptide-MHC complex with the TCR, thereby conferring specificity for the response. The T cell co-stimulatory and co-inhibitory pathways have a broad immunomodulatory role, controlling effector, memory and regulatory T cells and priming T cells. Therapeutic modulation of those pathways is turning into an effective new strategy for the treatment of cancer (for a review, see Schildberg et al, 44(5), Immunity, 2016). Ongoing research on the modulation of immune responses has identified a variety of immune pathways that can be targeted to develop cancer therapies. Those molecules are referred to herein as immune checkpoint co-activators or co-inhibitors (see Reviews Sharma et al, Cell,161(2),2015 and Pardoll, Nature Reviews Cancer,12(4), 2012).

Programmed cell death protein 1(PD-1, also known as CD279) is a cell surface protein molecule that belongs to the immunoglobulin superfamily. PD-1 is expressed on T and B lymphocytes and macrophages and plays a role in cell fate and differentiation. Two ligands for PD-1 have been identified, PD-L1 and PD-L2, which have been shown to down-regulate T cell activation upon binding to PD-1 (Freeman et al, (2000) J Exp Med 192: 1027-34; Latchman et al, (2001) Nat Immunol 2: 261-8; Carter et al, (2002) Eur J Immunol 32: 634-43). The interaction between PD-1 and its ligands results in a decrease in tumor infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancer cells. In particular, the PD1 junction reduces signals downstream of TCR stimulation on T cells, inhibiting T cell responses and resulting in reduced activation and cytokine production.

PD-1/PD-L1 therapy has been approved by the FDA as first and second line therapy for a wide range of hematological and solid cancers, but the objective responses, based on tumor size reduction of more than 30% as defined by RECIST criteria, vary widely between cancer subtypes.

High response rates were observed in refractory Hodgkin's lymphoma (65-85%) (Borcherding N et al, J Mol biol., 6.7.2018; 430(14): 2014-.

Intermediate objective response rates were observed in melanoma (24% to 44%) and non-small cell lung cancer patients (12.8% to 43.7%), with anti-PD-1 therapy used as first-line treatment. While only a subset of patients benefit from this therapy, PD-1/PD-L1 therapy improves overall survival compared to old standard of care chemotherapy.

In some solid tumors, low or zero clinical response was observed, particularly in pancreatic cancer, non-MSI colorectal cancer, gastric cancer, and some breast cancers (Borcherding N et al, J Mol biol., 6.7.2018; 430(14): 2014-.

Various mechanisms have been described and may explain this diverse efficacy and resistance to PD-1/PD-L1 checkpoint therapy, particularly where some are involved in T cell biology, e.g., (1) impaired formation of memory T cells; (2) impaired T cell infiltration; (3) insufficient production of tumor-specific T cells; (4) insufficient T cell function; and (5) regulatory T cell-induced immunosuppressive microenvironments. Combination therapy by targeting IL-7 signaling may be a good strategy to address patients resistant to PD-1 without stimulating regulatory T cell expansion and survival by stimulating T cell infiltration, maintaining T cell effector competence, and promoting a durable memory T cell response.

Interleukin-7 is an immunostimulatory cytokine member of the IL-2 superfamily that plays an important role in the adaptive immune system and promotes B-and T-cell mediated immune responses. This cytokine activates immune function through the survival and differentiation of T and B cells, the survival of lymphocytes, and the stimulation of Natural Killer (NK) cell activity. IL-7 also regulates lymph node development by Lymphoid Tissue Inducing (LTi) cells and promotes survival and division of naive or memory T cells. In addition, IL-7 enhances the immune response in humans by promoting the secretion of IL-2 and interferon-gamma. The receptor for IL-7 is a heterodimer and consists of IL-7R α (CD127) and a common γ chain (CD 132). The γ chain is expressed on all hematopoietic cell types, whereas IL-7 ra is predominantly expressed by lymphocytes (including B and T lymphoid precursors, naive T cells, and memory T cells). Low expression of IL-7 ra was observed on regulatory T cells compared to effector/naive T cells expressing higher levels, and therefore CD127 was used as a surface marker to distinguish between the two populations. IL-7R α is also expressed on innate lymphocytes NK and Gut Associated Lymphoid Tissue (GALT) -derived T cells. The IL-7R α (CD127) chain is shared with TSLP (tumor stromal lymphopoietin), and CD132 is shared with IL-2, IL-4, IL-9, IL-15, and interleukin 21. Two major signaling pathways are induced by the (1) Janus kinase/STAT pathway (i.e., Jak-STAT-3 and 5) and (2) phosphoinositide-3 kinase pathway (i.e., PI3K-Akt) of CD127/CD 132. IL-7 administration is well tolerated in patients and results in expansion of CD8 and CD4 cells and a relative reduction in CD4+ T regulatory cells. Recombinant naked IL-7 or IL-7 fused to the N-terminal domain of antibody Fc has been tested clinically, the rationale being to increase the half-life of IL-7 and improve the long-term sustained efficiency of therapy by fusion of the Fc domain. Targeting IL-7 signaling should be more promising than IL-2 signaling, since IL-2 acts on both tregs and T effector cells, whereas IL-7 selectively activates T effector cells.

To improve the efficacy of anti-PD 1 immunotherapy and overcome potential anti-PD-1 resistance in patients, developing a combination therapy targeting IL-7 signaling may be a good strategy to stimulate T cell infiltration, maintain T cell effector capacity, and promote a durable memory T cell response, without stimulating expansion and survival of regulatory T cells. Indeed, anti-PD-1 treatment increased the expression of CD127 on depleted T cells, thereby increasing its ability to respond to IL-7 and improving the co-production of interferon-gamma (IFN-. gamma.) and tumor necrosis factor-alpha (TNF-. alpha.) (Pauken et al, Science, 2016, 12/2/2016; 354(6316):1160-1165, Shi et al, Nat Commin, 2016, 8/2016; 7: 12335).

However, the validation and development of combination immunotherapy is strongly limited by the cost of biotherapeutics and the limited availability of such immunotherapies. Therefore, there remains a great need in the art for new and improved agents for safe immunotherapy, in particular for safe immunotherapy targeting T cells against cancer and having a potent positive impact on the adaptive immune response (in particular T cell immune response). The inventors have taken an important step forward with the invention disclosed herein.

Disclosure of Invention

The present inventors provide bifunctional molecules comprising an anti-hPD-1 antibody and human IL-7, which are promising for a variety of therapeutic applications, in particular for the treatment of cancer. The present invention is based on the development of antibodies specifically targeting human PD-1, which antibodies show high binding affinity for PD-1 and strongly compete with its ligands PDL-1 and/or PD-L2. Surprisingly, fusion of the N-terminal end of IL-7 to the C-terminal end of the Fc region of the anti-hPD-1 antibody allowed retention of its high affinity for CD127(IL7 receptor) to a similar extent as endogenous IL-7, indicating potent IL-7R activation. Fusion of the Fc domain to IL-7 also increases the half-life of the product. In addition, the bifunctional anti-PD 1/IL-7 molecules disclosed herein allow for accumulation of IL-7 in PD-1+ T cell infiltrates and relocation of IL-7 on PD-1+ T cells. In particular, the anti-PD-1/IL-7 bifunctional molecule induces the proliferation and activation of initially, partially depleted and fully depleted T cell subsets reflected by cytokine (e.g., IFN γ) secretion and integrin (e.g., α 4 and β 7 and LFA-1) expression). This anti-hPD-1/IL-7 bifunctional molecule has the potential to overcome the associated resistance mechanisms and improve the efficacy of anti-PD-1 immunotherapy.

In a first aspect, the present invention relates to a bifunctional molecule comprising:

(a) An anti-human PD-1 antibody or antigen-binding fragment thereof, comprising:

(i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2 and HCDR3, and

(ii) a light chain variable domain (VL) comprising LCDR1, LCDR2, and LCDR3, and

(b) human interleukin 7(IL-7) or a fragment or variant thereof,

wherein said antibody or fragment thereof is covalently linked to said human IL-7 or fragment or variant thereof as a fusion protein, preferably via a peptide linker.

In particular, the N-terminal end of the human IL-7 or fragment thereof is linked to the C-terminal end of the heavy chain or light chain or both of the anti-human PD-1 antibody or antigen-binding fragment thereof.

In one aspect, the antibody or antigen binding fragment thereof is a chimeric, humanized or human antibody.

In one particular aspect, the present invention relates to a bifunctional molecule comprising an anti-human PD-1 antibody, or an antigen-binding fragment thereof, comprising or consisting of:

(i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2 and HCDR3, and

(ii) a light chain variable domain (VL) comprising LCDR1, LCDR2 and LCDR3,

wherein:

-the heavy chain CDR1(HCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 1;

-the heavy chain CDR2(HCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 2;

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 3, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N and E;

-the light chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 12, wherein X is G or T;

-the light chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO:15,

-the light chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16.

In particular, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises or consists of: (a) a VH comprising or consisting of the amino acid sequence of SEQ ID NO 17, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N and E; (b) VL comprising or consisting of the amino acid sequence of SEQ ID NO. 26, wherein X is G or T.

More particularly, the present invention relates to a bifunctional molecule comprising an anti-human PD-1 antibody or an antigen-binding fragment thereof, which comprises or consists of: (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:24, and (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28.

Alternatively, the anti-PD 1 antibody is selected from pembrolizumab, nivolumab, pidilizumab, cimiraprizumab, PDR001, and monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4a11, 7D3, and 5F 4.

In particular, the IL-7 or variant thereof comprises or consists of an amino acid sequence having at least 75% identity to wild-type human IL-7 (wth-IL-7). In a particular aspect, the IL-7 comprises or consists of the amino acid sequence shown in SEQ ID NO. 51.

Alternatively, said IL-7 is an IL-7 variant, wherein said IL-7 variant exhibits at least 75% identity to wild-type human IL-7(wth-IL-7) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:51, wherein said variant comprises at least one amino acid mutation that i) reduces the affinity of said IL-7 variant for the IL-7 receptor as compared to the affinity of wth-IL-7 for IL-7R, and ii) improves the pharmacokinetics of a bifunctional molecule comprising said IL-7 variant as compared to a bifunctional molecule comprising IL-7.

In particular, the at least one mutation is an amino acid substitution or a group of amino acid substitutions selected from: (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F or W142Y, (iii) D74E, D74Q or D74N, (iv) Q11E, Y12F, M17L, Q22E and/or K81R; or any combination thereof.

In one aspect, the IL-7 variant comprises a set of amino acid substitutions selected from: C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, and C47S-C92S and C34S-C129S.

In another aspect, the IL-7 variant comprises an amino acid substitution selected from the group consisting of: W142H, W142F and W142Y.

In another aspect, the IL-7 variant comprises an amino acid substitution selected from the group consisting of: D74E, D74Q, and D74N.

Preferably, the IL-7 variant comprises or consists of the amino acid sequence shown in SEQ ID NO: 53-66. Even more preferably, the IL-7 variant comprises or consists of the amino acid sequence shown in SEQ ID NO 54, 56 or 63.

In a particular aspect, the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant domain (preferably an IgGl or IgG4 heavy chain constant domain).

In a more specific aspect, the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgGl heavy chain constant domain, optionally with a substitution or combination of substitutions selected from: T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A + M252Y/S254T/T256E; K322A and K444A, preferably selected from the following: N297A, optionally in combination with M252Y/S254T/T256E, and L234A/L235A.

In another more particular aspect, the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgG4 heavy chain constant domain, optionally with a substitution or combination of substitutions selected from the group consisting of: S228P; L234A/L235A, S228P + M252Y/S254T/T256E.17 and K444A.

Optionally, the antibody or fragment thereof is linked to IL-7 or a variant thereof via a linker sequence, preferably selected from (GGGGS)₃、(GGGGS)₄、(GGGGS)₂GGGGS, GGGS, GGG, GGS and (GGGS) ₃Even more preferably (GGGGS)₃Or (GGGS)₃。

In a very specific aspect, the IL-7 variant comprises an amino acid substitution set selected from the group consisting of: C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q and D74N; the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgGl heavy chain constant domain, optionally with a substitution or combination of substitutions selected from: T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A + M252Y/S254T/T256E; K322A and K444A, preferably selected from the following: N297A, optionally in combination with M252Y/S254T/T256E, and L234A/L235A; and the antibody or fragment thereof is linked via a linker (GGGGS)₃Linked to an IL-7 variant.

In another aspect, the present invention relates to an isolated nucleic acid molecule or set of isolated nucleic acid molecules encoding a bifunctional molecule as disclosed herein; a vector comprising a nucleic acid or a set of nucleic acid molecules disclosed herein; and/or a host cell comprising a vector, nucleic acid or set of nucleic acid molecules disclosed herein.

In another aspect, the present invention relates to a method for producing said bifunctional molecule comprising the step of culturing a host cell as disclosed herein and optionally the step of isolating said bifunctional molecule.

In another aspect, the present invention relates to a pharmaceutical composition comprising a bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector or host cell as disclosed herein and a pharmaceutically acceptable carrier.

Optionally, the pharmaceutical composition further comprises an additional therapeutic agent, preferably selected from the following: alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferative agents, antivirals, aurora kinase inhibitors, apoptosis-promoting agents (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T-cell cement) antibodies, antibody drug conjugates, biological response modifiers, Bruton's Tyrosine Kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, Heat Shock Protein (HSP) -90 inhibitors, Histone Deacetylase (HDAC) inhibitors, hormonal therapies, immunological drugs, inhibitors of apoptosis protein Inhibitors (IAPs), intercalating antibiotics, anti-viral agents, aurora kinase inhibitors, apoptosis-promoting agents (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T-cell cement) antibodies, antibody drug conjugates, biological response modifiers, and anti-inflammatory drugs, Kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target protein of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate) -ribose polymerase (PARP) inhibitors, platinum-based chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid/retinoid plant alkaloids (deltoids plants), small inhibitory ribonucleic acids (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines and the like, epitopes or neo-epitopes from tumor antigens, and combinations of one or more of these agents.

In particular, the pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell is used as a medicament.

The present invention finally relates to a pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell as disclosed herein for use as a medicament, preferably for use in the treatment of a disease of a cancer selected from: a hematologic malignancy or solid tumor that expresses PD-1 and/or PD-L1, such as a cancer selected from: lymphohematopoietic tumors, angioimmunoblastic T-cell lymphomas, myelodysplastic syndromes and acute myeloid leukemia, cancers induced by viruses or associated with immunodeficiency, such as cancers selected from: kaposi's sarcoma (e.g., associated with kaposi's sarcoma herpes virus); cervical cancer, anal cancer, penile cancer and vulvar squamous cell carcinoma and oropharyngeal cancer (e.g., associated with human papilloma virus); b-cell non-hodgkin's lymphoma (NHL) including diffuse large B-cell lymphoma, burkitt's lymphoma, plasmablast lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classical hodgkin's lymphoma, and lymphoproliferative disorders (e.g., associated with epstein-barr virus (EBV) and/or kaposi's sarcoma herpes virus); hepatocellular carcinoma (e.g., associated with hepatitis b and/or hepatitis c virus); merkel cell carcinoma (e.g., associated with merkel cell polyoma virus (MPV)); and cancers associated with Human Immunodeficiency Virus (HIV) infection; and a cancer selected from: metastatic or non-metastatic, melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, hodgkin's lymphoma, head and neck cancer, urothelial cancer, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic merkel cell cancer, gastric or esophageal cancer, and cervical cancer.

Optionally, the bifunctional molecule, the pharmaceutical composition, the isolated nucleic acid or the set of isolated nucleic acid molecules, the vector, or the host cell for use in combination with radiotherapy or another therapeutic agent, preferably selected from the group consisting of an alkylating agent, an angiogenesis inhibitor, an antibody, an antimetabolite agent, an antimitotic agent, an antiproliferative agent, an antiviral agent, an aurora kinase inhibitor, an apoptosis promoter (e.g., a Bcl-2 family inhibitor), a death receptor pathway activator, a Bcr-Abl kinase inhibitor, a BiTE (bispecific T-cell cement) antibody, an antibody drug conjugate, a biological response modifier, a Bruton's Tyrosine Kinase (BTK) inhibitor, a cyclin-dependent kinase inhibitor, a cell cycle inhibitor, a cyclooxygenase-2 inhibitor, a DVD, a pharmaceutical composition, a method of treating cancer, a method or a method for example, a method for use or a method for the use of a method for the like, Inhibitors of leukemia virus oncogene homolog (ErbB2) receptors, inhibitors of growth factors, inhibitors of Heat Shock Protein (HSP) -90, inhibitors of Histone Deacetylase (HDAC), hormone therapy, immunological drugs, inhibitors of Inhibitor of Apoptosis Proteins (IAP), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, inhibitors of mammalian target proteins of rapamycin, microRNA, inhibitors of mitogen-activated extracellular signal-regulated kinase, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), inhibitors of poly (ADP) -ribose polymerase (PARP), platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/retinoid-like plant alkaloids, Small inhibitory ribonucleic acids (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines and the like, epitopes or neo-epitopes from tumor antigens, and combinations of one or more of these agents.

The pharmaceutical compositions, bifunctional molecules, nucleic acids or sets of nucleic acid molecules, vectors, or host cells described herein are used to inhibit the suppressive activity of regulatory T cells, activate T effector cells, and/or stimulate the proliferation of naive, partially depleted, and fully depleted T cells.

The pharmaceutical compositions, bifunctional molecules, nucleic acids or sets of nucleic acid molecules, vectors, or host cells described herein may also be used to treat infectious diseases, preferably chronic infectious diseases, even more preferably chronic viral infections. Preferably, the infectious disease is caused by a virus selected from the group consisting of: HIV, hepatitis virus, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus and arbo encephalitis virus.

Drawings

FIG. 1: ELISA assay for binding to PD-1. Human recombinant PD-1(rPD1) protein was immobilized and different concentrations of antibody were added. The revealing was performed using an anti-human Fc antibody coupled to peroxidase. Colorimetric methods were determined at 450nm using TMB substrate. A. The binding of anti-PD 1(■), anti-PD 1VL-IL7(o), and anti-PD 1VH-IL7(●) was compared to recombinant PD1(rPD 1). B. Comparison of chimeric Bicki (●) versus humanized Bicki (■) form of anti-PD 1 antibody fused to IL7 on the heavy and/or light chain: anti-PD 1VH-IL7 (left panel), anti-PD 1VL-IL7 (middle panel) or anti-PD 1VH and VL-IL7 (right panel). Different concentrations of the molecule were added to the human PD1 recombinant protein coated plates. C. Bicki anti-PD-1/IL-7 binding constructed with Keytruda (●) and Opdivo (■) scaffolds was tested at various concentrations on plates coated with human PD1 recombinant protein.

FIG. 2: the Bicki anti-PD 1-IL7 molecule blocks the antagonistic ability of the PD-1/PD-L1 and PD1/PDL2 interactions. Elisa assay: PD-L1 was immobilized on a Maxisorp plate and complex antibody + biotinylated recombinant human PD-1 was added. The complex was generated with a fixed concentration of PD1 (0.6. mu.g/mL) and tested for various concentrations of anti-PD 1(■), anti-PD 1VH-IL7(●) or anti-PD 1VL-IL7(o) antibodies. B: affinity assessment of PD-1 recombinant proteins preincubated on human PD-L2 recombinant protein with anti-PD 1 antibody, anti-PD 1VH-IL7 or anti-PD 1VL-IL7 antibody was performed by Biacore. Human recombinant PD-L2 was immobilized on a CM5 biochip and complex antibody (200nM) + recombinant human PD-1(100nM) was added. Data are expressed as% relative response of interaction measured by Biacore: 100% ═ PD-1 relative response.

FIG. 3: the Bicki anti-PD 1-IL7 molecule stimulates the IL-7R signaling pathway as measured by ex vivo STAT5 phosphorylation in human PBMC. PBMCs isolated from peripheral blood of healthy volunteers were incubated with recombinant IL-7(rIL7) (Grey ●) +/-anti-PD 1 (Grey), anti-PD 1VH-IL7(■) or anti-PD 1VL-IL7(●) for 15 minutes. Cells were then fixed, permeabilized and stained with AF 647-labeled anti-pSTAT 5 (clone 47/Stat5(pY 694)). Data were obtained by calculating and normalizing the MFI pSTAT% pSTAT5+ population (100% ═ rIL-7(57.5nM)) and represent the average of 3 different donors in two independent experiments.

FIG. 4: bicki anti-PD 1-IL7 enhanced T cell activation in vitro. (A) Discover' x PD-1Path Hunter Bioassay: jurkat T cells stably expressing an engineered PD-1 receptor fused to a β -gal fragment (ED) and an engineered SHP1 fused to a complementary β -gal fragment (EA). Addition of anti-PD 1 antagonist antibodies blocked PD-1 signaling, resulting in loss of bioluminescent signal (RLU). anti-PD 1(■) or anti-PD 1VH-IL7(●) were tested at different molar concentrations. Data are expressed in RLU (relative luminescence signal). (B) Promega PD-1/PD-L1 bioassay: the (1) effector T cells (Jurkat stably expressing PD-1, NFAT-induced luciferase) were co-cultured with (2) activating target cells (CHO K1 cells stably expressing PDL1 and surface proteins designed to activate homologous TCRs in an antigen-independent manner). In the addition of BioGlo^TMAfter fluorescein, the luminescence was quantified and reflected in T cell activation. The series of molar concentrations of anti-PD 1 antibody +/-recombinant IL-7(rIL-7) or Bicki anti-PD 1VH-IL7 or anti-PD 1VL-IL7 antibody were tested. Each point represents EC50 for one experiment. (C) Bicki anti-PD-1/IL-7 constructed with Keytruda or Opdivo backbones has the ability to stimulate NFAT. T cell activation was also tested using the Promega PD-1/PD-L1 bioassay. Keytruda alone or Opdivo alone (●) was tested at different concentrations compared to pembrolizumab VH IL-7 or nivolumab VH IL-7 (. smallcircle.).

FIG. 5: the Bicki anti-PD 1-IL7 molecule enhances IFNg secretion. T cells isolated from peripheral blood of healthy volunteers were stimulated with plates coated with OKT3/PDL1 (2 and 5. mu.g/ml, respectively) in the presence of isotype control, anti-PD 1+/-rIL7, anti-PD 1VH-IL7, anti-PD 1VL-IL7, isotype VH-IL7 (final antibody concentration of 5. mu.g/ml). On day 5 post-stimulation, secreted IFN γ was quantified by sandwich ELISA. Results are representative of 4 donors (n ═ 2 experiments).

FIG. 6: bicki anti-PD 1-IL7 enhanced T cell proliferation to a similar extent as recombinant soluble IL-7. PBMC cells isolated from peripheral blood of healthy volunteers were stimulated with anti-CD 3/CD 28. 20 hours post-stimulation, PBMCs were harvested and restimulated on OKT3/PDL1 coated plates (2 and 5. mu.g/mL, respectively) in the presence of rIL-7 or Bicki anti-PD 1VH-IL 7. Fixed doses (29nM BiCKI or 3,2nM rIL-7) or (B) multiple doses of rIL-7 alone (□) were tested against PD1 or against PD1VH-IL7(●) or against PD1VL-IL7(■) or the isoform VH IL-7 (. tangle-solidup.). T cell proliferation was assessed by 3H thymidine incorporation at day 5 post-stimulation. Data were normalized (100% ═ rIL 710 nM) and represent the mean values obtained on 3 different donors. EC50(pM) refers to the concentration required to achieve 50% T cell proliferation.

FIG. 7 Bicki anti-PD 1VH-IL7 stimulates integrin expression on the surface of T cells. Human PBMC were incubated without any molecules (grey histogram) for 3 days, or with rIL-7/rIL-2 or rIL-7(50ng/mL) or anti-PD-1 or anti-PD 1VH-IL7 (5. mu.g/mL) for 3 days. A. Cell surface expression analysis of α 4 and β 7 integrins. FACS was analyzed by LSR and data expressed as fold change normalized to 1, which corresponds to median fluorescence of control (untreated) for each donor. B. LFA-1 cell surface expression analysis by FACS using LSR (CD11a and CD 18). The results are expressed as median fluorescence. Each dot represents one donor of 3 independent experiments.

Figure 8 modeling of chronic antigen stimulation of T cells leading to T cell depletion. Human PBMCs were stimulated repeatedly every 3 days on CD3 CD28 coated plates (3. mu.g/mL OKT3 and 3. mu.g/mL CD28.2 antibody). (A) 24 hours after stimulation, T cells were stained for PD-1, Lag3, and Tim3 inhibitory receptors. Expression was analyzed by flow cytometry using fluorochrome-labeled antibodies and FACS LSRII. Data are expressed as% positive cells from 3 donors (one donor-one curve). (B) T cell proliferative capacity was determined by thymidine 3H incorporation at day 5 after each stimulation. (C) The supernatant IFNg secretion was analyzed by ELISA (pg/ml) 24 hours after each stimulation.

Figure 9. T cell depleted IL7 pathway activation: the response of depleted T cells to 15 min incubation of cells with either rIL-7 or Bicki anti-PD 1VH-IL7 was measured by measuring phosphorylation of STAT5 48 hours after each stimulation. Cells were then fixed, permeabilized and stained with AF 647-labeled anti-pSTAT 5 (clone 47/Stat5(pY 694)). A. Grey histograms indicate cells treated with rIL7, and black histograms indicate cells treated with Bicki anti-PD 1VH-IL 7. Data were normalized (MFI pSTAT% pSTAT5 population) and represent 4 different donors. (B) ED50(pM) of pSTAT5 was determined for each stimulus, and it refers to the concentration of rIL-7(■ gray) or Bicki anti-PD 1VH-IL7(■ black) required to achieve 50% activation of pSTAT 5.

FIG. 10: proliferation of depleted T cells following IL-7 stimulation. Human PBMC were stimulated repeatedly on CD3 CD28 coated plates (3. mu.g/mL OKT3 and 3. mu.g/mL CD28.2 antibody). (A) T cells were re-stimulated 24 hours after each stimulation on OKT 3-coated plates (2. mu.g/mL) in the presence of anti-PD 1, rIL-7 or Bicki anti-PD 1-IL7 (anti-PD 1VH-IL7 or anti-PD 1VL-IL 7). H3 incorporation assays were performed on day 5 to determine T cell proliferation. Raw proliferation data for one donor after Stimulation (STIM)3, 4 and 5 are shown. (B) T cells after 3 stimulations were either not stimulated anymore (no Stim) or restimulated on plates (2 and 5. mu.g/mL, respectively) coated with anti-CD 3(StimOKT3), anti-CD 3+ recombinant PDL1(Stim OKT3/PDL1) or anti-CD 3+ recombinant PDL2(StimOKT3/PDL 2). H3 incorporation assays were performed on day 5 and the data were normalized (1 ═ H3 incorporation using isotype antibodies). n-4 donors and 2 different experiments.

Fig. 11 suppressive activity of tregs on CD8 effector T cell proliferation.

CD8+ effector T cells and CD4+ CD25 high CD127 low tregs were isolated from peripheral blood of healthy donors and stained with cell proliferation dye (CPDe 450 was used against CD8+ T cells). Treg/CD8+ Teff were then co-cultured on OKT 3-coated plates at a ratio of 1:1 for 5 days in the presence or absence of rIL-7, anti-PD-1 + rIL-7, anti-PD 1VH-IL 7. Proliferation of effector T cells was analyzed by cytofluorimetry. A. Data represent the following% proliferation: teff alone (black histogram) or Teff co-cultured with tregs (grey histogram) +/-SEM, based on the loss of CPD markers in the CD 8T effector cell population. (n-4 donors in 4 different experiments). B. After incubation with different equimolar doses of IL-7 (a), IL-2(●), IL-15(■) or anti-PD 1VH-IL7 (xxx), CPD proliferation dye was used to assess Treg proliferation.

FIG. 12: humanized mouse modelIn vivo efficacy of Bicki anti-PD 1-IL7 antibodies in type I. Human PBMCs were injected intraperitoneally into mice. Mice were treated with anti-PD 1 antibody alone or Bicki anti-PD 1VH-IL7(5mg/kg) every two weeks. Blood was collected and mice sacrificed on day 16 post injection. (A) Among the population of human CD45+ cells, the percentage of peripheral human CD 3T cells was analyzed by flow cytometry. Each dot represents one mouse. (B) Human IFNg in plasma was quantified by ELISA. Each dot represents one mouse. (C) Infiltration of human CD3+ cells in the colon, liver and lung was quantified by immunohistofluorescence. The proximal and distal colon, liver and lung were embedded in Tissue In OCT, and staining was performed for Dapi and human CD 3. Each point represents one mouse and for the colon each point represents the mean of CD3+ counts for 3 sections.

FIG. 13: immunophenotype of human tumor infiltrating lymphocytes. T-cells were extracted from renal cancer (. DELTA.), metastatic colorectal cancer (□), pancreatic cancer (. smallcircle.), hepatocellular carcinoma (●) (. diamond) and stained for CD3, CD4, CD8, PD-1, CD127 and CD 132. Immunofluorescence was analyzed by FACS LSRII. Data represent CD4+ CD3+ or CD8+ CD3+ populations.

FIG. 14: STAT5 activation of intratumoral regulatory T cells or effector T cells in ex vivo tumors. Cells were extracted from tumors from patients with schwannoma (T.X), renal cancer (O), hepatocellular carcinoma (□) (■), metastatic colorectal cancer (●) or pancreatic cancer (a-solidup), and treated with rIL-7 or Bicki anti-PD 1VH-IL7(29nM) for 15 min. Cells were then fixed, permeabilized and stained for pSTAT5 (clone 47/Stat5(pY694)), CD3 and Foxp 3. Histograms represent the median pSTAT5 fluorescence in CD3+ FoxP3+ population (regulatory T cells) or CD3+ FoxP3- (effector T cells).

FIG. 15 in vitro study of IFN γ secretion following treatment of human cancer biopsies with Bicki anti-PD 1-IL 7: human tumor biopsies were mashed in complete media to isolate cells. Cells were resuspended at a concentration of 5 μ g/mL in intact media containing isotype control, anti-PD 1, B12-IL7 isotype control antibody (isotype-VH IL-7), anti-PD-1 + recombinant IL-7, or anti-Bicki anti-PD 1VH-IL 7. After 48 hours, supernatants were collected and analyzed for IFN γ secretion using MSD technology (Meso scale Discovery). A. Results representing colorectal cancer cells, and B. results of individual tumors (CC: colorectal cancer biopsy, HCC: liver cancer biopsy, KC: renal cancer).

Figure 16 STAT5 activation of regulatory or effector T cells in tumors following treatment with Bicki anti-PD 1VH-IL 7. (A) Percentage of intratumoral FoxP3 Treg cells entering tumors of colorectal, schwannoma, renal cancer or hepatocellular carcinoma. (B) Cells from colorectal cancer (●), schwannomas (° o) and pancreatic cancer (□) were analyzed for STAT5 activation in FoxP3-CD3+ effector T cells compared to FoxP3-CD3+ Treg cells after treatment with rIL7 or with anti-PD 1VH-IL7(29nM) (15 min incubation). Cells were then fixed, permeabilized and stained for pSTAT5 (clone 47/Stat5(pY694)), CD3 and Foxp 3.

FIG. 17: ELISA assay for binding of bicki IL-7 mutants to PD-1. Human recombinant PD-1(rPD1) protein was immobilized and different concentrations of antibody were added. The revealing was performed using an anti-human Fc antibody coupled to peroxidase. Colorimetric methods were determined at 450nm using TMB substrate. A. PD-1 binding of a bifunctional molecule comprising an anti-PD 1 antibody and IL-7 mutated at amino acids D74, Q22, Y12F, M17, Q11, K81. B. PD-1 binding of a bifunctional molecule comprising IL-7 mutated at amino acid W142. C. PD-1 binding of bifunctional molecules mutated in the disulfide bond of IL-7(SS1, SS2 and SS3 mutants). All molecules tested in this figure were constructed with the IgG4m isotype and the GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains.

FIG. 18: ELISA assay for binding of IgG fused mutant IL-7 to CD 127. The PD-1 recombinant protein was immobilized on a plate, and then a bifunctional anti-PD-1 IL-7 molecule was preincubated with CD127 recombinant protein (histidine tag, Sino reference 10975-H08H) and added to the wells. The revealing was performed using a mixture of anti-histidine antibody coupled to biotin + streptavidin coupled to peroxidase. Colorimetric methods were determined at 450nm using TMB substrate. A. CD127 binding of a bifunctional molecule comprising IL-7 mutated at amino acids D74, Q22, M17, Q11, Y12F, K81. B. CD127 binding of a bifunctional molecule comprising IL-7 mutated at amino acid W142.

FIG. 19: the IL-7-7R signaling pathway of different bifunctional molecules as measured by STAT5 phosphorylation. Human PBMCs isolated from peripheral blood of healthy volunteers were incubated with bifunctional anti-PD-1 IL-7 molecules for 15 minutes. Cells were then fixed, permeabilized and stained with AF 647-labeled anti-pSTAT 5 (clone 47/Stat5(pY 694)). Data were obtained by calculating the MFI pSTAT5 in CD 3T cells. A. pSTAT5 activation of an anti-PD-1 IL-7 bifunctional molecule comprising IL-7 mutated at amino acids D74, Q22, M17, Y12F, Q11, K81. B. Activation of pSTAT against a PD-1 IL-7 bifunctional molecule comprising IL-7 mutated at amino acid W142. C. anti-PD-1 IL-7 bifunctional molecule pSTAT5 comprising IL-7, SS2(● black) and SS3 (. tangle-solidup.) mutated in the disulfide bond of IL-7, activated in comparison to anti-PD-1 IL-7 WT (● grey). All molecules tested in this figure were constructed with the IgG4m isotype and the GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains.

FIG. 20: pharmacokinetics of anti-PD-1 IL-7 bifunctional molecules in mice. Mice were injected intravenously with one dose of IgG fused IL-7 wild-type or mutant IL-7. At various time points after injection, the concentration of molecules in serum was assessed by ELISA. A. IgG4-G4S3 IL7 WT (■ gray) was injected; IgG4-G4S3 IL 7D 74E (● black). B. IgG4-G4S3 IL7 WT (■ gray) or IgG4-G4S3 IL 7W 142H (● black) was injected. C. IgG4-G4S3 IL7 WT (■ gray) was injected; IgG4-G4S3 IL7 SS2(●) or IgG4-G4S3 IL7 SS3 (a). D. Correlation between area under the curve (AUC) calculated from PK per molecule compared to ED50 pSTAT5 (nM). All molecules tested in this figure were constructed with the IgG4m isotype and the GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains.

FIG. 21: the addition of a disulfide bond between anti-PD-1 and IL-7 reduced pSTAT5 activation, while increasing drug exposure in vivo. A. IL7R signaling as measured by activation of pSTAT5 on human PBMCs following treatment with anti-PD-1 IL-7 bifunctional molecule WT (grey ●) or anti-PD-1 IL-7 bifunctional molecule with additional disulfide bonds (black ●). B. Pharmacokinetics in mice of anti-PD-1 IL-7 bifunctional molecule WT (grey ●) or anti-PD-1 IL-7 bifunctional molecule with additional disulfide bonds (black ●). Mice were injected intravenously with a dose of the anti-PD-1 IL7 bifunctional molecule. At various time points after injection, the concentration of molecules in serum was assessed by ELISA. All molecules tested in this figure were constructed with the IgG4m isotype and the GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains.

FIG. 22: ELISA assay for binding to PD-1. Human recombinant PD-1(rPD1) protein was immobilized and different concentrations of antibody were added. The revealing was performed using an anti-human Fc antibody coupled to peroxidase. Colorimetric methods were determined at 450nm using TMB substrate. A. PD-1 binding of an anti-PD-1 IL-7 WT bifunctional molecule with IgG4m (● grey), an anti-PD-1 IL-7 WT bifunctional molecule with IgG1m (. tangle-solidup.) and an anti-PD-1 IL-7D 74E bifunctional molecule with IgG1m isotype (■) or an anti-PD-1 IL-7W 142H bifunctional molecule with IgG1m (. diamond.). B. In another experiment, PD-1 binding was tested for an anti-PD-1 IL-7 SS2 bifunctional molecule (■) with IgG4m isotype or an anti-PD-1 IL-7 SS2 bifunctional molecule (a-solidup) with IgG1 m.

FIG. 23: CD127 binding ELISA assay of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1N298A or IgG4 isotype. The antibody backbone-targeted recombinant protein was immobilized and the antibody fused to IL-7 was then preincubated with CD127 recombinant protein (histidine tag, Sino ref 10975-H08H). The revealing was performed using a mixture of anti-histidine antibody coupled to biotin and streptavidin coupled to peroxidase. Colorimetric methods were determined at 450nm using TMB substrate. A. CD127 binding of an anti-PD-1 IL-7W 142H bifunctional molecule with IgG4m isotype (● grey), an anti-PD-1 IL-7W 142H bifunctional molecule with IgG1m (. tangle-solidup in black) or an anti-PD-1 IL-7 WT bifunctional molecule with IgG1m isotype (● black). B. CD127 binding of an anti-PD-1 IL-7 SS2 bifunctional molecule with IgG4m isotype (● grey), an anti-PD-1 IL-7 SS2 bifunctional molecule with IgG1m (. tangle-solidup in black) or an anti-PD-1 IL-7 WT bifunctional molecule with IgG1m (● black). C. CD127 binding of an anti-PD-1 IL-7 SS3 bifunctional molecule with IgG4m isotype (● grey), an anti-PD-1 IL-7 SS3 bifunctional molecule with IgG1m (. tangle-solidup in black) or an anti-PD-1 IL-7 WT bifunctional molecule with IgG1m (● black). D. CD127 binding of anti-PD-1W 142H bifunctional molecules with isotype IgG1m (● black) or isotype IgG1m + YTE (● grey). CD127 was also tested against the PD-1D74E bifunctional molecule with isotype IgG1m (black a) or isotype IgG1m + YTE (grey a). All molecules tested in this figure were constructed with a GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains.

FIG. 24: IL-7R signaling analysis of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1N298A or IgG4 isotype. Human PBMC or jurkat PD1+ CD127+ cells were incubated with the anti-PD-1 IL7 bifunctional molecule for 15 minutes. Cells were then fixed, permeabilized and stained with AF 647-labeled anti-pSTAT 5 (clone 47/Stat5(pY 694)). Data were obtained by calculating the% of pSTAT5 in CD 3T cells. A. pSTAT5 signaling on human PBMCs following treatment of bifunctional molecules with the mutation D74E against PD-1 IL-7 with IgG4m isotype (● grey) or IgG1m isotype (black a). B. pSTAT5 signaling on human PBMCs following treatment of anti-PD-1 IL-7 SS2 with IgG4m isotype (● gray) or anti-PD-1 IL-7 SS2 with IgG1m (black a). C. pSTAT5 signaling on human PBMCs following treatment with IgG4m isotype (● gray) or IgG1m (black a-solidup) anti-PD-1 IL-7 SS 3. (left panel) treatment of pSTAT5 signaling on jurkat PD1+ CD127+ cells following anti-PD-1 IL-7 WT constructed with IgG4m (● grey) or IgG1m (a black) isotype. (right panel) pSTAT5 signaling after treatment of anti-PD-1 IL-7 SS2 with IgG4m isotype (● grey) or anti-PD-1 IL-7 SS2 with IgG1m (a black).

FIG. 25: bifunctional molecules against PD-1 IL-7 mutations enhance T cell activation in vitro. Promega PD-1/PD-L1 bioassay: co-culture of (1) effector T cells (Jurkat stably expressing PD-1, NFAT-induced luciferase) and (2) activating target cells (CHO K1 cells stably expressing PDL1 and surface proteins designed to activate homologous TCRs in an antigen-independent manner). Addition of BioGlo ^TMAfter fluorescein, luminescence was quantified and reflected in T cell activation. A series of molar concentrations of anti-PD 1 antibody +/-recombinant IL-7(rIL-7) or anti-PD 1IL7 bifunctional molecule were tested. Each point represents an experiment. A. NFAT activation of anti-PD-1 IL-7 WT bifunctional molecule (● grey) or anti-PD-1 (. tangle-solidup.) or anti-PD-1 + rIL-7 (. smallcircle.) with IgG4m isotype. B. NFAT activation of anti-PD-1 IL-7D 74E IgG4m (●), PD-1 IL-7D 74E IgG1m (. tangle-solidup) and anti-PD-1 alone (black a). C. Having Iganti-PD-1 IL-7W 142H bifunctional molecule of G4m (●), PD-1 IL-7W 142H bifunctional molecule with IgG1m (dashed a), and NFAT activation alone against PD-1 (black a). D. anti-PD-1 IL-7 SS2 bifunctional molecule (●) with IgG4m and NFAT activation alone against PD-1 (black a).

FIG. 26: pharmacokinetics of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1m or IgG4m isotype. Mice were injected intravenously with a dose of IgG fused to IL-7 wild-type or mutant IL-7. Drug concentrations in serum were assessed by ELISA at various time points post-injection. A. Pharmacokinetics of anti-PD-1 IL-7 WT bifunctional molecule with IgG4m (● grey dotted line), anti-PD-1 IL-7 WT bifunctional molecule with IgG1m (● grey dotted line), anti-PD-1 IL-7D 74E bifunctional molecule with IgG1m (a black dotted line), anti-PD-1 IL-7W 142H bifunctional molecule with IgG4m (o black line), anti-PD-1 IL-7W 142H bifunctional molecule with IgG1m (o dotted black line), anti-PD-1 IL-7 SS3 with IgG4 (■ plain line), and anti-PD-1 IL-7 SS3 with IgG1m (■ dotted line). B. Pharmacokinetics of anti-PD-1 IL-7D 74E, D74Q, W142H, D74E + W142H mutant bifunctional molecules with IgG1 m.

FIG. 27 is a schematic view showing: pharmacokinetics of an anti-PD-1 IL-7 bifunctional molecule constructed with the IgG1N298A + K444A isotype. Mice were injected intravenously with a dose of anti-PD-1 IL 7D 74E bifunctional molecule of isotype IgG1N298A (■) or isotype IgG1m + K444A mutant isotype (●). At various time points after injection, the concentration of the antibody was assessed by ELISA.

FIG. 28: the length of the linker does not significantly affect pharmacokinetics, but reduces stimulation of IL-7R signaling. A. Pharmacokinetics of anti-PD-1 IL-7 WT bifunctional molecules constructed with different linkers ((GGGGS), (GGGGGGS) 2, (GGGGS) 3). B. Pharmacokinetics of anti-PD-1 IL-7D 74 bifunctional molecules constructed with different linkers ((GGGGS), (GGGGS)2, (GGGGS) 3). C. Pharmacokinetics of anti-PD-1 IL-7W 142H bifunctional molecules constructed with different linkers ((GGGGGGS) 2, (GGGGS) 3). Mice were injected intravenously with a dose of IgG fused to IL-7 wild-type or mutant IL-7. At various time points after injection, the concentration of IgG fused to IL-7 was assessed by ELISA. D. pSTAT5 signaling of anti-PD-1 IL-7 bifunctional molecules constructed without a linker or with a GGGGS, (GGGGS)2, (GGGGS)3 linker.

FIG. 29: the anti-PD-1 IL-7 mutant preferentially targets PD-1+ CD127+ cells over PD-1-CD127+ cells. Jurkat cells expressing CD127+ or co-expressing CD127+ and PD-1+ were stained with 45nM of anti-PD-1 IL-7 bifunctional molecule and revealed with anti-IgG-PE (Biolegend, clone HP 6017). Data represent the ratio of the median fluorescence on PD-1+ CD127+ Jurkat cells to the median fluorescence obtained on PD 1-cell CD127+ Jurkat cells. In this assay, anti-PD-1 IL-7 WT bifunctional molecule IgG1m, anti-PD-1 IL-7D 74E bifunctional molecule IgG1m, anti-PD-1 IL-7W 142H bifunctional molecule IgG1m, anti-PD-1 IL-7 SS2 bifunctional molecule IgG4m, and anti-PD-1 IL-7 SS3 bifunctional molecule IgG1m were tested.

FIG. 30: the anti-PD-1 IL-7 molecule enhances T cell proliferation and shows preclinical safety in cynomolgus monkeys. c targeting PD-1+ CD127+ cells relative to PD-1-CD127+ cells. Cynomolgus monkeys were injected intravenously with a dose of bicki anti-PD-1 IL-7 WT (6,87nM/kg (n ═ 2) or 34,35nM (n ═ 1)). Blood analysis was performed until day 15 or 4 hours post injection. A. Lymphocyte counts in peripheral blood were assessed at various time points with Bicki anti-PD-1 IL-7 WT (n-2) injected at 6,87 nM/kg. B. Proliferation of CD4/CD8 or B cells was assessed by flow cytometry using Ki67/CD4/CD8 and CD19 markers, with Bicki anti-PD-1 IL-7 WT (n-2) injected at 6,87 nM/kg. C. Pscki anti-PD-1 IL-7 WT (n ═ 2) was analyzed by FACS in CD3+ T cells at multiple time points for pSTAT5 injected at 6,87 nM/kg. D/E/F/G and H. Biochemical and cellular blood analyses were evaluated at multiple time points.

FIG. 31: description of the mechanism of action of Bicki anti-PD 1-IL-7 according to the invention.

Detailed description of the invention

Brief introduction to the drawings

The antibodies of the invention are bifunctional in that they combine the specific anti-PD-1 effect with the effect of human interleukin-7 fused to an anti-PD-1 antibody. Indeed, the present invention relates to a bifunctional molecule comprising an anti-PD-1 antibody and IL-7, said interleukin being covalently linked to the polypeptide chain of said anti-PD-1 antibody (the light chain or the heavy chain or both or a fragment thereof of said antibody). Preparing a chain of the anti-PD-1 antibody or fragment thereof and the IL-7 as a fusion protein. In this particular aspect, the N-terminal end of the IL-7 is linked, optionally via a peptide linker, to the C-terminal end of the chain of the anti-PD-1 antibody or fragment thereof.

As is known to those skilled in the art, T cells may not be able to adequately clear tumor cells due to a phenomenon known as T cell depletion observed in many cancers. For example, as described by Jiang, y., Li, y, and Zhu, B (Cell Death Dis 6, el792(2015)), depleted T cells in the tumor microenvironment can lead to overexpression of inhibitory receptors, effector cytokine production, and reduction of cytolytic activity, leading to failure of cancer elimination and often to cancer immune evasion. Restoring depleted T cells is therefore a clinical strategy envisaged for cancer therapy.

PD-1 is a major inhibitory receptor that regulates T cell depletion. In fact, T cells with high PD-1 expression have a reduced ability to eliminate cancer cells. anti-PD 1 therapeutic compounds, in particular anti-PD 1 antibodies, are used to block the inhibitory effect of the PD1-PDL1 interaction (PD1 on T cells and PDL1 on tumor cells) and T cell depletion in clinical cancer therapy. However, anti-PD 1 antibodies are not always effective enough to allow "heavy" activation of depleted T cells.

Applicants herein show that the bifunctional anti-PD 1-IL-7 molecule according to the present invention enhances activation of T cells (NFAT-mediated activation), in particular depleted T cells, compared to anti-PD-1 alone. In particular, the anti-PD 1-IL-7 bifunctional molecule induces the proliferation and activation of initially, partially depleted and fully depleted T cell subsets as reflected by cytokine (e.g., IFN γ) secretion. This anti-PD 1-IL-7 bifunctional molecule has the ability to overcome the associated resistance mechanisms and improve the efficacy of anti-PD-1 immunotherapy.

The applicant has in particular shown that the interaction of said anti-PD 1-IL-7 bifunctional molecule with a single T cell expressing i) PD1 and ii) IL-7 receptors results in an unexpected activation of the NFAT pathway (TCR signaling), with a positive effect on T cell activation, in particular on depleted T cell activation, promoting the ability of T cells to eliminate tumor cells.

This means that, in one aspect, the IL-7 of the bifunctional molecule of the invention targets the IL-7 receptor, activating the PSTAT5 pathway; in another aspect, the anti-PD 1 portion of the bifunctional molecule blocks the PD-1/PD-L1 interaction. The BICKI molecule targets both IL-7 and PD-1 on the same cell. This results in synergistic activation of tcr (nfat) signaling, which has never been observed when a combination of an anti-PD 1 antibody and IL-7 (as two separate compounds) was used alone. This activation cannot be provided by a bifunctional molecule targeting PD-L1. Indeed, it is known in the art that PD-L1 is expressed on tumor cells rather than on immune cells such as T cells.

Furthermore, the bifunctional anti-PD 1/IL-7 molecule allows for accumulation of IL-7 in PD-1+ T cell infiltration and relocation of IL-7 on PD-1+ T cells. Accumulation of IL-7 in the vicinity of such PD-1+ T cells is of particular interest in the context of depleted T cells that require high doses of IL-7 to activate or reactivate these T cells.

Synergistic effects on T cell activation were observed not only in the specific anti-PD-1 antibodies described in the present invention, but also in the other two anti-PD-1 references (i.e., Opdivo and Keytruda).

In addition, the bifunctional anti-PD 1/IL-7 molecule has the ability to promote T cell infiltration into tumors. Given that lack of T cell infiltration at the tumor site is a major obstacle to the efficacy of current anti-PD 1 antibody treatments, this ability is an advantage for optimizing anti-PD-1 antibody treatments.

In addition, the bifunctional anti-PD 1/IL-7 molecule can block Treg-mediated suppression. Thus, the bifunctional molecule is capable of specifically activating T effector cells but not T reg cells, whereas the anti-PD 1 antibody is not capable of inhibiting Treg inhibitory activity on T effector cells. The inventors then show that the bifunctional anti-PD 1-IL7 molecule biases T cell effectors towards T regulatory immune balance by stimulating effector T cell proliferation and survival while retaining (spark) regulatory T cells.

In addition, the IL7 anti-PD-1 bifunctional molecule has other advantages.

The inventors show that the anti-PD-1 bifunctional molecule of IL7 activates mainly T effector cells (Teff) but not T regulatory cells (Treg). The IL7 anti-PD-1 bifunctional molecule has the advantages of not promoting T Reg proliferation, inducing T Reg inactivation and inducing T cell (especially exhausted T cell) activation.

Furthermore, the IL7 anti-PD-1 bifunctional molecule allows the use of very favourable doses, showing a favourable therapeutic index (ratio between lethal dose (DL50) and therapeutically effective dose). In particular, for patients, IL7 in the range of 10 to 1500. mu.g/Kg, advantageously 200 and 1200. mu.g/Kg, can generally be used; patients can tolerate high doses of, for example, 1200 mug/Kg well. The IL7 anti-PD-1 bifunctional molecule then allows the production of therapeutic compounds at appropriate doses of the original compound and the final product. Indeed, a high dose tolerance of IL7 (e.g., about 1.2mg/kg of IL7) corresponds to an antibody amount of about 2mg/kg, which is a satisfactory dose for administration to a patient.

An advantage of the IL7 anti-PD-1 bifunctional molecule is that it can essentially target depleted T progenitor cells that are only partially depleted, which is a key goal to meet the above-mentioned medical needs. Additionally, chronic activation is important in the case of viral infections associated with similar depletion of T cells, and thus viral pathologies are included in the scope of the pathologies targeted by the new products of the present application.

Finally, in a particular aspect, the inventors have designed bifunctional molecules comprising IL-7 mutants or variants. The IL-7 mutant or variant is characterized by i) a reduced affinity for the IL-7 receptor (IL-7R) compared to the affinity of wild-type IL-7, and ii) an improved pharmacokinetics of the bifunctional molecule comprising the IL-7 variant compared to the bifunctional molecule comprising wild-type IL-7. First, the use of IL-7 variants in bifunctional molecules is important to increase the in vivo pharmacokinetics of the bifunctional molecule. Secondly, by reducing the affinity of the IL-7 variant for its receptor, it increases the ability of the bifunctional molecule to preferentially bind to the targeted T cells via the anti-PD-1 antibody part of the bifunctional molecule and to exhibit specific effects on these cells, while simultaneously exploiting the synergistic effects associated with the effects of the two parts of the bifunctional molecule on the same T cell. Bifunctional molecules with IL-7 variants have good binding and antagonistic activity against PD-1. Furthermore, the bifunctional molecule exhibits a suitable balance between its affinity for PD-1 and its affinity for IL-7R. Surprisingly, the invention It was observed that the bifunctional molecule with the constant domain of the IgG1 heavy chain had improved IL-7 variant activity (pStat5 signaling, synergy and CD127 binding) compared to the same molecule with the constant domain of the IgG4 heavy chain. In addition, a linker (GGGGS) was used between the antibody and IL-7₃The activity of the IL-7 variant (pStat5 signaling and CD127 binding) can be maximized.

The bifunctional molecules described herein have certain one or more of the following advantages:

-the bifunctional molecule induces the proliferation of an initial, partially depleted and fully depleted T cell subpopulation, and not only of a partially depleted T cell subpopulation as in anti-PD 1/PDL1 therapy. More particularly, it has a synergistic effect on T cell activation.

-the bifunctional molecule allows specific localization of IL-7 close to or on PD-1+ depleted T cells into tumors, targeting cells requiring higher concentrations of IL-7. It induces, inter alia, IL-7 accumulation in PD-1+ T cell infiltration and IL-7 relocation on PD-1+ T cells.

Although the inability of anti-PD-1 blockers to reprogram depleted T cells to active memory T cells limits the long-term clearance of tumors, the bifunctional molecule promotes the formation, survival and proliferation of memory T cells through the presence of IL-7. The bifunctional molecule then induces a durable anti-tumor immunity through a sustained and expanded memory T cell response.

Although the anti-PD 1/PD-L1 therapeutic efficacy is associated with pre-existing T cell infiltration and T cell effector functions, in particular IFN γ characteristics, the bifunctional molecule increases the proliferation of effector T cells and their ability to secrete IFN γ, with a synergistic effect.

The bifunctional molecule may reduce the immunosuppressive microenvironment by reducing the T reg population and inhibiting the secretion of TGF β (an inhibitory cytokine). More particularly, the bifunctional molecule specifically stimulates effector T cells without stimulating T regs.

-said bifunctional molecule, wherein IL-7 may be fused to the C-terminal part of said heavy and/or light chain, and wherein IL7 retains high affinity for CD127, similar to naked/native IL-7. The bifunctional molecule may be more effective in IL-7R activation and half-life.

It is produced in high yield as a bifunctional molecule.

-the bifunctional molecule decreases the immunosuppressive activity of Treg cells on the tumor microenvironment by decreasing the number of T regs. More particularly, the bifunctional molecule specifically stimulates effector T cells without stimulating T regs.

-the bifunctional compound increases the expression of integrins (i.e. α 4 and/or β 7 and LFAT) compared to the anti-PD 1 response alone, promoting infiltration of T cells into tissues and/or tumors. In particular, the bifunctional compounds promote T cell migration and tumor infiltration.

The bifunctional molecule may comprise IL-7 variants or mutants as recognized by the present inventors to maximize pharmacokinetics in vivo while maintaining IL-7 activity and antagonist activity of anti-DP-1 antibodies with a suitable affinity balance between IL-7 and IL-7R and anti-PD-1 and PD-1.

Definition of

In order that the invention may be more readily understood, certain terms are defined below. Additional definitions are set forth throughout the detailed description.

Unless defined otherwise, all technical terms, symbols, and other scientific terms used herein are intended to have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. In some instances, terms having commonly understood meanings are defined herein for clarity and/or ease of reference, and such definitions contained herein should not be construed as representing differences from those commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed by those skilled in the art using conventional methodologies.

As used herein, the terms "interleukin-7," "IL-7," and "IL-7" refer to mammalian endogenous secreted glycoproteins, particularly IL-7 polypeptides, derivatives and analogs thereof, that have substantial amino acid sequence identity and substantially equivalent biological activity as wild-type mammalian IL-7, e.g., in a standard bioassay or assay for IL-7 receptor binding affinity. For example, IL-7 refers to the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence: i) a naturally occurring or naturally occurring allelic variant of an IL-7 polypeptide, ii) a biologically active fragment of an IL-7 polypeptide, iii) a biologically active polypeptide analog of an IL-7 polypeptide, or iv) a biologically active variant of an IL-7 polypeptide. IL-7 may or may not contain its peptide signal. Alternative names for this molecule are "precursor B cell growth factor" and "lymphopoietin-1". Preferably, the term "IL-7" refers to human IL-7. For example, the human IL-7 amino acid sequence is about 152 amino acids (in the absence of a signal peptide) and has Genbank accession number NP-000871.1, which is located on chromosome 8q 12-13. Human IL-7 is described in UniProtKB-P13232.

As used herein, the terms "wild-type interleukin-7", "wt-IL-7" and "wt-IL 7" refer to mammalian endogenous secreted glycoproteins, particularly IL-7 polypeptides, derivatives and analogs thereof, which have substantial amino acid sequence identity and substantially equivalent biological activity as wild-type mammalian IL-7, e.g., in a standard bioassay or assay for IL-7 receptor binding affinity. For example, IL-7 refers to the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence: i) a naturally occurring or naturally occurring allelic variant of an IL-7 polypeptide, ii) a biologically active fragment of an IL-7 polypeptide, iii) a biologically active polypeptide analog of an IL-7 polypeptide, or iv) a biologically active variant of an IL-7 polypeptide. IL-7wt may or may not contain its peptide signal. Alternative names for this molecule are "precursor B cell growth factor" and "lymphopoietin-1". Preferably, the term "wt-IL-7" refers to human IL-7(wth-IL 7). For example, the human wt-IL-7 amino acid sequence is about 152 amino acids (in the absence of a signal peptide) and has Genbank accession number NP-000871.1, which is located on chromosome 8q 12-13. Human IL-7 is described in UniProtKB-P13232.

As used herein, the terms "programmed death 1", "programmed cell death 1", sequences PD1 "," PD-1 "," PDCD1 "," PD-1 antigen "," human PD-1 "," hPD-1 ", and" hPD1 "are used interchangeably to refer to the programmed death-1 receptor, also known as CD279, and include variants and subtypes of human PD-1, as well as analogs having at least one common epitope with PD-1. PD-1 is a key regulator of immune response threshold and peripheral immune tolerance. It is expressed on activated T cells, B cells, monocytes and dendritic cells and binds to its ligands PD-L1 and PD-L2. Human PD-1 is encoded by the PDCD1 gene. For example, the amino acid sequence of human PD-1 is disclosed under GenBank accession No. NP _ 005009. PD1 has four splice variants expressed on human Peripheral Blood Mononuclear Cells (PBMCs). Thus, the PD-1 protein includes full-length PD-1, as well as alternative splice variants of PD-1, such as PD-1Aex2, PD-1Aex3, PD-1Aex2,3, and PD-1Aex2,3, 4. Unless otherwise indicated, the term includes any variant and subtype of human PD-1 expressed naturally by PBMC or by cells transfected with the PD-1 gene.

As used herein, the term "antibody" describes one immunoglobulin molecule type and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. Unless specifically stated otherwise, the term "antibody" includes intact immunoglobulins as well as "antibody fragments" or "antibody antigen-binding fragments" (e.g., Fab ', F (ab')2, Fv), single chains (scFv), mutants thereof, molecules comprising an antibody portion, diabodies, linear antibodies, single chain antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site with the desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody. Preferably, the term "antibody" refers to a humanized antibody.

As used herein, an "antigen-binding fragment" of an antibody refers to a portion of an antibody that exhibits antigen-binding ability to PD-1, i.e., a molecule that corresponds to a portion of the structure of an antibody of the invention, possibly in its native form; such fragments in particular exhibit the same or substantially the same antigen binding specificity for the antigen as compared to the antigen binding specificity of the corresponding four-chain antibody. Advantageously, the antigen binding fragment has a similar binding affinity as the corresponding 4 chain antibody. However, antigen binding fragments having reduced antigen binding affinity relative to the corresponding 4 chain antibody are also encompassed within the invention. Antigen binding capacity can be determined by measuring the affinity between the antibody and the target fragment. These antigen binding fragments may also be referred to as "functional fragments" of an antibody. An antigen-binding fragment of an antibody is a fragment comprising the hypervariable domains of the portion thereof referred to as the CDRs (complementarity determining regions) or which comprise the antigen recognition site (i.e., the extracellular domain of PD 1), thereby defining the antigen recognition specificity.

The "Fab" fragment comprises the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F (ab ') fragments are generated by cleavage of disulfide bonds at the hinge cysteines of F (ab')2 pepsin digestion products. Other chemical couplings of antibody fragments are well known to those of ordinary skill in the art. Fab and F (ab')2 fragments lack the Fc fragment of intact antibodies, clear more rapidly from the circulation of the animal, and may have less non-specific tissue binding than intact antibodies (see, e.g., Wahl et al, 1983, J.Nucl. Med.24: 316).

An "Fv" fragment is the smallest fragment of an antibody that contains the entire target recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer targeted binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) may have the ability to recognize and bind the target, albeit with less affinity than the entire binding site.

"Single chain Fv" or "scFv" antibody binding fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, Fv polypeptides further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for target binding.

A "single domain antibody" consists of a single VH or VL domain that exhibits sufficient affinity for PD-1. In a particular embodiment, the single domain antibody is a camelized antibody (see, e.g., Riechmann,1999, Journal of Immunological Methods 231: 25-38).

Structurally, an antibody may have a heavy chain (H) and a light chain (L) that are linked to each other by disulfide bonds. There are two types of light chains, λ and κ. Each heavy and light chain contains a constant region and a variable region ("domain"). The light and heavy chain variable regions comprise a "framework" region interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). The extent of the framework regions and CDRs has been defined (see Kabat et al, Sequences of Proteins of Immunological Interest, and U.S. department of Health and Human Services,1991, which is incorporated herein by reference). Preferably, the CDRs are defined according to the Kabat method. The framework regions serve to form a scaffold, positioning the CDRs in the correct orientation by interchain non-covalent interactions. The CDRs are primarily responsible for binding to an epitope of antigen. The CDRs of each chain are commonly referred to as "complementarity determining region 1" or "CDR 1", "CDR 2", and "CDR 3", numbered sequentially from the N-terminus. The VL and VH domains of antibodies according to the invention may comprise four framework regions or "FRs", which are referred to in the art and herein as "framework region 1" or "FR 1", "FR 2", "FR 3" and "FR 4", respectively. These framework regions and complementarity determining regions are preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from the amino terminus to the carboxyl terminus). As used herein, the term "antibody framework" refers to the portion of a variable domain, VL and/or VH, that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain.

As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody conformation. The CDRs of antibody heavy chains are commonly referred to as "HCDR 1", "HCDR 2" and "HCDR 3". The framework regions of antibody heavy chains are commonly referred to as "HFR 1", "HFR 2", "HFR 3" and "HFR 4".

As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in an antibody conformation, and kappa and lambda light chains refer to the light chain isotypes of the two major antibodies. The CDRs of the antibody light chain are commonly referred to as "LCDR 1", "LCDR 2" and "LCDR 3". The framework regions of antibody light chains are commonly referred to as "LFR 1", "LFR 2", "LFR 3", and "LFR 4".

With respect to binding of an antibody to a target molecule, the term "binding" refers to peptides, polypeptides, proteins, fusion proteins, molecules, and antibodies (including antibody fragments) that recognize and contact an antigen. Preferably, it refers to antigen-antibody type interactions. The terms "specifically binds," specifically binds to, "" specifically targets, "" selectively binds, "and" selectively targets "a particular antigen (e.g., PD-1) or an epitope on a particular antigen (e.g., PD-1)" refer to an antibody that recognizes and binds to a particular antigen, but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically (or preferentially) binds to a PD-1 or PD-1 epitope is an antibody that binds to that PD-1 epitope, e.g., with higher affinity, avidity, more readily, and/or for longer duration than to other PD-1 epitopes or non-PD-1 epitopes. Preferably, the term "specific binding" means between an antibody and an antigen at or below 10 ^-7Binding affinity of M. In certain aspects, the antibody is equal to or less than 10^-8M、10^-9M or 10^-10Affinity binding of M.

As used herein, "PD-1 antibody," "anti-PD-1 antibody," "PD-1 Ab," "PD-1 specific antibody," "anti-PD-1 Ab," are used interchangeably and refer to an antibody as described herein that specifically binds to PD-1, particularly human PD-1. In some embodiments, the antibody binds to an extracellular domain of PD-1. In particular, an anti-PD-1 antibody is an antibody that is capable of binding to the PD-1 antigen and inhibiting the PD-1 mediated signaling pathway, thereby enhancing immune responses such as T cell activation.

As used herein, the terms "bifunctional molecule", "bifunctional compound", "bifunctional protein", "Bicki antibody", "bifunctional antibody" and "bifunctional checkpoint inhibitor molecule" have the same meaning and are used interchangeably. These terms refer to an antibody that recognizes an antigen by containing at least one region specific for the antigen (e.g., a variable region derived from an antibody) and at least a second region that is a polypeptide. More specifically, the bifunctional molecule is a fusion protein of an antibody or a part thereof, preferably an antigen-binding fragment thereof with another polypeptide or a polypeptide fragment thereof.

As used herein, the term "chimeric antibody" refers to an antibody or antigen-binding fragment of which a portion of the heavy and/or light chain is derived from one species and the remainder of the heavy and/or light chain is derived from a different species. In an illustrative example, the chimeric antibody can comprise a constant region derived from a human and a variable region derived from a non-human species (e.g., mouse).

As used herein, the term "humanized antibody" is intended to refer to an antibody in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences (e.g., a chimeric antibody comprising minimal sequences derived from a non-human antibody). "humanized antibody" (e.g., a non-human antibody) also refers to an antibody that has undergone humanization. Humanized antibodies are typically human immunoglobulins (recipient antibody) in which residues from one or more CDRs are replaced by residues from at least one CDR of the non-human antibody (donor antibody) while retaining the desired specificity, affinity, and capacity of the original antibody. The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken or non-human primate antibody having the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the acceptor antibody are replaced with framework region residues from the donor antibody. Alternatively, selected framework region residues of the common antibody are replaced by framework region residues from a human or humanized antibody. Additional framework region modifications can be made within the human framework sequences. Thus, humanized antibodies may also comprise residues not found in either the recipient or donor antibody. Such amino acid modifications can be made to further improve antibody function and/or to increase the humanization process. Herein, "amino acid change" or "amino acid modification" refers to a change in the amino acid sequence of a polypeptide. "amino acid modifications" include substitutions, insertions, and/or deletions in the polypeptide sequence. An "amino acid substitution" or "substitution" herein refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. "amino acid insertion" or "insertion" refers to the addition of an amino acid at a particular position in a parent polypeptide sequence. "amino acid deletion" or "deletion" refers to the removal of an amino acid at a particular position in a parent polypeptide sequence. Amino acid substitutions may be conservative. A conservative substitution is one in which a given amino acid residue is replaced with another residue having a side chain ("hydrophobic group") of similar chemical nature (e.g., charge, volume, and/or hydrophobicity). As used herein, "amino acid position" or "amino acid position number" are used interchangeably and refer to the position of a particular amino acid in an amino acid sequence, usually specified in the single letter code for the amino acid. The first amino acid in the amino acid sequence (i.e., from the N-terminus) should be considered to have position 1.

A conservative substitution is one in which a given amino acid residue is replaced with another residue having a side chain ("R-group") of similar chemical nature (e.g., charge, volume, and/or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. Conservative substitutions and corresponding rules are well described in the prior art. For example, conservative substitutions may be defined by substitutions within the amino acid groups reflected in the following table:

TABLE A amino acid residues

Amino acid radical	Amino acid residue
		Acidic residue	ASP and GLU
Basic residue	LYS, ARG and HIS
		Hydrophilic uncharged residues	SER, THR, ASN and GLN
Aliphatic uncharged residues	GLY, ALA, VAL, LEU and ILE
		Non-polar uncharged residues	CYS, MET and PRO
Aromatic residue	PHE, TYR and TRP

TABLE B-replacement of conservative amino acid residue substitution groups

TABLE C-alternative physical and functional Classification of amino acid residues

As used herein, an "isolated antibody" is an antibody that is isolated and/or recovered from a component of its natural environment. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the natural environment of the antibody is not present. In some embodiments, the antibody is purified to homogeneity and/or purity of 90%, 95%, or 99% as determined by, for example, electrophoresis (e.g., SDS-PAG, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) under reducing or non-reducing conditions.

As used herein, the term "derived from" refers to a compound having a structure derived from the parent compound or protein structure, and which structure is sufficiently similar to those disclosed herein and, based on that similarity, one of skill in the art would expect it to exhibit the same or similar properties, activities, and utilities as the claimed compound. For example, a humanized antibody derived from a mouse antibody refers to an antibody or antibody fragment that shares similar properties with the mouse antibody, e.g., recognizes the same epitope, shares similar VH and VL with residues that are involved in and/or increase the modification of antibody humanization.

The term "treatment" refers to any act aimed at improving the health status of a patient, such as the treatment, prevention, prophylaxis and delay of progression of a disease or disease symptoms. It refers to curative and/or prophylactic treatment of a disease. Curative treatment is defined as treatment that results in a cure or treatment that reduces, ameliorates and/or eliminates, reduces and/or stabilizes a disease or a symptom of a disease or the pain it causes directly or indirectly. Prophylactic treatment includes treatment that results in the prevention of disease and treatment that reduces and/or delays the progression and/or onset of disease or the risk of its onset. In certain embodiments, the term refers to amelioration or eradication of a disease, disorder, infection, or symptom associated therewith. In other embodiments, the term refers to minimizing the spread or progression of cancer. Treatment according to the invention does not necessarily mean 100% or complete treatment. Rather, there are varying degrees of treatment in which one of ordinary skill in the art would consider a potential benefit or therapeutic effect. Preferably, the term "treatment" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a condition/disease, such as a condition/disease associated with a signaling pathway mediated by PD-1.

As used herein, the term "disorder" or "disease" refers to an organ, part, structure or system of abnormal bodily function due to genetic or developmental errors, infection, toxicant, nutritional deficiency or imbalance, toxicity or the influence of adverse environmental factors. Preferably, these terms refer to monitoring a condition or disease, for example, a disease that disrupts normal physical or mental function. More preferably, the term disorder refers to an immune and/or inflammatory disease, such as cancer, affecting animals and/or humans.

As used herein, the term "immune disease" refers to a condition in a subject characterized by cellular, tissue and/or organ damage caused by the subject's immune response to its own cells, tissues and/or organs. The term "inflammatory disease" refers to a condition characterized by inflammation (e.g., chronic inflammation) in a subject. Autoimmune disorders may or may not be associated with inflammation. Accordingly, inflammation may or may not be caused by autoimmune disorders.

As used herein, the term "cancer" is defined as a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

As used herein, the term "disease associated with or associated with PD-1", "PD-1 positive cancer" or "PD-1 positive infectious disease" is intended to refer to a cancer or infectious disease (e.g., caused by viruses and/or bacteria) that is caused by PD-1 expression or has symptoms and/or features of PD-1 expression, i.e., caused by increased or decreased PD-1 expression or activity, causes an exacerbation, or any condition associated therewith.

As used herein, the term "subject", "host", "individual" or "patient" refers to a human, including an adult or a child.

As used herein, "pharmaceutical composition" refers to a formulation of one or more active agents, such as comprising a bifunctional molecule according to the present invention, and optionally other chemical components, such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of the active agent to the organism. The compositions of the invention may be in a form suitable for any conventional route of administration or use. In one embodiment, a "composition" generally refers to a combination of an active agent (e.g., a compound or composition) and a naturally-occurring or non-naturally-occurring carrier that is inert (e.g., a detection agent or label) or active, such as adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, and the like, and includes a pharmaceutically acceptable carrier. As referred to herein, an "acceptable carrier" or "acceptable carrier" is any known compound or combination of compounds known to those of skill in the art that can be used to formulate pharmaceutical compositions.

As used herein, "effective amount" or "therapeutically effective amount" refers to the amount of active agent required to confer a therapeutic effect on a subject, e.g., the amount of active agent required to treat a disease or disorder of interest or to produce a desired effect, alone or in combination with one or more other active agents. The "effective amount" will vary according to: one or more agents, the disease and its severity, the characteristics of the subject to be treated, including age, physical condition, physical constitution, sex and weight, duration of treatment, the nature of concomitant therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of a health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed by routine experimentation. It is generally preferred to use the maximum dose of the individual components or combinations thereof, i.e. the highest safe dose according to sound medical judgment.

As used herein, the term "drug" refers to any substance or composition having the property of curing or preventing a condition or disease.

As used herein, the term "in combination" refers to the use of more than one therapy (e.g., prophylactic and/or therapeutic agents). The use of the term "in combination" does not limit the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to a subject having a disease or disorder.

The terms "polynucleotide", "nucleic acid" and "nucleic acid sequence" are equivalent and refer to a polymeric form of nucleotides of any length, such as RNA or DNA or analogs thereof. A nucleic acid of the invention (e.g., a component or portion of a nucleic acid) can be naturally occurring, modified or engineered, isolated, and/or non-natural. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids.

An "isolated nucleic acid encoding an anti-PD 1 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such one or more nucleic acid molecules in a single vector or separate vectors, as well as such one or more nucleic acid molecules present at one or more locations in a host cell. As used herein, the terms "nucleic acid construct," "plasmid," and "vector" are equivalent and refer to a nucleic acid molecule used to transfer a passenger nucleic acid sequence, such as DNA or RNA, into a host cell.

As used herein, the term "host cell" is intended to include any individual cell or cell culture that may be or has been a vector, an exogenous nucleic acid molecule, and a polynucleotide encoding an antibody construct of the invention; and/or the receptor of the antibody construct itself. The corresponding material can be introduced into cells by transformation, transfection, or the like. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Host cells include, for example, bacterial, microbial, plant, and animal cells.

As used herein, "immune cell" refers to a cell associated with innate immunity and adaptive immunity, e.g., white blood cells (leukocytes), such as stem cells (HSCs) derived from hematopoietic stem cells produced in bone marrow, lymphocytes (T cells, B cells, Natural Killer (NK) cells, and natural killer T cells (NKTs) and cells of myeloid origin (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells) T helper 17 type T cells and suppressor T cells.

As used herein, the terms "T effector cell", "T eff" or "effector cell" describe a group of immune cells that includes several T cell types that respond positively to stimulation (e.g., co-stimulation). It includes in particular T cells having the function of eliminating antigens (for example, by producing cytokines that regulate the activation of other cells or by cytotoxic activity). It includes, inter alia, CD4+, CD8+, Treg cells, cytotoxic T cells and helper T cells (Th1 and Th 2).

As used herein, the term "regulatory T cells", "Treg cells" or "tregs" refers to a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs have immunosuppressive effects, typically inhibiting or down-regulating the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25 and are believed to be derived from the same lineage as the naive CD4 cells.

The term "depleted T cells" refers to a population of T cells that are in a dysfunctional state (i.e., "depleted"). T cell depletion is characterized by a gradual loss of function, a change in the transcriptional profile, and a sustained expression of inhibitory receptors. Depleted T cells lose their cytokine-producing capacity, their high proliferative capacity and their cytotoxic potential, eventually leading to their depletion. Depleted T cells generally indicate a combination of higher levels of CD43, CD69, and inhibitory receptors, with lower expression of CD62L and CD 127.

The term "immune response" refers to the effect of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or liver (including antibodies, cytokines, and complement) to selectively damage, destroy, or eliminate from the body cells or tissues invading, infected, cancerous, or normal human cells or tissues in the case of autoimmune or pathological inflammation.

As used herein, the term "antagonist" refers to a substance that blocks or reduces the activity or function of another substance. In particular, the term refers to an antibody that binds to a cellular receptor (e.g., PD-1) that serves as a reference substance (e.g., PD-L1 and/or PD-L2), preventing it from producing all or part of the usual biological effects (e.g., establishing an immunosuppressive microenvironment). Antagonist activity of the antibodies according to the invention can be assessed by competitive ELISA.

As used herein, the term "isolated" means that the material (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially separated or enriched relative to other materials with which it is found in nature. In particular, an "isolated" antibody is one that has been identified and isolated and/or recovered from a component of the natural environment. For example, the isolated antibody is purified (1) by greater than 75% of the antibody weight as determined by the Lowry method, or (2) by SDS-PAGE under reducing or non-reducing conditions. Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the antibody's natural environment will not be present. However, typically an isolated antibody will be prepared by at least one purification step.

As used herein, the term "and/or" will be considered a specific disclosure of each of two specific features or components, with or without the other. For example, "a and/or BA shall be considered a specific disclosure of each of (i) a, (ii) B, and (iii) a and B, as if each were individually listed.

The term "a" or "an" can refer to one or more of the elements that it modifies (e.g., "an agent" can mean one or more of the agent), unless the context clearly dictates otherwise.

As used herein, the term "about" in combination with any and all values (including lower and upper limits of a numerical range) refers to any value having an acceptable range up to +/-10% deviation (e.g., +/-0.5%, +/-1%, +/-1.5%, +/-2%, +/-2.5%, +/-3%, +/-3.5%, +/-4%, +/-4.5%, +/-5%, +/-5.5%, +/-6%, +/-6.5%, +/-7%, +/-7.5%, +/-8%, +/-8.5%, +/-9%, +/-9.5%). The use of the term "about" in the beginning of a string of values modifies each of the stated values (i.e., "about 1, 2, and 3" refers to about 1, about 2, and about 3). Further, when a list of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate and fractional values thereof (e.g., 54%, 85.4%).

anti-PD-1 antibodies

The bifunctional molecule according to the present invention comprises a first entity comprising an anti-hPD-1 antibody or antigen-binding fragment thereof.

Provided herein is an antibody that specifically binds to human PD-1. In some aspects, the antibody specifically binds to human PD-1 (preferably to the extracellular domain of human PD-1). In some aspects, the antibody selectively binds to one or more of full-length human PD-1, PD-1Aex2, PD-1Aex3, PD-1Aex2,3, and PD-1Aex2,3, 4.

In some aspects, the anti-PD 1 antibody is an isolated antibody, particularly a non-natural isolated antibody. Such isolated anti-PD 1 antibodies can be prepared by at least one purification step. In some embodiments, the isolated anti-PD 1 antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, the isolated anti-PD 1 antibody is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight of the antibody, the remainder of the weight comprising the weight of other solutes dissolved in the solvent.

Preferably, such antibodies have the ability to block or inhibit the interaction between PD-1 and at least one of its ligands (e.g., PD-L1 and/or PD-L2). As used herein, the ability to "block binding" or "block interaction" or "inhibit interaction" refers to the ability of an antibody or antigen-binding fragment to prevent a binding interaction between two molecules (e.g., PD-1 and its ligands PD-L1 and/or PD-L2) to any detectable degree.

Preferably, the anti-PD 1 antibody or antigen-binding fragment thereof is an antagonist of human PD-L1 and/or PD-L2 binding to human PD-1, more preferably an antagonist of human PD-L1 and PD-L2 binding to PD-1.

In certain embodiments, the anti-hPD 1 antibody or antigen-binding fragment thereof inhibits binding interaction between PD-1 and at least one of its ligands (e.g., PD-L1 and/or PD-L2, preferably PD-L1 and PD-L2) by at least 50%. In certain embodiments, the inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

The anti-hPD 1 antibodies according to the invention can comprise any class of immunoglobulin (e.g., IgD, IgE, IgG, IgA, or IgM (or subclasses thereof)), including immunoglobulin chains or fragments thereof (e.g., Fv, Fab ', F (ab')2, scFv, or other antigen-binding subsequences of antibodies) derived from minimal sequences of non-human (e.g., mouse) immunoglobulins targeted to human PD-1. Preferably, the anti-hPD-1 antibody according to the invention is derived from IgG1, IgG2, IgG3 or IgG4, preferably from IgG4 or IgG 1.

In one embodiment, the antigen-binding fragment of the antibody comprises a heavy chain comprising a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR 3; and a light chain comprising a light chain variable domain comprising LCDR1, LCDR2, and LCDR 3; and fragments of the heavy chain constant domain. By fragment of a heavy chain constant domain, it is understood that the antigen binding fragment thus comprises at least a portion of the entire heavy chain constant domain. For example, the heavy chain constant domain may comprise or consist of: at least the CH1 domain of the heavy chain, or at least the CH1 and CH2 domains of the heavy chain, or at least the up to CH1, CH2 and CH3 domains of the heavy chain. A fragment of a heavy chain constant domain may also be defined as comprising at least a portion of the Fc domain of said heavy chain. Thus, an antigen-binding fragment of an antibody encompasses the Fab portion of an intact antibody, the F (ab ')2 portion of an intact antibody, the Fab' portion of an intact antibody. The heavy chain constant domain may also comprise or consist of an entire heavy chain constant domain, such as described in the present specification, wherein several entire heavy chain constant domains are described. In a particular embodiment of the invention, and when the antigen-binding fragment of the antibody comprises a fragment of a heavy chain constant domain (which fragment comprises or is present in a portion of the complete heavy chain constant domain), said heavy chain constant domain fragment may consist of at least 10 amino acid residues; or may consist of 10 to 300 amino acid residues, in particular 210 amino acid residues.

Preferably, the antibody to human PD-1 is a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope. Preferably, such monoclonal antibodies (mabs) are from mammals, such as mice, rodents, rabbits, goats, primates, non-human primates or animals. Techniques for making such monoclonal antibodies can be found in, for example, Stits et al (eds.), BASIC AND CLINICAL IMMUNOLOGY (4 th edition), Lange Medical Publications, Los Altos, Calif., and references cited therein; harlow and Lane (1988), ANTIBODIES: ALABORATORY MANUAL CSH Press; goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2 nd edition), Academic Press, New York, NY.

In certain embodiments, the anti-hPD 1 antibodies provided herein are chimeric antibodies. In one embodiment, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (e.g., monkey)) and a human constant region. In another embodiment, the chimeric antibody is a "class switch" antibody, wherein the class or subclass has been altered from that of the parent antibody. The chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the anti-hPD 1 antibody is a humanized antibody. Humanized antibodies typically comprise one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human or humanized antibody sequences. Alternatively, some FR residues may be substituted to restore or improve antibody specificity, affinity and/or humanization. The humanized antibody optionally will also comprise at least a portion of a human or humanized constant region (Fc). Methods for humanizing antibodies are well known in the art, see, e.g., Winter and Milstein, Nature,1991,349: 293-; riechmann et al, Nature, 332, p.323 (1988); verhoeyen et al, Science, 239, page 1534 (1988), Rader et al, Proc. nat. Acad. Sci. U.S.A.,1998,95: 8910-; steinberger et al, J.biol.chem.,2000,275: 36073-36078; queen et al, Proc.Natl.Acad.Sci.U.S.A.,1989,86: 10029-10033; almagro, j.c. and Fransson, j., front. biosci.13(2008) 1619-; kashmiri, S.V. et al, Methods 36(2005)25-34 (describing SDR (a-CDR) grafting); padlan, e.a., mol.immunol.28(1991)489-498 (describing "resurfacing"); dall' Acqua, w.f. et al, Methods 36(2005)43-60 (describing "FR rearrangement"); and Osbourn, J, et al, Methods 36(2005) 61-68; and Klimka, A. et al, Br.J. cancer 83(2000)252- "260 (the" guided selection "method describing FR rearrangement); and U.S. Pat. nos. 5,585,089,5,693,761,5,693,762,5,821,337, 7,527,791,6,982,321, and 7,087,409; and 6,180,370.

Preferably, the humanized antibody directed against human PD-1 is a monoclonal antibody.

In particular, the humanized antibody is a humanized antibody having a T20 humanization score of at least 80% or at least 85%, more preferably at least 88%, even more preferably at least 90%, most preferably comprising a T20 humanization score of between 85% and 95%, preferably between 88% and 92%.

"humanization" is typically measured using a T20 scoring analyzer to quantify humanization of the variable regions of monoclonal antibodies as described in Gao S H, Huang K, Tu H, Adler a S., BMC Biotechnology 2013:13: 55. The T20 humanization score is a parameter commonly used in the field of antibody humanization, which was first disclosed by Gao et al (BMC biotechnol.,2013,13, 55). The T20 humanization score is commonly used in patent applications (e.g., WO15161311, WO17127664, WO18136626, WO18190719, WO19060750, or WO19170677) to define humanized antibodies.

A web-based tool was provided to calculate the T20 score for antibody sequences using the T20 cutoff value human database http:// abanalyzer. In calculating the T20 score, input VH, VK or VL variable region protein sequences are first assigned Kabat numbering and CDR residues are identified. The blastp protein-protein BLAST algorithm was used to compare full-length sequences or only the framework sequences (CDR residues removed) to each sequence in the corresponding antibody database. Sequence identity between pairwise comparisons is isolated and after each sequence in the database is analyzed, the sequences are ranked from high to low based on sequence identity to the input sequence. The percent identity of the top 20 matched sequences was averaged to obtain a T20 score.

For each chain type (VH, VK, VL) and sequence length (full length or only framework) in the "whole human database", each antibody sequence was scored with its corresponding database using a T20 scoring analyzer. T20 scores were obtained for the first 20 matching sequences after excluding the input sequence itself (since sequence 1 was always the input antibody itself, the percentage identity of sequences 2 to 21 was averaged). The T20 scores for each group were ranked from high to low. The score reduction is approximately linear for most sequences; however, the T20 score for the last-15% antibody began to drop dramatically. Thus, the last 15% of the sequences were removed and the remaining sequences were formed into a T20 cut-off database, where the T20 score cut-off indicates the lowest T20 score for the sequences in the new database.

Thus, the humanized anti-PD 1 antibody according to the invention comprised in the bifunctional molecule has a T20 humanization score of at least 80% or at least 85%, more preferably at least 88%, even more preferably at least 90%, most preferably a T20 humanization score of between 85% and 95%, preferably between 88% and 92%.

In one embodiment, the anti-PD 1 antibody may be selected from pembrolizumab (also known as Keytruda Lamborrelizumab, MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), pidilizumab (CT-011), cimeprinizumab (Libtayo), cimeprimab, AUNP12, AMP-224, AGEN-2034, BGB-A317 (tirezolidumab), PDR001 (Spalelizumab), MK-3477, SCH-900475, PF-06801591, JNJ-63723283, geminosumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103(HX-008), MEDI-0680 (also known as 514-514) MEDI0608, JS (see AMP-001 et al, J.ang Hepol. 136, CBT-2012018, CBT-36501, and Oncoll-754091 (III-36501), INCSAR 1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (armor), STI-1110 (see WO 2014/194302), AGEN2034 (see WO 2017/040790), MGA012 (see WO 2017/19846), or IBI308 (see WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540), monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in WO 2006/121168. Bifunctional or bispecific molecules targeting PD-1 are also known, such as RG7769(Roche), mAb20717(Xencor), MEDI5752(AstraZeneca), FS118(F-star), SL-279252(Takeda) and mAb23104(Xencor)

In a particular embodiment, the anti-PD 1 antibody may be pembrolizumab (also known as Keytruda Lamborrelizumab, MK-3475) or nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538).

One specific example of a humanized anti-hPD 1 antibody is described below by its CDRs, framework regions, and Fc and hinge regions.

CDR

"complementarity determining regions" or "CDRs" are well known in the art and refer to discontinuous sequences of amino acids within the variable region of an antibody that confer antigen specificity and binding affinity. The exact amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including Kabat et al, (Sequences of Proteins of Immunological Interest 5 th edition (1991) "Kabat" numbering scheme); Al-Lazikani et Al, 1997, J.mol.biol.273: 927-948 ("Chothia" numbering scheme); MacCallum et al, 1996, J.mol.biol.262:732-745 ("Contact" numbering scheme); lefranc et al, Dev. Comp. Immunol.,2003,27:55-77 ("IMGT" numbering scheme); and those described by Honegge and Pluckthun, J.Mol.biol,2001,309:657-70 ("AHo" numbering scheme). Unless otherwise indicated, the numbering scheme used to identify a particular CDR herein is the Kabat numbering scheme.

In one embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or antigen-binding fragment thereof. The CDR regions of the humanized antibody can be derived from a mouse antibody and have been optimized to i) provide a safe humanized antibody with a very high level of humanization (better than 85%); and ii) improved antibody properties, in particular higher producibility when produced in mammalian cells and higher yields in mammalian cells (e.g. COS and HCO cells), while retaining antagonist activity (i.e. inhibition of binding of human PD-L1 to human PD-1) as they have a binding affinity (KD) for human PD-1 of less than 10^-7M, preferably less than 10^-8M。

In a very particular embodiment, the bifunctional molecule comprises an anti-human PD-1 antibody or antigen-binding fragment thereof, preferably a humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprising:

(i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and

(ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3,

wherein:

-the heavy chain CDR1(HCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 1, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than position 3 of SEQ ID No. 1 and any combination thereof;

-the heavy chain CDR2(HCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 2, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 13, 14 and 16 of SEQ ID No. 2 and any combination thereof;

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 3, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N, E; optionally one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 3, and any combination thereof;

-the light chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 12, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID No. 12 and any combination thereof; and is

-the light chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 15, optionally with any combination of one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions and any combination thereof; and

-the light chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 16, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 1, 4 and 6 of SEQ ID No. 16 and any combination thereof.

In one aspect, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or antigen-binding fragment thereof comprising

(i) A heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and

(ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3,

wherein:

-the heavy chain CDR1(HCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 1, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than position 3 of SEQ ID No. 1 and any combination thereof;

-the heavy chain CDR2(HCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 2, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 13, 14 and 16 of SEQ ID No. 2 and any combination thereof;

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 3, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N, E; optionally one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 3, and any combination thereof;

-the light chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 12, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID No. 12 and any combination thereof; and is

-the light chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 15, optionally with any combination of one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions and any combination thereof; and

-the light chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 16, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 1, 4 and 6 of SEQ ID No. 16 and any combination thereof.

In another embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or antigen-binding fragment thereof comprising

(i) A heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and

(ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3,

wherein:

-the heavy chain CDR1(HCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 1, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than position 3 of SEQ ID No. 1 and any combination thereof;

-the heavy chain CDR2(HCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 2, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 13, 14 and 16 of SEQ ID No. 2 and any combination thereof;

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 4, 5, 6, 7, 8, 9, 10 or 11, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 4, 5, 6, 7, 8, 9, 10 or 11 and any combination thereof; and is

-the light chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 13 or SEQ ID No. 14, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID No. 13 or SEQ ID No. 14 and any combination thereof;

-the light chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 15, optionally with a modification selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof;

-the light chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 16, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 1, 4 and 6 of SEQ ID No. 16 and any combination thereof.

In another aspect, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or antigen-binding fragment thereof comprising:

(i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2 and HCDR3, and

(ii) a light chain variable domain (VL) comprising LCDR1, LCDR2 and LCDR3,

wherein:

(a) the light chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 13, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID No. 13 and any combination thereof;

(b) The light chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 15, optionally with any combination of one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof;

(c) the light chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 16, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 1, 4 and 6 of SEQ ID No. 16, and any combination thereof;

(d) the heavy chain CDR1(HCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 1, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than position 3 of SEQ ID No. 1 and any combination thereof;

(e) the heavy chain CDR2(HCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 2, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 13, 14 and 16 of SEQ ID No. 2 and any combination thereof; and is

(f) The heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 4, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 4 and any combination thereof;

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 5, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 5 and any combination thereof; or

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 6, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 6 and any combination thereof; or

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 7, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 7 and any combination thereof; or

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 8, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 8 and any combination thereof; or

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 9, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 9 and any combination thereof; or

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 10, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 10 and any combination thereof; or

-said heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 11, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 11 and any combination thereof.

In another aspect, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or antigen-binding fragment thereof comprising:

(i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2 and HCDR3, and

(ii) a light chain variable domain (VL) comprising LCDR1, LCDR2 and LCDR3,

wherein:

(a) the light chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 14, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID No. 14, and any combination thereof;

(b) the light chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 15, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof;

(c) the light chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 16, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 1, 4 and 6 of SEQ ID No. 16, and any combination thereof;

(d) The heavy chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 1, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than position 3 of SEQ ID No. 1 and any combination thereof;

(e) the heavy chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 2, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 13, 14 and 16 of SEQ ID No. 2 and any combination thereof; and is

(f) The heavy chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 4, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 4, and any combination thereof;

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 5, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 5 and any combination thereof; or

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 6, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 6 and any combination thereof; or

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 7, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 7 and any combination thereof; or

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 8, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 8 and any combination thereof; or

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 9, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 9 and any combination thereof; or

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 10, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 10 and any combination thereof; or

-said heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 11, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 11 and any combination thereof.

In particular aspects, the modification is a substitution, particularly a conservative substitution.

In one embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises the following: (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2 and CDR3 of SEQ ID NO:3, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N and E; and (ii) a light chain comprising CDR1 of SEQ ID NO:12 (wherein X is G or T), CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16.

In one embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises the following: (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2 and CDR3 of SEQ ID NO:3, wherein X1 is D and X2 is selected from T, H, A, Y, N and E, preferably from H, A, Y, N and E; or wherein X1 is E, and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N, E and S; and (ii) a light chain comprising CDR1 of SEQ ID NO:12 (wherein X is G or T), CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16.

In one embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises the following: (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2 and CDR3 of SEQ ID NO:3, wherein X1 is D and X2 is selected from T, H, A, Y, N and E, preferably from H, A, Y, N and E; and (ii) a light chain comprising CDR1 of SEQ ID NO:12 (wherein X is G or T), CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16.

In one embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises the following: (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2 and CDR3 of SEQ ID NO:3, wherein X1 is E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N, E and S; and (ii) a light chain comprising the CDRL of SEQ ID NO:12 (wherein X is G or T), CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16.

In another embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises or consists essentially of: (i) a heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2, and CDR3 of SEQ ID NO. 4, 5, 6, 7, 8, 9, 10, or 11; and (ii) a light chain comprising CDR1 of SEQ ID NO:13 or SEQ ID NO:14, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16.

In another embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises or consists essentially of:

(i) a heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 4; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 5; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 6; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 7; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 8; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 9; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 10; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 11; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

In another embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises or consists essentially of:

(i) a heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 4; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 5; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 6; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 7; (ii) a light chain comprising CDR1 of SEQ ID NO. 14, CDR2 of SEQ ID NO. 15 and CDR3 of SEQ ID NO. 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 8; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 9; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 10; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16; or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 11; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO: 16.

Frame structure

In one embodiment, the anti-PD 1 antibody or antigen-binding fragment according to the invention comprises framework regions, in particular the heavy chain variable region framework region (HER) HFR1, HFR2, HFR3 and HFR4, and the light chain variable region framework region (LFR) LFR1, LFR2, LFR3 and LFR 4.

Preferably, the anti-PD 1 antibody or antigen-binding fragment according to the invention comprises human or humanized framework regions. For purposes herein, a "human acceptor framework" is a framework comprising an amino acid sequence derived from a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework of a human immunoglobulin framework or a human consensus framework as defined below. The human acceptor framework derived from a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence thereof, or it may comprise amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence. A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a series of human immunoglobulin VL or VH framework sequences.

In particular, the anti-PD 1 antibody or antigen-binding fragment comprises a heavy chain variable region framework region (HFR) HFR1, HFR2, HFR3, and HFR4 comprising the amino acid sequences of SEQ ID NOs 41, 42, 43, and 44, respectively, optionally with one, two, or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combinations thereof, at any position other than positions 27, 29, and 32 of HFR3 (i.e., SEQ ID NO: 43). Preferably, the anti-PD 1 antibody or antigen-binding fragment comprises HFR1 of SEQ ID NO:41, HFR2 of SEQ ID NO:42, HFR3 of SEQ ID NO:43, and HFR4 of SEQ ID NO: 44.

Alternatively or additionally, the anti-PD 1 antibody or antigen-binding fragment comprises light chain variable region framework region (LFR) LFR1, LFR2, LFR3, and LFR4, which comprise the amino acid sequences of SEQ ID NOs 45, 46, 47, and 48, respectively, optionally with one, two, or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof. Preferably, the humanized anti-PD 1 antibody or antigen-binding fragment comprises LFR1 of SEQ ID NO:45, LFR2 of SEQ ID NO:46, LFR3 of SEQ ID NO:47, and LFR4 of SEQ ID NO: 48.

VH-VL

The VL and VH domains of an anti-hPD 1 antibody according to the invention may comprise four framework regions interrupted by three complementarity determining regions, preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from the amino terminus to the carboxyl terminus).

In a first embodiment, the anti-human PD-1 humanized antibody or antigen-binding fragment thereof comprised in a bifunctional molecule comprises:

(a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:17, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N and E; optionally having one, two or three modifications selected from the group consisting of one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 17, and any combination thereof;

(b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26.

In a second embodiment, said anti-human PD-1 humanized antibody or antigen-binding fragment thereof comprised in said bifunctional molecule comprises:

(a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:17, wherein X1 is D and X2 is selected from T, H, A, Y, N, E, preferably H, A, Y, N, E; or X1 is E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N, E and S; optionally one, two or three modifications selected from the group consisting of one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 17, and any combination thereof;

(b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO: 26.

In a third embodiment, said anti-human PD-1 humanized antibody or antigen-binding fragment thereof comprised in said bifunctional molecule comprises:

(a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:17, wherein X1 is D and X2 is selected from T, H, A, Y, N, E, preferably H, A, Y, N, E; optionally one, two or three modifications selected from the group consisting of one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 17, and any combination thereof;

(b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO: 26.

In another embodiment, said anti-human PD-1 humanized antibody or antigen-binding fragment thereof comprised in said bifunctional molecule comprises:

(a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:17, wherein X1 is E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N, E and S, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:17 and any combination thereof;

(b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO: 26.

In another embodiment, said anti-human PD-1 humanized antibody or antigen-binding fragment thereof comprised in said bifunctional molecule comprises:

(a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:18, 19, 20, 21, 22, 23, 24 or 25, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:18, 19, 20, 21, 22, 23, 24 or 25, respectively;

(b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27 or SEQ ID No. 28, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID No. 27 or SEQ ID No. 28, and any combination thereof.

In another embodiment, said anti-human PD-1 humanized antibody or antigen-binding fragment thereof comprised in said bifunctional molecule comprises:

(a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:18 or consisting of SEQ ID NO:18, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 18; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 27; or

(a) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:19 or consisting of SEQ ID NO:19, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 19; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 27; or

(a) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:20 or consisting of SEQ ID NO:20, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 20; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 27; or

(a) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:21 or consisting of SEQ ID NO:21, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 21; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 27; or

(a) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:22 or consisting of SEQ ID NO:22, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 22; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 27; or

(a) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:23 or consisting of SEQ ID NO:23, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 23; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 27; or

(a) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:24 or consisting of SEQ ID NO:24, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 24; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 27; or

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:25, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:25, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 27; or

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:18, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:18, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 28, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 28; or

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:19, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:19, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 28, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 28; or

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:20, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:20, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 28, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 28; or

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:21, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:21, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 28, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 28; or

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:22, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:22, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 28, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 28; or

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID No. 23, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 23, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 28, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 28; or

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID No. 24, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 24, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 28, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID No. 28; or

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:25, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:25, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 28.

In particular aspects, the modification is a substitution, particularly a conservative substitution.

CH-CL

In one embodiment, the heavy Chain (CH) and light Chain (CL) comprise VL and VH sequences as described above.

In a particular embodiment, said anti-human PD-1 antibody or antigen-binding fragment thereof comprised in said bifunctional molecule comprises:

(a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 29, 30, 31, 32, 33, 34, 35 or 36, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112, respectively, selected from SEQ ID No. 29, 30, 31, 32, 34, 35 or 36; and

(b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO 37 or SEQ ID NO 38, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO 37 or SEQ ID NO 38, and any combination thereof.

In another embodiment, said anti-human PD-1 humanized antibody or antigen-binding fragment thereof comprised in said bifunctional molecule comprises:

(a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 29, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 29 and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID No. 37, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID No. 37, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 30, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 30, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID No. 37, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID No. 37, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 31, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 31, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID No. 37, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID No. 37, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 32, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 32, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID No. 37, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID No. 37, and any combination thereof;

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 33, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 33, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID No. 37, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID No. 37, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 34, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 34, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID No. 37, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID No. 37, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 35, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 35 and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID No. 37, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID No. 37, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 36, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 36, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID No. 37, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID No. 37, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 29, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 29 and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:38, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:38, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 30, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 30, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:38, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29 of SEQ ID NO:38, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105, and any combination thereof, of SEQ ID NO: 38; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 31, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 31, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:38, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:38, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 32, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 32, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:38, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:38, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 33, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 33, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:38, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:38, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 34, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 34, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:38, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:38, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 35, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 35 and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:38, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:38, and any combination thereof; or

(a) A heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID No. 36, optionally having one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID No. 36, and any combination thereof; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:38, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:38, and any combination thereof.

Preferably, the modification is a substitution, in particular a conservative substitution.

Fc and hinge region

Several studies to develop therapeutic antibodies have led to engineering the Fc region to optimize antibody properties to produce molecules more tailored to their desired pharmacological activity. The Fc region of an antibody mediates its serum half-life and effector functions such as Complement Dependent Cytotoxicity (CDC), Antibody Dependent Cellular Cytotoxicity (ADCC), and Antibody Dependent Cellular Phagocytosis (ADCP). Several mutations located at the interface between CH2 and CH3 domains, such as T250Q/M428L, and M252Y/S254T/T256E + H433K/N434F, have been shown to increase the affinity for binding to FcRn and increase the half-life of IgG1 in vivo. However, there is not always a direct correlation between increasing FcRn binding and improving half-life. One way to improve the effectiveness of therapeutic antibodies is to increase their serum persistence, thereby allowing higher circulating levels, reducing the frequency of administration, and lowering the dosage. Engineering the Fc region may be desirable to reduce or increase the effector function of the antibody. For antibodies that target cell surface molecules, particularly those on immune cells, elimination of effector functions is desirable. Conversely, for antibodies intended for oncological use, increasing effector function may improve therapeutic activity. The four human IgG isotypes bind with different affinities to the first component of activating Fc γ receptors (Fc γ RI, Fc γ RIIa, Fc γ RIIIa), inhibitory Fc γ RIIb receptors and complement (C1q), resulting in very different effector functions. IgG binding to Fc γ R or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for Fc γ R and C1q binding and have unique sequences in IgG2 and IgG 4.

The antibody according to the invention optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically an immunoglobulin constant region of a mammalian immunoglobulin, even more preferably an immunoglobulin constant region of a human or humanized immunoglobulin. Preferably, the Fc region is part of an anti-hPD-1 antibody described herein. The anti-hPD 1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule of the invention may comprise a constant region of an immunoglobulin, or a fragment, analog, variant, mutant or derivative of said constant region. As is well known to those skilled in the art, the choice of the IgG isotype of the heavy chain constant domain focuses on whether a specific function is required and whether an appropriate half-life in vivo is required. For example, antibodies designed to selectively eradicate cancer cells generally require an active isotype that allows for complement activation and effector-mediated cell killing mediated through antibody-dependent cell-mediated cytotoxicity. Both the human IgG1 and IgG3 (shorter half-life) isotypes meet these criteria, particularly the human IgG1 isotype (wild-type and variant). In particular, depending on the IgG isotype of the heavy chain constant domain (particularly the human wild-type and variant IgG1 isotypes), the anti-hPD 1 antibodies of the invention can be cytotoxic to PD-1 expressing cells through CDC, ADCC and/or ADCP mechanisms. Indeed, the fragment crystallizable (Fc) region interacts with a variety of accessory molecules to mediate indirect effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

In preferred embodiments, the constant region is derived from a human immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, or other classes. In a further aspect, the human constant region is selected from the following: IgG1, IgG2, IgG2, IgG3, and IgG 4. Preferably, the anti-PD 1 antibody comprises an IgG1 or IgG4 Fc region. Even more preferably, the anti-hPD 1 antibody comprises an IgG4 Fc region with S228P that stabilizes the IgG 4.

In one embodiment, the anti-PD 1 antibody comprises a truncated Fc region or fragment of an Fc region. In one embodiment, the constant region comprises a CH2 domain. In another embodiment, the constant region comprises the CH2 and CH3 domains or comprises the hinge-CH 2-CH 3. Alternatively, the constant region may comprise all or a portion of the hinge region, the CH2 domain, and/or the CH3 domain. In a preferred embodiment, the constant region comprises the CH2 and/or CH3 domains derived from the heavy chain of human IgG 4. In some embodiments, the constant region comprises a CH2 and/or CH3 domain derived from the heavy chain of human IgG 4.

In another embodiment, the constant region comprises a CH2 domain and at least a portion of a hinge region. The hinge region may be derived from an immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, or other classes. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable class, mutated or unmutated. More preferably, the hinge region is derived from the heavy chain of human IgG 1. In one embodiment, the constant region comprises a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In a particular embodiment, the CH2 domain is derived from a human IgG2 or IgG4 heavy chain, and the hinge region is derived from an altered human IgG1 heavy chain.

In one embodiment, the constant region contains a mutation that reduces affinity for an Fc receptor, reducing Fc effector function. For example, the constant region may contain a mutation that eliminates a glycosylation site within the IgG heavy chain constant region.

In another embodiment, the constant region comprises a CH2 domain and at least a portion of a hinge region. The hinge region may be derived from an immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, or other classes. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable class. The IgG1 hinge region has three cysteines, two of which are involved in disulfide bonding between the two heavy chains of an immunoglobulin. These same cysteines allow for efficient and consistent disulfide bond formation between the Fc portions. Thus, a preferred hinge region of the invention is derived from IgG1, more preferably, from human IgG 1. In some embodiments, the first cysteine in the hinge region of human IgG1 is mutated to another amino acid, preferably serine. The IgG2 isotype hinge region has four disulfide bonds that tend to promote oligomerization and possibly incorrect disulfide bonds during secretion in recombinant systems. Suitable hinge regions may be derived from the IgG2 hinge; the first two cysteines are each preferably mutated to another amino acid. The hinge region of IgG4 is known to be ineffective in forming interchain disulfide bonds. However, suitable hinge regions for the present invention may be derived from the IgG4 hinge region, preferably containing mutations that enhance the correct formation of disulfide bonds between heavy chain derived portions (Angal S, et al, (1993) mol. Immunol.,30: 105-8). More preferably, the hinge region is derived from the heavy chain of human IgG 4.

In one embodiment, the constant region comprises a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In a particular embodiment, the CH2 domain is derived from a human IgG4 heavy chain and the hinge region is derived from an altered human IgG1 heavy chain.

According to the invention, the constant region may contain a CH2 and/or CH3 domain and a hinge region, which are derived from different antibody isotypes, i.e. hybrid constant regions. For example, in one embodiment, the constant region comprises a CH2 and/or CH3 domain derived from IgG2 or IgG4 and a mutated hinge region derived from IgG 1. Alternatively, a mutated hinge region from another IgG subclass is used in the hybrid constant region. For example, a mutated form of the IgG4 hinge that allows efficient disulfide bonding between the two heavy chains may be used. The mutant hinge may also be derived from an IgG2 hinge, in which the first two cysteines are each mutated to another amino acid. The assembly of such hybrid constant regions has been described in U.S. patent publication No. 20030044423, the disclosure of which is incorporated herein by reference.

In one embodiment, the constant region may comprise CH2 and/or CH3 having one of the mutations described in table D below, or any combination thereof.

Table D: suitable human engineered Fc domains of antibodies. Numbering of residues in the heavy chain constant region is based on EU numbering (Edelman, g.m., et al, proc.natl.acad.usa,63,78-85 (1969);www.imgt.org/ IMGTScientificChart/Numbering/Hu_IGHGnber.html#refs)。

in a particular aspect, the bifunctional molecule (preferably the binding moiety) comprises a human IgG1 heavy chain constant domain or an IgG1 Fc domain, optionally with a substitution or combination of substitutions selected from: T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A + M252Y/S254T/T256E; K322A and K444A, preferably selected from the following: N297A, optionally in combination with M252Y/S254T/T256E, and L234A/L235A.

In another aspect, the binding moiety comprises a human IgG4 heavy chain constant domain or an IgG4 Fc domain, optionally with a substitution or combination of substitutions selected from: S228P; L234A/L235A, S228P + M252Y/S254T/T256E and K444A. Even more preferably, said bifunctional molecule (preferably said binding moiety) comprises an IgG4 Fc region with S228P stabilizing said IgG 4.

In certain embodiments, amino acid modifications can be introduced into the Fc region of the antibodies provided herein to produce Fc region variants. In certain embodiments, the Fc region variant has some, but not all, effector functions. Such antibodies may be useful, for example, in applications where the in vivo half-life of the antibody is important but certain effector functions are unnecessary or detrimental. Examples of effector functions include Complement Dependent Cytotoxicity (CDC) and antibody-directed complement-mediated cytotoxicity (ADCC). Numerous substitutions or deletions to alter effector function are well known in the art.

In one embodiment, the constant region comprises a mutation that reduces affinity for an Fc receptor or reduces Fc effector function. For example, the constant region may comprise a mutation that eliminates a glycosylation site within the IgG heavy chain constant region. Preferably, the CH2 domain comprises a mutation that eliminates the glycosylation site within the CH2 domain.

In one embodiment, anti-hPD 1 according to the present invention has the heavy chain constant domain of SEQ ID No.39 or 52 and/or the light chain constant domain of SEQ ID No. 40, in particular the heavy chain constant domain of SEQ ID No.39 or 52 and the light chain constant domain of SEQ ID No. 40.

In another embodiment, anti-hPD 1 according to the present invention has the heavy chain constant domain of SEQ ID NO. 52 and/or the light chain constant domain of SEQ ID.40, in particular the heavy chain constant domain of SEQ ID NO. 52 and the light chain constant domain of SEQ ID.40.

Table E: examples of heavy and light chain constant domains suitable for the humanized antibodies according to the present invention.

Amino acid changes near the binding of the Fc portion to the non-Fc portion can significantly increase the serum half-life of the Fc fusion protein (PCT publication No. WO 01/58957). Thus, the junction region of the proteins or polypeptides of the invention may comprise alterations preferably located within about 10 amino acids of the junction relative to the naturally occurring sequences of the immunoglobulin heavy chain and erythropoietin. These amino acid changes can cause an increase in hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which a C-terminal lysine residue is replaced. Preferably, the C-terminal lysine of the IgG sequence is replaced with a non-lysine (e.g., alanine or leucine) to further increase serum half-life.

All subclasses of human IgG carry C-terminal lysine residues of the antibody heavy chain that are cleaved in the circulation (K444). This blood-borne cleavage can impair the biological activity of the bifunctional molecule by releasing IL-7. To circumvent this problem, the K444 amino acid in the IgG1 or IgG4 domain may be substituted with alanine to reduce proteolytic cleavage, a mutation commonly used in antibodies. Then, in one embodiment, the anti-PD 1 antibody comprises at least one additional amino acid substitution consisting of K444A.

In one embodiment, the anti-PD 1 antibody comprises an additional cysteine residue in the C-terminal domain of IgG to create an additional disulfide bond and potentially limit the flexibility of the bifunctional molecule.

In certain embodiments, the antibody can be altered to increase, decrease, or eliminate the degree of glycosylation.

Checkpoint inhibitors

The inventors herein show that the bifunctional molecules according to the present invention bind the effect of IL-7 variants or mutants on the IL-7 receptor and block the PD-1 inhibitory effect and are suitable for optimizing the effect of checkpoint inhibitors (e.g. anti-PD-1 antibodies). In particular, a synergistic effect on T cell (especially depleted T cells) activation, more particularly on TCR signalling, has been shown. The inventors have shown in particular that activation of the same cells is provided by the binding of an anti-PD-1 antibody to IL-7 contained in a bifunctional molecule on the same immune cell. This synergy has never been observed using IL-7 and anti-PD-1 antibodies as individual compounds. It is then envisaged that any molecule other than PD-1 expressed on immune cells expressing IL-7R may be triggered by the bifunctional construct according to the invention (in particular the depleting factor). Then, in embodiments, the bifunctional molecule comprises an antibody or antigen-binding fragment thereof directed against a target (other than PD-1) expressed on an immune cell. For example, the target may be a receptor expressed on the surface of an immune cell (particularly a T cell). The receptor may be an inhibitor receptor. Alternatively, the receptor may be an activating receptor.

As used herein, the term "target" refers to a peptide, polypeptide, protein, antigen, or epitope expressed on the outer surface of an immune cell. With respect to expression of a target on the surface of an immune cell, the term "expression" refers to a target that is present at or presented at the outer surface of the cell. The term "specifically expressed" means that the target is expressed on immune cells, but not substantially expressed by other cell types, in particular, for example, tumor cells.

In one embodiment, the target is specifically expressed by immune cells in a healthy subject or in a subject with a disease (in particular, e.g., cancer). This means that the expression level of the target in the immune cells is higher than in other cells, or the ratio of immune cells expressing the target in the total immune cells is higher than that of other cells expressing the target in the total other cells. Preferably, the expression level or ratio is 2, 5, 10, 20, 50 or 100 fold higher. More specifically, specific types of immune cells, such as T cells, more specifically CD8+ T cells, effector T cells, or depleted T cells, or in a specific context, such as a subject with a disease such as cancer or infection, can be identified.

In one aspect, the target is an immune checkpoint. Preferably, the target is selected from PD-1, CD28, CD80, CTLA-4, BTLA, TIG IT, CD160, CD40L, ICOS, CD27, 0X40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, Tim3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD 8. Such targets are described in more detail in table F below.

Table f. examples of targets of interest.

Then, in this aspect, the antibody or antigenic fragment thereof comprised in the bifunctional molecule according to the invention binds to a peptide selected from the group consisting of CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, Tim3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD 8.

In a preferred aspect, the antibody or antigen binding fragment thereof comprised in the bifunctional molecule according to the invention is selected from CTLA-4, BTLA, TIGIT, LAG3 and TIM 3.

Antibodies to TIM3 and bifunctional or bispecific molecules targeting TIM3 are also known, e.g., Sym023, TSR-022, MBG453, LY3321367, INCACGN 02390, BGTB-A425, LY3321367, RG7769 (Roche). In some embodiments, the TFM-3 antibody is as disclosed in international patent application publication nos. WO2013006490, WO2016/161270, WO 2018/085469, or WO 2018/129553, WO 2011/155607, u.s.8,552,156, EP 2581113, and u.s 2014/044728.

Antibodies against CTLA-4 and bifunctional or bispecific molecules targeting CTLA-4 are also known, e.g., ipilimumab, tremelimumab, MK-1308, AGEN-1884, XmAb20717(Xencor), MEDI5752 (AstraZeneca). anti-CTLA-4 antibodies are also disclosed in WO18025178, WO19179388, WO19179391, WO19174603, WO19148444, WO19120232, WO19056281, WO19023482, WO18209701, WO18165895, WO18160536, WO18156250, WO18106862, WO18106864, WO 18068168168182, WO18035710, WO18025178, WO17194265, WO17106372, WO17084078, WO17087588, WO16196237, WO16130898, WO16015675, WO12120125, WO09100140 and WO 07008463.

Antibodies to LAG-3 and bifunctional or bispecific molecules targeting LAG-3, such as BMS-986016, IMP701, MGD012 or MGD013 (bispecific PD-1 and LAG-3 antibodies), are also known. anti-LAG-3 antibodies are also disclosed in WO2008132601, EP2320940, WO 19152574.

Antibodies to BTLA are also known in the art, for example hu Mab8D5, hu Mab8A3, hu Mab21H6, hu Mab19a7, or hu Mab4C 7. Antibody TAB004 against BTLA is currently undergoing clinical trials in subjects with advanced malignancies. anti-BTLA antibodies are also disclosed in WO08076560, WO10106051 (e.g. BTLA8.2), WO11014438 (e.g. 4C7), WO17096017 and WO17144668 (e.g. 629).

Antibodies against TIGIT are also known in the art, for example, BMS-986207 or AB154, BMS-986207CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.560.560.9, CHA.9.547.547.9.547.9.547.9.9.547.9.9.9.9.9, CHA.9.9.9.7, CHA.541.9.560, CHA.9.9.9.9.9, CHA.9.9.560.9.9.9.9, CHA.9.9.9, CHA.560, CHA.9.9.9.9.9.560, CHA.9.9.560, CHA.9.9.9.9, CHA.9.9.9.9.9, CHA.9, CHA.560, CHA.9.9.9, CHA.560, CHA.9.9, CHA.9.9.9.9.9, CHA.9.9, CHA.560, CHA.9.560, CHA.9.9, CHA.9.9.9.9.9.9.9.9, CHA.9.9, CHA.9.9.9.9.9.9, CHA.560, CHA.9.9.9.9, CHA.9.9.9, CHA.9.9, CHA.9.560, CHA.560, CHA.9.9.9.9, CHA.9.9.9, CHA.9, CHA.9.9.9.9.9.9.560, CHA.9, CHA.9.9.9.9, CHA.9, CHA.9.9.9.9.9.9.9.9.9.9.9, CHA.9, CHA.9.9.9, CHA.9, CHA.9.9.9, CHA.9.9, CHA.9, CHA.9.9.9, CHA.9.9, CHA.9, CHA.9.9, CHA.9, CHA.9.9.9.9.9.9, CHA.9.9.9.9, CHA.9.9.9.9.9.9, CHA.9.9, CHA.9, CHA.9.9.9, CHA.9. anti-TIGIT antibodies are also disclosed in WO16028656, WO16106302, WO16191643, WO17030823, WO17037707, WO17053748, WO17152088, WO18033798, WO18102536, WO18102746, WO18160704, WO18200430, WO18204363, WO19023504, WO19062832, WO19129221, WO19129261, WO19137548, WO19152574, WO19154415, WO19168382 and WO 19215728.

Antibodies against CD160 are also known in the art, e.g., CL1-R2 CNCM I-3204 as disclosed in WO06015886, or other antibodies as disclosed in WO10006071, WO10084158, WO 18077926.

In a particular aspect, the bifunctional molecule according to the invention comprises an anti-CTLA-4 antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-CTLA-4 antibody or antigen-binding fragment thereof. Preferably, the antibody is an antagonist of CTLA-4. Thus, the bifunctional molecule binds to the blockade of the effects of IL-7wt, variants or mutants thereof on the IL-7 receptor and on CTLA-4 inhibition and may have a synergistic effect on the activation of T cells, especially depleted T cells, more particularly on TCR signaling.

In another particular aspect, the bifunctional molecule according to the present invention comprises an anti-BTLA antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-BTLA antibody or antigen-binding fragment thereof. Preferably, the antibody is an antagonist of BTLA. Thus, the bifunctional molecule binds to the blocking of the effect of IL-7wt, variants or mutants thereof on the IL-7 receptor and on BTLA inhibition and may have a synergistic effect on the activation of T cells (especially depleted T cells), more particularly on TCR signaling.

In another particular aspect, the bifunctional molecule according to the invention comprises an anti-TIGIT antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-TIGIT antibody or antigen-binding fragment thereof. Preferably, the antibody is an antagonist of TIGIT. Thus, the bifunctional molecule binds to the blockade of the effects of IL-7wt, variants or mutants thereof on the IL-7 receptor and on TIGIT inhibition and may have a synergistic effect on the activation of T cells, especially depleted T cells, more particularly on TCR signaling.

In another particular aspect, the bifunctional molecule according to the present invention comprises an anti-LAG-3 antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-LAG-3 antibody or antigen-binding fragment thereof. Preferably, the antibody is an antagonist of LAG-3. Thus, the bifunctional molecule binds to IL-7wt, its variants or mutants, blocks the effect of the IL-7 receptor and the inhibitory effect of LAG-3, and may have a synergistic effect on the activation of T cells (especially depleted T cells), more particularly on TCR signaling.

In another particular aspect, the bifunctional molecule according to the present invention comprises an anti-TIM 3 antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-TIM 3 antibody or antigen-binding fragment thereof. Preferably, the antibody is an antagonist of TIM 3. Thus, the bifunctional molecule binds to the blocking of the effect of the IL-7 variant or mutant on the IL-7 receptor and on the inhibition of TIM3, and may have a synergistic effect on the activation of T cells (especially depleted T cells), more particularly on TCR signaling.

Peptide linker

The invention includes bifunctional molecules which may comprise a peptide linker between the anti-PD-1 antibody or fragment thereof and IL-7. The peptide linker is generally of sufficient length and flexibility to ensure that the two protein elements connected to the linker between them have sufficient degrees of freedom in space to perform their function and to avoid the formation of alpha helices and beta sheets affecting the stability of the recombinant bifunctional molecule.

In one aspect of the disclosure, the anti-hPD 1 antibody is preferably linked to IL-7 through a peptide linker. In other words, the present invention relates to a bifunctional molecule comprising an anti-PD 1 antibody or antigen-binding fragment thereof as detailed herein, a chain thereof, e.g. a light chain or a heavy chain or a fragment thereof, preferably a heavy chain or a fragment thereof, linked to IL-7 by a peptide linker. The term "linker" as used herein refers to a sequence linking IL-7 and at least one amino acid of said anti-PD-1 immunoglobulin sequence part. Such linkers can be used to prevent steric hindrance. The linker is typically 3-44 amino acid residues in length. Preferably, the linker has 3-30 amino acid residues. In some embodiments, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues.

In one embodiment, the invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or antigen-binding fragment thereof as defined above and IL-7, wherein the chain of said antibody, e.g. the light chain or the heavy chain, preferably the heavy chain, even more preferably the C-terminus of the heavy chain or the light chain, is linked to IL-7, preferably to the N-terminus of IL-7, by a peptide linker.

In a particular aspect, the present invention relates to a bifunctional molecule comprising an anti-hPD-1 antibody or antigen-binding fragment thereof as defined above, wherein IL-7 is linked, preferably by a peptide linker, to the C-terminal end of the heavy chain of said antibody (e.g. the C-terminal end of the heavy chain constant domain).

In one embodiment, the invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or antigen-binding fragment thereof as defined above, wherein IL-7 is preferably linked to the C-terminal end of the light chain of said antibody (e.g. the C-terminal end of said light chain constant domain) via a peptide linker.

The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to which the bifunctional molecule is administered. A useful set of linker sequences is derived from the heavy chain as described in WO 96/34103 and WO 94/04678A linker of the hinge region of the antibody. Other examples are poly-alanine linker sequences. Other preferred examples of linker sequences are Gly/Ser linkers of different lengths, including (Gly4Ser)₄、(Gly4Ser)₃、(Gly4Ser)₂Gly4Ser, Gly3Ser, Gly3, Gly2Ser and (Gly3Ser2)₃In particular (Gly4Ser) ₃. Preferably, the linker is selected from (Gly4Ser)₄、(Gly4Ser)₃And (Gly3Ser2)₃。

In one embodiment, said linker comprised in said bifunctional molecule is selected from (Gly4Ser)₄、(Gly4Ser)₃、(Gly4Ser)₂Gly4Ser, Gly3Ser, Gly3, Gly2Ser and (Gly3Ser2)₃Preferably (Gly4Ser)₃. Preferably, the linker is selected from (Gly4Ser)₄、(Gly4Ser)₃And (Gly3Ser2)₃. Even more preferably, the linker is (GGGGS)₃。

In one embodiment, the invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or fragment thereof as defined above, wherein said antibody or fragment thereof is linked to IL-7 via a linker sequence, preferably selected from the group consisting of (GGGGS)₃、(GGGGS)₄、(GGGGS)₂GGGGS, GGGS, GGG, GGS and (GGGS)₃Even more preferably (GGGGS)₃. Preferably, the linker is selected from (GGGGS)₃、(GGGGS)₄And (GGGS)₃。

Preferably, the heavy chain of the anti-PD-1 antibody, preferably the C-terminus of the heavy chain of the anti-PD-1 antibody, is linked via a flexible (Gly4Ser)₃The linker is genetically fused to the N-terminus of IL-7. At the fusion junction, the C-terminal lysine residue of the antibody heavy chain may be mutated to alanine to reduce proteolytic cleavage.

Preferably, the heavy chain of the anti-PD-1 antibody, preferably the C-terminus of the light chain of the anti-PD-1 antibody, is linked via a flexible (Gly4Ser) ₃The linker is genetically fused to the N-terminus of IL-7. At the fusion junction, the C-terminal lysine residue of the antibody light chain may be mutated to alanine to reduce proteolytic cleavage.

IL-7

The bifunctional molecule according to the present invention comprises a further or second entity comprising interleukin 7 or a variant or fragment thereof.

Preferably, the IL-7 protein is human IL-7 or a variant thereof. Thus, the IL-7 or variant thereof has an amino acid sequence which is at least 75% identical to wild-type IL-7, in particular to the protein of SEQ ID No: 51.

In one embodiment, the bifunctional molecule comprises a typical wild-type IL-7 human protein of 152 amino acids (SEQ ID NO: 51). Preferably, the IL-7 protein is the protein of SEQ ID No: 51. The IL-7 protein may or may not comprise its peptide signal.

A "variant" of an IL-7 protein is defined as an amino acid sequence that is altered by one or more amino acids. Variants may have "conservative" modifications or "non-conservative" modifications. Such modifications may include amino acid substitutions, deletions and/or insertions. Guidance in determining which and how many amino acid residues can be substituted, inserted, or deleted without disrupting a biological property (e.g., activity, binding ability, and/or structure) can be found using computer programs well known in the art, such as software for molecular modeling or for generating alignments. In a particular aspect, the variant IL-7 proteins included in the invention specifically include IL-7 proteins that retain substantially equivalent biological properties of IL-7 as compared to wild-type IL-7. In another aspect, the invention includes the variant IL-7 protein includes specifically including compared to wild type IL-7 does not retain substantially equal biological characteristics (such as activity, binding capacity and/or structure) of IL-7 protein. Variants of IL-7 also include altered IL-7 polypeptide sequences (e.g., oxidized, reduced, deaminated, or truncated forms). In particular, truncated forms or fragments of IL-7 that retain biological properties comparable to full-length IL-7 proteins are included within the scope of the invention. In one embodiment, interleukin 7 is any biologically active fragment thereof. More preferably, variants of IL-7 include naturally occurring allelic variants arising from natural polymorphisms, including SNPs, splice variants, and the like.

The biological activity of the IL-7 protein can be measured using an in vitro cell proliferation assay. Preferably, the IL-7 variants according to the invention retain at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60% of the biological activity compared to wild-type human IL-7, preferably at least 80%, 90%, 95% and even more preferably 99% of the biological activity compared to wild-type IL-7.

Variant IL-7 proteins also include polypeptides having at least about 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more sequence identity to wild-type IL-7, particularly to the protein of SEQ ID No. 51.

Preferred IL-7 according to the invention are human IL-7 polypeptides comprising or consisting of an amino acid sequence as described in SEQ ID No:51, EP 314415 or WO2004/018681A2, and any natural variants and homologues thereof.

In one aspect, the IL-7 polypeptide used in the present invention is a recombinant IL-7. The term "recombinant" as used herein means that the polypeptide is obtained or derived from a recombinant expression system, i.e., from a culture of a host cell (e.g., a microorganism or insect or plant or mammal) or from a transgenic plant or animal engineered to have obtained a nucleic acid molecule encoding an IL-7 polypeptide. Preferably, the recombinant IL-7 is a human recombinant IL-7 (e.g., a human IL-7 produced in a recombinant expression system).

The invention also provides bifunctional molecules comprising an IL-7 protein having enhanced biological activity compared to a wild-type IL-7 protein. For example, IL-7 proteins with disulfide bond patterns of Cys2-Cys92, Cys34-Cys129, and Cys47-141 are more active in vivo than wild-type recombinant IL-7 proteins, as described in US 7960514. As described in EP1904635, high glycosylation of IL-7 also increases the bioactivity of IL-7 (e.g., IL-7 protein), where Asn116 is not glycosylated but Asn70 and Asn91 are glycosylated.

Alternatively, the invention provides bifunctional molecules comprising an IL-7 protein having reduced immunogenicity as compared to a wild-type IL-7 protein, in particular by removing T-cell epitopes within IL-7 that can stimulate an immune response. Examples of such IL-7 are described in WO 2006061219.

In particular aspects, the disclosure also provides bifunctional molecules comprising IL-7 variants or mutants. The terms "interleukin-7 mutant", "mutant IL-7", "IL-7 mutant", "IL-7 variant", "IL-7 m" or IL-7v "are used interchangeably herein.

In this case, the IL-7 variant or mutant does not retain substantially equivalent biological properties (e.g., activity, binding capacity and/or structure) as compared to wild-type IL-7. The IL-7 mutant or variant comprises at least one mutation. In particular, the at least one mutation reduces the affinity of the IL-7 variant or mutant for the IL-7 receptor (IL-7R), but does not result in a loss of recognition of IL-7R. Thus, an IL-7 mutant or variant retains the ability to activate IL-7R, e.g., as measured by pStat5 signaling (e.g., as disclosed in Bitar et al, front. immunol.,2019, volume 10). The biological activity of the IL-7 protein can be measured using an in vitro cell proliferation assay or by measuring P-Stat5 into T cells by ELISA or FACS. Preferably, the IL-7 variant according to the invention has a reduction in biological properties (e.g. activity, binding capacity and/or structure) of at least 2, 5, 10, 20, 30, 40, 50, 100, 250, 500, 750, 1000, 2500, 5000 or 8000 fold compared to the wild type IL-7, preferably wth-IL 7. More preferably, the IL-7 variant has reduced binding to the IL-7 receptor but retains the ability to activate IL-7R. For example, binding to the IL-7 receptor can be reduced by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60% compared to wild-type IL-7, and retain at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the ability to activate the IL-7R compared to wild-type IL-7.

In one aspect, the IL-7 variant or mutant differs from wt-IL-7 by at least one amino acid mutation that i) reduces the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wt-IL-7 for IL-7R, and ii) improves the pharmacokinetics of the IL7 variant compared to wt-IL 7. More particularly, the IL-7 variants or mutants further retain the ability to activate IL-7R, particularly by pStat5 signaling.

In another aspect, a bifunctional molecule comprising an IL-7 variant or mutant differs from wt-IL-7 by at least one amino acid mutation, which i) has a reduced affinity for IL-7R compared to the affinity of the bifunctional molecule comprising wt-IL-7 for IL-7 receptor (IL-7R), and ii) has an improved pharmacokinetics of a bifunctional molecule comprising an IL7 variant or mutant thereof compared to the bifunctional molecule comprising wt-IL 7. More particularly, bifunctional molecules comprising a variant of IL7 or a mutant thereof further retain the ability to activate IL-7R, particularly by pStat5 signaling. For example, a bifunctional molecule comprising an IL-7 variant or mutant may have at least 10%, 20%, 30%, 40%, 50%, 60% less binding to the IL-7 receptor as compared to a bifunctional molecule comprising wild-type IL-7, and at least 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% ability to activate IL-7R as compared to a bifunctional molecule comprising wild-type IL-7.

In particular aspects, the IL-7 variant or mutant has a reduced affinity for the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 for IL-7R. In particular, the IL-7 variant or mutant has a reduced affinity for CD127 and/or CD132 compared to the affinity of wth-IL-7 for CD127 and/or CD132, respectively. Preferably, the IL-7 variant or mutant has a reduced affinity for CD127 compared to the affinity of wth-IL-7 for CD 127.

Preferably, the at least one amino acid mutation reduces the affinity of the IL-7 variant or mutant for IL-7R, in particular CD132 or CD127, by at least a factor of 10, 100, 1000, 10,000 or 100,000 compared to the affinity of wt-IL-7 for IL-7R. Such affinity comparisons may be performed by any method known to those skilled in the art, such as ELISA or Biacore.

Preferably, the at least one amino acid mutation reduces the affinity of the IL-7 variant or mutant for IL-7R compared to IL-7wt, but does not reduce the biological activity of the IL-7 variant or mutant (particularly as measured by pStat5 signaling).

Alternatively, the at least one amino acid mutation reduces the affinity of the IL-7 variant or mutant for IL-7R compared to IL-7wt, but does not significantly reduce the biological activity of IL-7m (particularly as measured by pStat5 signaling).

Additionally or alternatively, the IL-7 variant or mutant improves the pharmacokinetics of the bifunctional molecule comprising the IL-7 variant or mutant compared to the bifunctional molecule comprising wild-type IL-7. In particular, the IL-7 variant or mutant according to the present invention improves the pharmacokinetics of the bifunctional molecule comprising the IL-7 variant or mutant by at least 10, 100 or 1000 fold compared to the bifunctional molecule comprising wth-IL-7. The comparison of pharmacokinetic profiles may be performed by any method known to those skilled in the art, such as in vivo drug injection and ELISA of drug doses in serum at various time points, for example as shown in example 9.

As used herein, the terms "pharmacokinetics" and "PK" are used interchangeably to refer to the home of administration of a compound, substance or drug to a living body. Pharmacokinetics includes, inter alia, ADME or ladem regimens, representing release (i.e., release of a substance from a composition), absorption (i.e., entry of a substance into the blood circulation), distribution (i.e., dispersal or dissemination of a substance through the body), metabolism (i.e., conversion or degradation of a substance), and excretion (i.e., removal or clearance of a substance from an organism). The two stages of metabolism and excretion can also be grouped under the heading elimination. One skilled in the art can monitor various pharmacokinetic parameters such as elimination half-life, elimination constant rate, clearance (i.e., the volume of plasma that clears the drug per unit time), Cmax (maximum serum concentration), and drug exposure (as determined by the area under the curve) (Scheff et al, Pharm res.,2011,28, 1081-9).

Then, improving the pharmacokinetics by using IL-7 variants or mutants refers to improving at least one of the above parameters. Preferably, it refers to an improvement of the elimination half-life of the bifunctional molecule, i.e. an increase of the half-life duration or Cmax.

In a specific embodiment, the at least one mutation of the IL-7 variant or mutant increases the elimination half-life of the bifunctional molecule comprising the IL-7 variant or mutant compared to the bifunctional molecule comprising IL-7 wt.

In one embodiment, the IL-7 variant or mutant exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a wild-type human IL-7(wth-IL-7) protein of amino acid 152 as disclosed, for example, in SEQ ID NO: 51. Preferably, the IL-7 variant or mutant exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO 51.

In particular, the at least one mutation occurs at amino acid position 74 and/or 142 of IL-7. Additionally or alternatively, the at least one mutation occurs at amino acid positions 2 and 141, 34 and 129, and/or 47 and 92. These positions refer to the positions of the amino acids shown in SEQ ID NO: 51.

In particular, the at least one mutation is an amino acid substitution or a group of amino acid substitutions selected from: C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, Q11E, Y12F, M17L, Q22E, K81R, D74E, D74Q and D74N, or any combination thereof. These mutations refer to the positions of the amino acids shown in SEQ ID NO 51. The mutation W142H then represents, for example, the replacement of the tryptophan of wth-IL7 by histidine to obtain IL-7m with a histidine in amino acid position 142. Such mutants are described, for example, in SEQ ID No. 56.

In one embodiment, the IL-7 variant or mutant comprises a set of substitutions to disrupt the disulfide bond between C2 and C141, C47 and C92, and C34-C129. In particular, the IL-7 variants or mutants comprise two sets of substitutions to disrupt the disulfide bond between C2 and C141, and C47 and C92, C2 and C141, and C34-C129, or C47 and C92, and C34-C129. For example, cysteine residues may be replaced by serine to prevent disulfide bond formation. Thus, amino acid substitutions may be selected from C2S-C141S and C47S-C92S (referred to as "SS 2"), C2S-C141S and C34S-C129S (referred to as "SS 1"), and C47S-C92S and C34S-C129S (referred to as "SS 3"). These mutations refer to the positions of the amino acids shown in SEQ ID NO 51. Such IL-7 variants or mutants are described in particular under the sequences shown in SEQ ID Nos 53 to 55 (SS 1, SS2 and SS3, respectively). Preferably, the IL-7 variant or mutant comprises the amino acid substitutions C2S-C141S and C47S-C92S. Even more preferably, the IL-7 variant or mutant exhibits the sequence shown in SEQ ID NO 54.

In another embodiment, the IL-7 variant or mutant comprises at least one mutation selected from the group consisting of W142H, W142F, and W142Y. Such IL-7 variants or mutants are described in detail under the sequences shown in SEQ ID NOS: 57 to 58, respectively. Preferably, the IL-7 variant or mutant comprises the mutation W142H. Even more preferably, the IL-7 variant or mutant exhibits the sequence shown in SEQ ID NO 56.

In another embodiment, the IL-7 variant or mutant comprises at least one mutation selected from the group consisting of D74E, D74Q and D74N, preferably D74E and D74Q. Such IL-7 variants or mutants are described in detail under the sequences shown in SEQ ID NO 63 to 65, respectively. Preferably, the IL-7 variant or mutant comprises the mutation D74E. Even more preferably, the IL-7 variant or mutant exhibits the sequence shown in SEQ ID NO 63.

In another embodiment, the IL-7 variant or mutant comprises at least one mutation selected from Q11E, Y12F, M17L, Q22E and/or K81R. These mutations refer to the positions of the amino acids shown in SEQ ID NO 51. Such IL-7 variants or mutants are specifically described under the sequences shown in SEQ ID NOs 59, 60, 61, 62 and 66, respectively.

In one embodiment, the IL-7 variant or mutant comprises at least one mutation, which is present in: i) W142H, W142F or W142Y and/or ii) D74E, D74Q or D74N, preferably D74E or D74Q and/or iii) C2S-C141S and/or C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

In one embodiment, the IL-7 variant or mutant comprises the W142H substitution and at least one mutation consisting of: i) D74E, D74Q or D74N, preferably D74E or D74Q and/or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

In one embodiment, the IL-7 variant or mutant comprises the D74E substitution and at least one mutation consisting of: i) W142H, W142F or W142Y and/or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

In one embodiment, the IL-7 variant or mutant comprises the mutations C2S-C141S and C47S-C92S and at least one substitution, consisting of: i) W142H, W142F or W142Y and/or ii) D74E, D74Q or D74N, preferably D74E or D74Q.

In one embodiment, the IL-7 variant or mutant comprises i) a D74E and W142H substitution and ii) mutations C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

The IL-7 variant or mutant may or may not comprise a peptide signal thereof.

In one embodiment the bifunctional molecule according to the present invention comprises an IL-7 variant comprising or consisting of the amino acid sequence shown in SEQ ID NO 53-58 or 63-65. Even more preferably, the bifunctional molecule according to the present invention comprises an IL-7 variant comprising or consisting of the amino acid sequence shown in SEQ ID NO 54, 56 or 63.

Bifunctional molecules or "Bicki"

The present invention provides, inter alia, a bifunctional molecule comprising or being present in an anti-hPD 1 antibody or an antibody fragment thereof as disclosed above and IL-7, or in an anti-hPD 1 antibody or an antibody fragment thereof as disclosed above and IL-7, said anti-hPD 1 antibody or an antibody fragment thereof being covalently linked to IL-7, preferably via a peptide linker as disclosed above, in particular as a fusion protein.

In particular, the bifunctional molecule according to the present invention comprises two entities: a first entity comprising, or consisting essentially of, an anti-hPD 1 antibody or fragment thereof; a second entity comprising or consisting essentially of interleukin 7(IL-7), preferably human IL-7, optionally connected by a peptide linker.

In particular, the bifunctional molecules according to the present invention comprise one, two, three or four IL-7 molecules. In particular, the bifunctional molecule may comprise only one molecule of IL-7 linked to only one light or heavy chain of the anti-PD-1 antibody. The bifunctional molecule may further comprise two molecules of IL-7 linked to the light or heavy chain of the anti-PD-1 antibody. The bifunctional molecule may further comprise two molecules of IL-7, a first molecule linked to the light chain of the anti-PD-1 antibody and a second molecule linked to the heavy chain of the anti-PD-1 antibody. The bifunctional molecule may also comprise three IL-7 molecules, two of which are linked to the light or heavy chain of the anti-PD-1 antibody and the last of which is linked to the other chain of the anti-PD-1 antibody. Finally, the bifunctional molecule may further comprise four IL-7 molecules, two molecules linked to the light chain of the anti-PD-1 antibody and two molecules linked to the heavy chain of the anti-PD-1 antibody. Thus, the bifunctional molecule comprises one to four IL-7 molecules as disclosed herein.

In one embodiment, only one light chain comprises one IL-7 molecule (e.g., the bifunctional molecule comprises one IL-7 molecule), only one heavy chain comprises one IL-7 molecule (e.g., the bifunctional molecule comprises one IL-7 molecule), each light chain comprises one IL-7 molecule (e.g., the bifunctional molecule comprises two IL-7 molecules), each heavy chain comprises one IL-7 molecule (e.g., the bifunctional molecule comprises two IL-7 molecules), only one light chain and only one heavy chain comprises one IL-7 molecule (e.g., the bifunctional molecule comprises two IL-7 molecules), each light chain comprises one IL-7 molecule, and only one heavy chain comprises one IL-7 molecule (e.g., the bifunctional molecule comprises three IL-7 molecules), each heavy chain comprises one IL-7 molecule and only one light chain comprises one IL-7 molecule (e.g. the bifunctional molecule comprises three IL-7 molecules), or both light and heavy chains comprise one IL-7 molecule (e.g. the bifunctional molecule comprises four IL-7 molecules).

In one embodiment, the bifunctional molecule according to the present invention comprises or consists of:

(a) an anti-human PD-1 antibody or antigen-binding fragment thereof comprising (i) a heavy chain, and (ii) a light chain; and

(b) human interleukin (IL-7) or a fragment or variant thereof,

wherein the antibody heavy and/or light chain or fragment thereof is covalently linked to IL-7 via a peptide linker, preferably as a fusion protein.

Preferably, the bifunctional molecule according to the present invention comprises or consists of:

(a) a humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprising (i) a heavy chain, and (ii) a light chain; and

(b) human interleukin (IL-7) or a variant or fragment thereof,

wherein the antibody heavy or light chain or fragment thereof is covalently linked to IL-7 via a peptide linker, preferably as a fusion protein.

Preferably, such bifunctional molecule comprises at least one peptide linker connecting the N-terminus of IL-7 to the C-terminus of the heavy or light chain or both of said anti-human PD-1 antibody, said peptide linker preferably being selected from the group consisting of (GGGGS)₃、(GGGGS)₄、(GGGGS)₂GGGGS, GGGS, GGG, GGS and (GGGS)₃Even more preferably (GGGGS)₃。

Preferably, the N-terminal end of the IL-7 is linked to the C-terminal end of the heavy or light chain or both of the anti-human PD-1 antibody by at least one peptide linker. Alternatively, the C-terminal end of IL-7 is linked to the N-terminal end of the heavy or light chain or both of said anti-human PD-1 antibody by at least one peptide linker.

In one embodiment, the bifunctional molecule according to the present invention comprises or consists of:

(a) an anti-human PD-1 antibody or antigen-binding fragment thereof comprising (i) a heavy chain, and (ii) a light chain,

(b) human interleukin (IL-7) or a variant or fragment thereof, and

(c) a peptide linker connecting the N-terminal end of IL-7 with the C-terminal end of the heavy chain or the light chain or both of said anti-human PD-1 antibody, said peptide linker preferably being selected from the group consisting of (GGGGS)₃、(GGGGS)₄、(GGGGS)₂GGGGS, GGGS, GGG, GGS and (GGGS)₃Even more preferably (GGGGS)₃。

In a particular embodiment, the bifunctional molecule according to the present invention comprises or consists of:

(a) an anti-human PD-1 antibody or antigen-binding fragment thereof, comprising:

(i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

(ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3,

wherein:

-the heavy chain CDR1(HCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 1, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than position 3 of SEQ ID No. 1 and any combination thereof;

-the heavy chain CDR2(HCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 2, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 13, 14 and 16 of SEQ ID No. 2 and any combination thereof;

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 3, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N and E; optionally one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 3, and any combination thereof;

-the light chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 12, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID No. 12 and any combination thereof;

-the light chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 15, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions and any combination thereof; and is

-the light chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 16, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 1, 4 and 6 of SEQ ID No. 16 and any combination thereof; and (b) human interleukin 7 of SEQ ID No:51 or a variant or fragment thereof,

wherein the antibody heavy and/or light chain or fragment thereof is covalently linked to IL-7 as a fusion protein, preferably via a peptide linker.

In another embodiment, the bifunctional molecule according to the present invention comprises or consists of: (a) an anti-human PD-1 antibody or antigen-binding fragment thereof, comprising:

(i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

(ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3,

Wherein:

-the heavy chain CDR1(HCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 1, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than position 3 of SEQ ID No. 1 and any combination thereof;

-the heavy chain CDR2(HCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 2, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 13, 14 and 16 of SEQ ID No. 2 and any combination thereof;

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 3, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N and E; optionally one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 3, and any combination thereof;

-the light chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 12, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID No. 12 and any combination thereof;

-the light chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 15, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions and any combination thereof; and is

-the light chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 16, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 1, 4 and 6 of SEQ ID No. 16 and any combination thereof; and (b) human interleukin 7 of SEQ ID No:51 or a variant or fragment thereof,

wherein the antibody heavy and/or light chain or fragment thereof is covalently linked to IL-7 as a fusion protein, preferably via a peptide linker.

In another embodiment, the bifunctional molecule according to the present invention comprises or consists of: (a) a humanized anti-human PD-1 antibody or antigen-binding fragment thereof, comprising:

-the heavy chain CDR1(HCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 1, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than position 3 of SEQ ID No. 1 and any combination thereof;

-the heavy chain CDR2(HCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 2, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 13, 14 and 16 of SEQ ID No. 2 and any combination thereof;

-the heavy chain CDR3(HCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 4, 5, 6, 7, 8, 9, 10 or 11, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 2, 3, 7 and 8 of SEQ ID No. 4, 5, 6, 7, 8, 9, 10 or 11 and any combination thereof;

-the light chain CDR1(LCDR1) comprises or consists of the amino acid sequence of SEQ ID No. 13 or SEQ ID No. 14, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID No. 13 or SEQ ID No. 14 and any combination thereof;

-the light chain CDR2(LCDR2) comprises or consists of the amino acid sequence of SEQ ID No. 15, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions and any combination thereof; and is

-the light chain CDR3(LCDR3) comprises or consists of the amino acid sequence of SEQ ID No. 16, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 1, 4 and 6 of SEQ ID No. 16 and any combination thereof; and (b) human interleukin 7 of SEQ ID No:51 or a variant or fragment thereof,

wherein the antibody heavy and/or light chain or fragment thereof is covalently linked to IL-7 as a fusion protein, preferably via a peptide linker.

Preferably, the peptide linker is selected from (GGGGS)₃、(GGGGS)₄、(GGGGS)₂GGGGS, GGGS, GGG, GGS and (GGGS)₃Even more preferably (GGGGS)₃。

In another embodiment, the present invention relates to a bifunctional molecule comprising:

(a) a humanized anti-human PD-1 antibody or antigen-binding fragment thereof, comprising:

(i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:17, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably selected from H, A, Y, N, E, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:17 and any combination thereof;

(ii) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:26, and

(b) human interleukin 7 of SEQ ID No 51 or a variant or fragment thereof,

(c) a peptide linker between the light and/or heavy chain of the anti-hPD 1 antibody and the human IL-7 or variant or fragment thereof, selected from (GGGGS)₃、(GGGGS)₄、(GGGGS)₂GGGGS, GGGS, GGG, GGS and (GGGS)₃Even more preferably (GGGGS)₃。

Preferably, the N-terminal end of the IL-7 is linked to the C-terminal end of the heavy or light chain or both of the anti-human PD-1 antibody by at least one peptide linker. Alternatively, the C-terminal end of IL-7 is linked to the N-terminal end of the heavy or light chain or both of said anti-human PD-1 antibody by at least one peptide linker.

In another embodiment, the invention relates to a bifunctional molecule comprising or consisting of:

(a) A humanized anti-human PD-1 antibody comprising:

(i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:17, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably selected from H, A, Y, N, E, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:17 and any combination thereof;

(ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:26, and

(b) human interleukin 7 of SEQ ID No 51 or a variant or fragment thereof,

Wherein the C-terminal end of the heavy and/or light chain of the antibody or antigen-binding fragment thereof is preferably via (GGGGS)₃The peptide linker is covalently linked to the N-terminal end of IL-7.

In another embodiment, the invention relates to a bifunctional molecule comprising or consisting of: a) a humanized anti-human PD-1 antibody comprising:

(i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:18, 19, 20, 21, 22, 23, 24 or 25, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof, at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:18, 19, 20, 21, 22, 23, 24 or 25, respectively;

(ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:28, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:27 or SEQ ID NO:28 and any combination thereof,

(b) Human interleukin 7 of SEQ ID No 51 or a variant or fragment thereof,

wherein the C-terminal end of the heavy and/or light chain of the antibody or antigen-binding fragment thereof is preferably by (GGGGS)₃A peptide linker covalently attached to the N-terminal end of IL-7.

In a preferred embodiment, the C-terminal end of the heavy chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminal end of IL-7 to form a fusion protein. Preferably, only the heavy chain of the antibody or antigen binding protein thereof is covalently linked to IL-7.

In another embodiment, the invention relates to a bifunctional molecule comprising or consisting of: a) a humanized anti-human PD-1 antibody comprising:

(i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:17, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably selected from H, A, Y, N, E, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO:17 and any combination thereof;

(ii) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, optionally with one, two or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO:26, and

(b) human interleukin 7 of SEQ ID No 51 or a variant or fragment thereof,

wherein the C-terminal end of the heavy chain of said antibody or antigen-binding fragment thereof is preferably preceded by (GGGGS)₃A peptide linker covalently linked to the N-terminal end of IL-7 to form a fusion protein.

In another embodiment, the invention relates to a bifunctional molecule comprising or consisting of: a) a humanized anti-human PD-1 antibody comprising:

(i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 24;

(ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28,

(b) human interleukin 7 of SEQ ID No 51 or a variant or fragment thereof,

Wherein the C-terminal end of the heavy chain of said antibody or antigen-binding fragment thereof is preferably preceded by (GGGGS)₃A peptide linker covalently linked to the N-terminal end of IL-7 to form a fusion protein.

Preferably, the antibody or antigen fragment thereof has an IgGl or IgG4 Fc domain.

In one aspect, the antibody or antigenic fragment thereof has an IgGl Fc domain, optionally with a substitution or combination of substitutions selected from: T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A + M252Y/S254T/T256E; K322A and K444A, preferably selected from the following: N297A, optionally in combination with M252Y/S254T/T256E, and L234A/L235A, even more preferably an IgGl Fc domain with mutation N297A, e.g. as described above.

In another aspect, the antibody or antigenic fragment thereof has an IgG4 Fc domain, optionally with a substitution or combination of substitutions selected from: S228P; L234A/L235A, S228P + M252Y/S254T/T256E and K444A, even more preferably an IgG4 Fc domain with a mutation S228P such as described above.

Optionally, in any of the embodiments specified above, the IL-7 is an IL-7 variant or mutant.

More particularly, the IL-7 variant or mutant exhibits at least 75% identity to wild-type human IL-7(wth-IL-7) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:51, such IL-7 variant comprising at least one amino acid mutation that i) reduces the affinity of the IL-7 variant for the IL-7 receptor as compared to the affinity of wth-IL-7 for IL-7R, and ii) improves the pharmacokinetics of the bifunctional molecule comprising the IL-7 variant as compared to the bifunctional molecule comprising IL-7. More preferably, such mutation i) reduces the affinity of the IL-7 variant for the IL-7 receptor compared to the affinity of wth-IL-7 for IL-7R, ii) retains the ability to activate IL-7R; and iii) improving the pharmacokinetics of the bifunctional molecule comprising the IL-7 variant compared to the bifunctional molecule comprising IL-7.

More specifically, the IL-7 variant or mutant may exhibit at least 75% identity to wild-type human IL-7(wth-IL-7) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:51, such IL-7 variant comprising at least one amino acid mutation selected from the group consisting of: (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F or W142Y, (iii) D74E, D74Q or D74N, preferably D74E or D74Q; (iv) Q11E, Y12F, M17L, Q22E and/or K81R; or any combination thereof.

The IL-7 variant or mutant may comprise at least one set of substitutions selected from: C2S-C141S and C47S-C92S (referred to as "SS 2"), C2S-C141S and C34S-C129S (referred to as "SS 1"), and C47S-C92S and C34S-C129S (referred to as "SS 3"). These mutations refer to the positions of the amino acids shown in SEQ ID NO 51. Such IL-7 variants or mutants are described in particular under the sequences shown in SEQ ID Nos 53 to 55 (SS 1, SS2 and SS3, respectively). Preferably, the IL-7 variant or mutant comprises the amino acid substitutions C2S-C141S and C47S-C92S. Even more preferably, the IL-7 variant or mutant exhibits the sequence shown in SEQ ID NO 54.

The IL-7 variant or mutant may comprise at least one mutation selected from: W142H, W142F and W142Y. Such IL-7 variants or mutants are described in particular under the sequences shown in SEQ ID NOS: 57 to 58, respectively. Preferably, the IL-7 variant or mutant comprises the mutation W142H. Even more preferably, the IL-7 variant or mutant exhibits the sequence shown in SEQ ID NO 56.

The IL-7 variant or mutant may comprise at least one mutation selected from: D74E, D74Q and D74N, preferably D74E or D74Q. Such IL-7 variants or mutants are described in particular under the sequences shown in SEQ ID NOS: 63 to 65, respectively. Preferably, the IL-7 variant or mutant comprises the mutation D74E. Even more preferably, the IL-7 variant or mutant exhibits the sequence shown in SEQ ID NO 63.

The IL-7 variant or mutant may comprise at least one mutation selected from: Q11E, Y12F, M17L, Q22E and/or K81R. These mutations refer to the positions of the amino acids shown in SEQ ID NO 51. Such IL-7 variants or mutants are specifically described under the sequences shown in SEQ ID NOs 59, 60, 61, 62 and 66, respectively.

The IL-7 variant or mutant may comprise at least one mutation present in: i) W142H, W142F or W142Y and/or ii) D74E, D74Q or D74N, preferably D74E or D74Q and/or iii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

The IL-7 variant or mutant may comprise the W142H substitution and at least one mutation consisting of: i) D74E, D74Q or D74N, preferably D74E or D74Q and/or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

The IL-7 variant or mutant may comprise the D74E substitution and at least one mutation consisting of: i) W142H, W142F or W142Y and/or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

The IL-7 variant or mutant may comprise the mutations C2S-C141S and C47S-C92S and at least one substitution consisting of: i) W142H, W142F or W142Y and/or ii) D74E, D74Q or D74N.

The IL-7 variant or mutant may comprise i) D74E and W142H substitutions and ii) mutations C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

The variant or mutant IL-7 may comprise or consist of the amino acid sequence shown in SEQ ID NO 53, 54, 55, 56, 57, 58, 63, 64 or 65.

In a particular aspect, said IL-7 is an IL-7 variant according to the invention and said antibody or antibody fragment thereof has an IgGl Fc domain, optionally with a substitution or a combination of substitutions selected from: T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A + M252Y/S254T/T256E; K322A and K444A, preferably selected from the following: N297A, optionally in combination with M252Y/S254T/T256E, and L234A/L235A, even more preferably, an IgGl Fc domain with, for example, the mutation N297A described above. Preferably, the antibody or fragment thereof is produced by a method selected from (GGGGS)₃、(GGGGS)₄And (GGGS)₃Even more preferably by (GGGGS) ₃Linked to IL-7 or a variant thereof. Preferably, the IL-7 variant comprises a set of amino acid substitutions selected from: C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q and D74N. More preferably, the IL-7 variant comprises an amino acid substitution set selected from the group consisting of: C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q and D74N. Still more preferably, the IL-7 variant comprises an amino acid substitution set selected from the group consisting of: C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H and D74E.

In a particular aspect, the bifunctional molecule according to the present invention is a fusion protein comprising or consisting of:

(a) an antibody or antibody fragment thereof as described above which specifically binds to a target expressed on the surface of an immune cell, preferably a T cell, more preferably a target selected from the group consisting of: PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD8, more preferably PD-1, TIM3, CD244, LAG-3, BTLA, TIGIT and CD 160.

(b) Human interleukin 7 of SEQ ID No 51 or a variant or fragment thereof, and

(c) optionally a peptide linker selected from (GGGGS)₃、(GGGGS)₄、(GGGGS)₂GGGS, GGG, GGS and (GGGS)₃Preferably (GGGGS)₃。

All or any of the specific aspects and embodiments disclosed above for the bifunctional anti-PD-1 molecule may be applied to these alternative bifunctional molecules.

In a particular aspect, the antibody or antigenic fragment thereof as described above, which specifically binds to a target expressed on the surface of an immune cell, preferably a T cell, more preferably a target selected from the group consisting of: PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD8, more preferably PD-1, TIM3, CD244, LAG-3, BTLA, TIGIT and CD 160; the IL-7 is an IL-7 variant according to the invention and the antibody or antibody fragment thereof has an IgG1 Fc domain, optionally with a substitution or a combination of substitutions selected from: T250Q/M428L; M252Y/S254T/T256E + H433K/N434F; E233P/L234V/L235A/G236A + A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; N297A + M252Y/S254T/T256E; k3 22A and K444A, preferably selected from the following: N297A, optionally in combination with M252Y/S254T/T256E, and L234A/L235A, even more preferably, an IgGl Fc domain with, for example, the mutation N297A described above. Preferably, the antibody or fragment thereof is produced by a method selected from (GGGGS)₃、(GGGGS)₄And (GGGS)₃Even more preferably by (GGGGS)₃Linked to IL-7 or a variant thereof. Preferably, the IL-7 variant comprises a set of amino acid substitutions selected from: C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q and D74N. More preferably, the IL-7 variant comprises an amino acid substitution set selected from the group consisting of: C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q and D74N. Still more preferably, the IL-7 variant comprises an amino acid substitution set selected from the group consisting of: C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H and D74E.

Binding of the bifunctional molecule to its specific target can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or western blot assay. Each of these assays typically detects the presence of a protein-antibody complex of particular interest by using a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the anti-hPD-1 antibody/IL-7 complex can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds IL-7 or the IL-7 receptor.

In some examples, the bifunctional molecule described herein suppresses the PD-1 signaling pathway by at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold. Preferably, such bifunctional molecules have the ability to block or inhibit the interaction between PD-1 and its ligands (e.g. PD-L1 and/or PD-L2). In certain embodiments, the bifunctional molecule inhibits the interaction between PD-1 and its ligand (e.g., PD-L1 and/or PD-L2) by at least 50%. In certain embodiments, the inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

In some embodiments, the bifunctional molecule described herein suppresses the PD-1 signaling pathway by at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold.

In some examples, the bifunctional molecules described herein stimulate IFN γ secretion and/or α 4 and β 7.

In another example, the bifunctional molecules described herein promote T cell infiltration in tumors.

In some examples, a bifunctional molecule described herein stimulates the IL-7R signaling pathway by at least 10%, at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold.

In other aspects, the bifunctional molecules described herein retain substantially equivalent IL-7 biological properties compared to wild-type IL-7. For example, it retains biological properties comparable to full-length IL-7 protein. The biological activity of the L-7 protein can be measured using an in vitro cell proliferation assay or by measuring the P-Stat5 entry into T cells by ELISA or FACS. Preferably, the IL-7 bifunctional molecule described herein retains at least 10%, 20%, 30%, 40%, 50%, 60% of the biological activity compared to wild-type human IL-7, preferably at least 80%, 90%, 95% and even more preferably 99% of the biological activity compared to wild-type IL-7. For example, biological activity can be assessed by measuring the ability of a bifunctional molecule described herein to bind to IL-7R and/or compete with wild-type IL-7 for binding to IL-7R.

In another example, the bifunctional molecules described herein induce cytokine secretion, and/or the proliferation of an initial, partially depleted, and/or fully depleted T cell subpopulation.

Preparation of bifunctional molecules-nucleic acid molecules encoding said bifunctional molecules, recombinant expression vectors and host cells comprising same

To produce the bifunctional molecules of the present invention, the anti-hPD 1 antibodies of the present invention are functionally linked to IL-7 or variants thereof.

Both entities of the bifunctional molecule are encoded in the same vector and produced as a fusion protein. Thus, also disclosed herein are nucleic acids encoding any of the bifunctional molecules described herein, vectors (e.g., expression vectors) or recombinant viruses comprising these nucleic acids, and host cells comprising the nucleic acids and/or vectors.

In order to produce a bifunctional fusion protein which is secreted by mammalian cells in a stable form, according to the invention the nucleic acid sequence encoding the bifunctional molecule is subcloned into an expression vector which is typically used for transfection of mammalian cells. General techniques for producing molecules comprising Antibody sequences are described in Coligan et al (eds.), Current protocols in immunology, pp. 10.19.1-10.19.11 (Wiley Interscience 1992), the contents of which are incorporated herein by reference, and in "Antibody engineering: a practical guide" (1992) from W.H.Freeman and Company, with comments relating to the production of molecules interspersed throughout the text.

Generally, the method comprises the steps of:

(1) transfecting or transforming a suitable host cell with a polynucleotide encoding a recombinant bifunctional molecule of the invention or a variant thereof or a vector comprising said polynucleotide;

(2) culturing the host cell in a suitable medium; and

(3) optionally isolating or purifying the protein from the culture medium or host cell.

The invention further relates to a nucleic acid encoding the above bifunctional molecule, a vector, preferably an expression vector, comprising a nucleic acid according to the invention, a genetically engineered host cell transformed with a vector according to the invention or directly with a sequence encoding the recombinant bifunctional molecule, and a method for producing a protein according to the invention by recombinant techniques.

The nucleic acids, the vectors and the host cells are described in more detail below.

Nucleic acid sequences

The present invention also relates to a nucleic acid molecule encoding a bifunctional molecule as defined above; or a set of nucleic acid molecules encoding a bifunctional molecule as defined above.

Antibody DNA sequences can be amplified, for example, from RNA of cells that synthesize immunoglobulins, synthesized using PCR with cloned immunoglobulins, or synthesized by oligonucleotides encoding known signal peptide amino acid sequences. Preferably, for VH and/or CH, the signal peptide comprises or consists of an amino acid sequence selected from SEQ ID NO. 49; and/or for VL and/or CL the signal peptide comprises or consists of an amino acid sequence selected from SEQ ID NO: 50. In particular, the signal peptide is at the N-terminus of CH, VH, CL and/or VL.

Such nucleic acids may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH of an antibody (e.g., a light chain and/or a heavy chain of an antibody). Such nucleic acids can be readily isolated and sequenced using conventional procedures.

In particular, said nucleic acid molecule encoding a bifunctional molecule as defined above comprises:

-a first nucleic acid molecule encoding the variable heavy chain domain of an anti-hPD-1 antibody as disclosed herein, optionally having the peptide signal of SEQ ID No.49, and

-a second nucleic acid molecule encoding the variable light chain domain of an anti-hPD-1 antibody as disclosed herein, optionally having the peptide signal of SEQ ID NO:50, and

-a third nucleic acid encoding IL-7 or a variant thereof, preferably human IL-7 or a variant thereof, operably linked to the first nucleic acid or the second nucleic acid or both by a nucleic acid encoding a peptide linker.

Preferably, said nucleic acid molecule encoding a bifunctional molecule as defined above comprises:

-a first nucleic acid molecule encoding the variable heavy chain domain of SEQ ID NO 17, wherein X1 is D or E, and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N and E; optionally a peptide signal having SEQ ID NO.49, and

-a second nucleic acid molecule encoding the variable light chain domain of SEQ ID No. 26, wherein X is G or T; optionally a peptide signal having SEQ ID NO:50, and

-a third nucleic acid molecule encoding human IL-7 of SEQ ID nos 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or 63 or a variant or fragment thereof, said human IL-7 or variant or fragment thereof being operably linked to said first nucleic acid or said second nucleic acid or both, optionally via a nucleic acid encoding a peptide linker.

Preferably, the nucleic acid molecule encoding a bifunctional molecule as defined above comprises:

-a first nucleic acid molecule encoding the variable heavy domain of the amino acid sequence set forth in SEQ ID No. 18, 19, 20, 21, 22, 23, 24 or 25; optionally a peptide signal having SEQ ID NO.49, and

-a second nucleic acid molecule encoding the variable light chain domain of the amino acid sequence set forth in SEQ ID No. 27 or SEQ ID No. 28; optionally a peptide signal having SEQ ID No.50, and

-a third nucleic acid molecule encoding human IL-7 of SEQ ID nos 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or 63 or a variant thereof, said human IL-7 or variant thereof being operably linked to said first nucleic acid or said second nucleic acid or both, optionally via a nucleic acid encoding a peptide linker.

In a very particular embodiment, said nucleic acid molecule encoding the variable heavy domain has the sequence shown in SEQ ID NO. 73 and/or said nucleic acid molecule encoding the variable light domain has the sequence shown in SEQ ID NO. 74.

Operably linked means that the nucleic acid encodes a protein fusion comprising a variable heavy or light chain domain, optionally a peptide linker, and IL-7. Preferably, the linker is selected from (GGGGS)₃、(GGGGS)₄、(GGGGS)₂GGGGS, GGGS, GGG, GGS and (GGGS)₃Even more preferably (GGGGS)₃。

In one embodiment, the nucleic acid molecule is an isolated, in particular non-natural, nucleic acid molecule.

The nucleic acid molecule or set of nucleic acid molecules encoding the bifunctional molecule according to the present invention is preferably comprised in a vector or set of vectors.

Carrier

In another aspect, the invention relates to a vector comprising a nucleic acid molecule or a set of nucleic acid molecules as defined above.

As used herein, a "vector" is a nucleic acid molecule that serves as a carrier, transferring genetic material into a cell. The term "vector" encompasses plasmids, viruses, cosmids, and artificial chromosomes. In general, engineered vectors contain an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is usually a nucleotide sequence, usually a DNA sequence, which contains an insert (transgene) and a larger sequence, which serves as the "backbone" of the vector. In addition to the transgene insert and backbone, modern vectors may also contain other features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags. Vectors, referred to as expression vectors (expression constructs), are specifically used for expressing transgenes in target cells and typically have control sequences.

In one embodiment, the heavy chain coding sequence and the light chain coding sequence and/or the constant region of the anti-PD 1 antibody are contained in one expression vector. Each of the heavy chain coding sequence and the light chain coding sequence may be operably linked to a suitable promoter, the heavy chain and/or the light chain being operably linked to an immunotherapeutic agent as described herein. Alternatively, expression of both the heavy and light chains may be driven by the same promoter. In another embodiment, each of the heavy and light chains of the antibody is cloned into a separate vector, one or both of the heavy and light chains, the heavy and/or light chain being operably linked to an immunotherapeutic agent according to the invention. In the latter case, expression vectors encoding the heavy and light chains may be co-transfected into a host cell to express both chains, which may be assembled in vivo or in vitro to form a complete antibody. Alternatively, expression vectors encoding the heavy chain and encoding the light chain may be introduced into different host cells to express both the heavy and light chains, which may then be purified and assembled in vitro to form the intact antibody.

One skilled in the art can clone a nucleic acid molecule encoding a humanized anti-PD-1 antibody or antibody fragment thereof into a vector, which is then transformed into a host cell. Accordingly, the present invention also provides a recombinant vector comprising a nucleic acid molecule encoding the anti-PD-1 antibody of the present invention or a fragment thereof. In a preferred embodiment, the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretion signal peptide, and optionally at least one drug resistance gene for screening.

Suitable expression vectors typically contain (1) prokaryotic DNA elements encoding a bacterial origin of replication and an antibiotic resistance marker to provide for growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control transcription initiation, such as promoters; and (3) DNA elements that control transcript processing, such as transcription termination/polyadenylation sequences.

Methods well known to those skilled in the art can be used to construct expression vectors comprising nucleic acid sequences of the anti-bifunctional molecules described herein and appropriate regulatory components for transcription/translation. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence is operably linked to a suitable promoter in an expression vector to direct the synthesis of mRNA. The expression vector may also contain a ribosome binding site for initiating translation, transcription terminator and the like.

The expression vector may be introduced into the host cell using a variety of techniques, including calcium phosphate transfection, liposome-mediated transfection, electroporation, and the like. Preferably, the transfected cells are selected and propagated, wherein the expression vector is stably integrated into the host cell genome to produce stable transformants. Techniques for introducing vectors into eukaryotic cells and for selecting stable transformants using dominant selectable markers are described below: sambrook, Ausubel, Bebbington, "Expression of Antibody Genes in Nonlymphoid Mammarian Cells," 2METHODS: A companion to METHODS in enzymology 136(1991), and Murray (eds.), Gene transfer and Expression protocols (Humana Press 1991). Suitable cloning vectors are described below: sambrook et al (eds.), Molecula clone: A Laboratory Manual, second edition (Cold Spring Harbor Press 1989) (hereinafter "Sambrook"); ausubel et al (eds.), Current promoters IN MOLECULAR BIOLOGY (Wiley Interscience 1987) (hereinafter "Ausubel"); and Brown (eds.), MOLECULAR BIOLOGY LABFAX (Academic Press 1991).

Host cell

In another aspect, the present invention relates to a host cell comprising a vector or a nucleic acid molecule or a set of nucleic acid molecules as defined above for the purpose of bifunctional molecule production.

As used herein, the term "host cell" is intended to include any individual cell or cell culture that may be or has been a recipient for a vector, an exogenous nucleic acid molecule, and a polynucleotide encoding an antibody construct of the invention and/or the antibody construct or bifunctional molecule itself. The corresponding substance can be introduced into the cells by transformation, transfection, or the like. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, and also include, but are not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells, e.g., mouse, rabbit, macaque, or human.

In one embodiment, the host cell comprises (e.g., is transformed with): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising the VL of an antibody and/or comprising an amino acid sequence of the VH of an antibody and/or a constant region of an antibody, or (2) a first vector comprising nucleic acids encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising nucleic acids encoding an amino acid sequence comprising the VH of an antibody.

In another embodiment, the host cell comprises (e.g. has been transformed with) a vector comprising both entities of the bifunctional molecule. Preferably, the host cell comprises (e.g., has been transformed with) a vector comprising a first nucleic acid molecule encoding the variable heavy chain domain of an anti-hPD-1 antibody as disclosed herein and a second nucleic acid molecule encoding the variable light chain domain of an anti-hPD-1 antibody as disclosed herein, said first and second nucleic acid molecules being operably linked to a third nucleic acid encoding IL-7 or a variant or mutant thereof, preferably human IL-7 or a variant thereof.

Also provided herein is a method of producing a humanized anti-PD 1 antibody. The method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium). In particular, for recombinant production of humanized anti-PD 1 antibodies, nucleic acids encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.

The bifunctional molecule of the present invention is preferably expressed in a eukaryotic cell (e.g., a mammalian cell, a plant cell, an insect cell, or a yeast cell). Mammalian cells are particularly preferred eukaryotic hosts because mammalian cells provide suitable post-translational modifications, such as glycosylation. Preferably, such suitable eukaryotic host cells may be fungi, such as Pichia pastoris (Pichia pastoris), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe); insect cells, such as oriental armyworm (Mythimna separate); plant cells, such as tobacco, and mammalian cells, such as BHK cells, 293 cells, CHO cells, NSO cells, and COS cells. Other examples of useful mammalian host cell lines are CV-1 derived cells (COS cells) having the SV40 gene, monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney cell lines (293 or 293 cells as described, for example, in Graham, f.l. et al, j.gen virol.36(1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, J.P., biol. reprod.23(1980) 243-252); human renal epithelial cells (HEK cells); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather, J.P. et al, Annals N.Y.Acad.Sci.383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR "CHO cells (Urlaub, G., et al, Proc. Natl. Acad. Sci. USA 77 (1980)) 4216-; and myeloma cell lines, such as Y0, NSO and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, p. and Wu, a.m., Methods in Molecular Biology, vol.248, Lo, b.k.c. (eds.), Humana Press, Totowa, NJ (2004), pp.255-268. For example, mammalian cell lines suitable for growth in suspension may be useful.

In particular, the host cell of the invention is selected from the following: CHO cells, COS cells, NSO cells and HEK cells.

For mammalian hosts, the transcriptional and translational regulatory signals of the expression vector may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, etc., where the regulatory signals are associated with specific genes having high expression levels. Suitable transcriptional and translational regulatory sequences may also be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.

A variety of methods can be used to identify stable transformants that produce the bifunctional molecules according to the invention. After the molecule-producing cell is identified, the host cell is cultured under conditions (e.g., temperature, culture medium) suitable for its growth and expression of the bifunctional molecule. The humanized antibody is then isolated and/or purified by any method known in the art. These methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (e.g., salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and combinations thereof. Bifunctional molecular separation techniques may specifically include affinity chromatography using protein a sepharose, size exclusion chromatography and ion exchange chromatography, as described for example by Coligan. Protein a is preferably used for isolating the bifunctional molecules according to the present invention.

Pharmaceutical compositions and methods of administration thereof

The present invention also relates to a pharmaceutical composition comprising any of the bifunctional molecules described herein, such as the nucleic acid molecule, set of nucleic acid molecules, vector and/or host cell described above, preferably being an active ingredient or compound. The formulation may be sterilized and, if desired, mixed with adjuvants, such as pharmaceutically acceptable carriers and excipients, which do not deleteriously interact with the bifunctional molecules, nucleic acids, vectors and/or host cells described herein. Optionally, the pharmaceutical composition may further comprise an additional therapeutic agent as described in detail below.

Preferably, the pharmaceutical composition according to the present invention may comprise a bifunctional molecule as described herein, such as a nucleic acid molecule, set of nucleic acid molecules, vector and/or host cell as described above, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, excipients, salts and antioxidants as described below. Ideally, a pharmaceutically acceptable form is used which does not adversely affect the desired immunopotentiation effect of the bifunctional molecule according to the present invention. For ease of administration, the bifunctional molecules as described herein may be formulated into a pharmaceutical composition for in vivo administration. Methods for preparing such compositions have been described in The art (see, e.g., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21 st edition (2005)).

In particular, the pharmaceutical compositions according to the invention may be formulated for any conventional route of administration, including topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like. Preferably, the pharmaceutical composition according to the invention is formulated for enteral or parenteral administration route. Compositions and formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier (carder) compounds and other pharmaceutically acceptable carriers or excipients.

The pharmaceutical composition can be prepared by mixingThe agent of the desired purity is prepared by mixing with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16 th edition, Osol, a. eds. (1980)), either as a lyophilized formulation or as an aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl esters such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, e.g. TWEEN ^TM、PLURONICS^TMOr polyethylene glycol (PEG).

A solid pharmaceutically acceptable carrier may include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweetening agents, preservatives, dyes, coatings or tablet disintegrants. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinyl pyrrolidine, low melting waxes and ion exchange resins. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Except insofar as any conventional media or agent is incompatible with the active compound, it is contemplated that it will be used in the pharmaceutical compositions of the invention.

The bifunctional molecule according to the present invention may be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, organic solvents, ethanol, polyols (e.g. glycerol, propylene glycol and liquid polyethylene glycol) and the like, mixtures of both or pharmaceutically acceptable oils or fats and suitable mixtures thereof. The liquid carrier may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, wetting agents, thickeners, colorants, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and enteral administration include water (partially containing additives as described above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the carrier may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for enteral administration. The liquid carrier for the pressurized composition may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

The pharmaceutical compositions of the present invention may also comprise one or more pharmaceutically acceptable salts. "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not produce any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphoric and the like, and those derived from non-toxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkali or alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as those derived from non-toxic organic amines, such as Ν, Ν' -dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbyl palmitate, Butyl Hydroxyanisole (BHA), Butyl Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

For ease of delivery, any of the bifunctional molecules, or a nucleic acid encoding it, may be conjugated to a chaperone agent (chaperon agent). The chaperone agent may be a naturally occurring substance, such as a protein (e.g., human serum albumin, low density lipoprotein, or globulin), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or a lipid. It may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include Polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-ethylene glycol) copolymer, divinyl ether-maleic anhydride copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), N-isopropylacrylamide polymer, and polyphosphazene. In one embodiment, the chaperone agent is a micelle, lipid, nanoparticle, or microsphere. Methods for preparing such micelles, liposomes, nanoparticles or microspheres are well known in the art. See, e.g., U.S. Pat. nos. 5,108,921; 5,354,844, respectively; 5,416,016; and 5,527,5285.

Pharmaceutical compositions must generally be sterile and stable under the conditions of manufacture and storage. The pharmaceutical composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration and/or suitable for injection. The appropriate mobile phase may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersants and by the use of surfactants.

In one embodiment, the pharmaceutical composition is an injectable composition, which may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, the water-soluble antibody may be administered by instillation, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may comprise, for example, 5% dextrose, 0.9% saline, ringer's solution, or other suitable excipients. Intramuscular preparations, e.g., sterile preparations of the antibody in the form of a suitable soluble salt, may be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% dextrose solution.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or more of the ingredients enumerated above, as required, followed by sterile microfiltration. Generally, dispersants are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

Prevention of the presence of microorganisms can be ensured by sterilization procedures and inclusion of various antibacterial and antifungal agents, such as chlorobutanol, sorbic acid phenol, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

It will be appreciated by those skilled in the art that the formulations of the invention may be isotonic with human blood, i.e. the formulations of the invention have substantially the same osmotic pressure as human blood. Such isotonic formulations typically have an osmotic pressure of from about 250mOSm to about 350 mOSm. Isotonicity can be measured by, for example, vapor pressure or freezing type osmometers. The tonicity of the formulation is adjusted by the use of a tonicity adjusting agent. "tonicity adjusting agents" are those pharmaceutically acceptable inert substances which may be added to the formulation to provide isotonicity of the formulation. Tonicity adjusting agents suitable for use in the present invention include, but are not limited to, sugars, salts, and amino acids.

The pharmaceutical composition according to the invention may be formulated to release the active ingredient (e.g. the bifunctional molecule according to the invention) substantially immediately after administration or at any predetermined time or time period after administration. In some aspects, the pharmaceutical compositions may employ delayed release, and sustained release delivery systems such that delivery of the composition occurs prior to sensitization of the site to be treated and for sufficient time to cause sensitization of the site to be treated. Methods known in the art can be used to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed release of the composition. Such a system may avoid repeated administration of the composition, thereby increasing convenience for the subject and the physician.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of the composition which produces a therapeutic effect.

Subject, regimen and administration

The present invention relates to bifunctional molecules as described herein; a nucleic acid or vector, host cell or pharmaceutical composition encoding the same, a nucleic acid, vector or host cell for use as a medicament, or for treating a disease or for administration in a subject or for use as a medicament. It also relates to the use of a pharmaceutical composition, a nucleic acid, a vector or a host cell of the invention or a bifunctional molecule comprising an anti-PD 1 antibody or antibody fragment thereof and IL-7 or a variant thereof for the preparation of a medicament for the treatment of a disease in a subject. Finally, it relates to a method for treating a disease or disorder in a subject comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition or a bifunctional molecule comprising an anti-PD 1 antibody or antibody fragment thereof and IL-7 or a variant thereof. Examples of treatments are described in more detail in the "methods and uses" section below.

The subject to be treated may be a human, in particular a human in the prenatal stage, a newborn, a child, an infant, an adolescent or an adult, in particular an adult of at least 30 years, 40 years, preferably at least 50 years, further more preferably at least 60 years, even more preferably at least 70 years.

In particular, the subject suffers from a disease that may involve at least one of the PD-1/PDL-1 pathway, in particular a ligand (e.g., PDL-1 and/or PDL-2) or PD-1 in which PD-1 is expressed, in particular overexpressed. Preferably, the subject has cancer, even more preferably PD1, PD-L1 and/or PD-L2-positive cancer or PD-1-positive cancer. Examples of diseases and cancers are described in more detail below in the "methods and uses" section.

In a particular embodiment, the subject has received at least one line of treatment, preferably several lines of treatment, prior to administration of the bifunctional molecule comprising an anti-PD 1 antibody or antibody fragment thereof according to the invention and IL-7 or a variant thereof or the pharmaceutical composition according to the invention.

The bifunctional molecule or the pharmaceutical composition disclosed herein can be administered to a subject using conventional methods well known to those of ordinary skill in the art, depending on the type of disease or the site of disease to be treated. The composition may be administered by conventional routes, for example, orally, parenterally, enterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intratumoral, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. When administered parenterally, the pharmaceutical composition of the present invention is preferably administered by intravenous route of administration. When administered enterally, the pharmaceutical compositions of the present invention are preferably administered by the oral route of administration. The composition may also be administered topically.

The form, route of administration and dosage of administration of the pharmaceutical composition according to the invention or of the bifunctional molecule can be adjusted by the person skilled in the art according to the type and severity of the infection as well as the patient, in particular his age, weight, sex and general physical condition. The compositions of the present invention may be administered in a variety of ways depending on whether local or systemic treatment is desired.

Preferably, the treatment with said bifunctional molecule or with the pharmaceutical composition according to the invention is administered periodically, preferably between every day, week or month, more preferably between every day and every week, every two weeks, every three weeks or every four weeks. In a particular embodiment, the treatment is administered several times a day, preferably 2 or 3 times a day.

The duration of the treatment with the bifunctional molecule or with the pharmaceutical composition according to the invention is preferably between 1 day and 20 weeks, more preferably between 1 day and 10 weeks, still more preferably between 1 day and 4 weeks, even more preferably between 1 day and 2 weeks. Alternatively, treatment may continue as long as the disease persists.

The bifunctional molecules disclosed herein may be provided in an effective dose in the range of about 1ng/kg body weight to about 30mg/kg body weight, 1 μ g/kg to about 20mg/kg, 10 μ g/kg to about 10mg/kg, or from 100 μ g/kg to 5mg/kg, optionally every one week, two weeks, three weeks or four weeks, preferably by parenteral or oral administration, in particular by intravenous or intradermal administration.

In particular, the bifunctional molecule according to the present invention may be administered at a sub-therapeutic dose. As used herein, the term "subtherapeutic dose" refers to a dose below the level of an effective monotherapy dose typically used to treat a disease, or a dose not currently used for effective monotherapy using an anti-hPD 1 antibody.

Method and use

Use in the treatment of diseases

The bifunctional molecules, nucleic acids, vectors, host cells, compositions, and methods described herein have a variety of in vitro and in vivo utilities and applications. For example, the bifunctional molecules, nucleic acids, vectors, host cells and/or pharmaceutical compositions described herein may be used as therapeutic agents, diagnostic agents and medical research. In particular, any bifunctional molecule, nucleic acid molecule, group of nucleic acid molecules, vector, host cell or pharmaceutical composition provided herein may be used in a method of treatment and/or for therapeutic purposes. In particular, the bifunctional molecules, nucleic acids, vectors or pharmaceutical compositions provided herein may be used to treat any disease or condition, preferably involving PD-1, such as cancer, autoimmune diseases and infections, or other diseases associated with immunodeficiency, such as T cell dysfunction.

Even more preferably, the present invention relates to a method of treating a disease and/or disorder selected from the group consisting of: cancer, infectious disease and chronic viral infection, said method comprising administering to said subject an effective amount of a bifunctional molecule or a pharmaceutical composition as defined above. Examples of such diseases are described more specifically below.

In particular, the bifunctional molecule according to the present invention is referred to as "bifunctional checkpoint inhibitor" because it targets the PD-1/PD-L1/PD-L2 and IL7 pathways.

The invention particularly relates to bifunctional molecules, nucleic acids, nucleic acid sets or vectors encoding the same, or pharmaceutical compositions comprising the same for use in the treatment of pathologies, diseases and/or disorders that can be prevented or treated by inhibiting the binding of PD-L1 and/or PD-L2 to PD-1.

The bifunctional molecules according to the present invention target CD127+ immune cells, in particular CD127+ T cells. Such cells can be found in the following regions of particular interest: resident lymphocytes in lymph nodes (mainly in the paracortical, occasionally cells in the follicles), tonsils (interfollicular region), spleens (mainly in the periarteriolar lymph sheath (PALS) of the white marrow and some scattered cells in the red marrow), thymus (mainly in the medulla; also in the cortex), bone marrow (scattered distribution), GALT (gut associated lymphoid tissue, mainly in the interfollicular region and lamina propria) of the entire digestive tract (stomach, duodenum, jejunum, ileum, cecum colon, rectum), MALT (mucosa associated lymphoid tissue) of the gall bladder. The bifunctional molecules of the present invention are therefore of particular interest for the treatment of diseases located in or involving these regions, in particular cancer.

Thus, disclosed herein is a method for the treatment of a disease specifically associated with the PD-1 and/or PD-1/PD-L1 and/or PD-1/PD-L2 signaling pathway, comprising administering to a subject in need of treatment an effective amount of any of the bifunctional molecules or pharmaceutical compositions described herein. The appropriate dosage must also be determined in view of the patient's physiological data (e.g., age, size and weight) and the route of administration in order to administer a therapeutically effective amount to the patient.

In another aspect, the bifunctional molecules described herein may be administered to a subject, e.g., in vivo, to enhance immunity, preferably to treat a disorder and/or disease. Thus, in one aspect, the present invention provides a method of altering an immune response in a subject comprising administering to the subject a bifunctional molecule, nucleic acid, vector or pharmaceutical composition according to the present invention, such that the immune response in the subject is altered. Preferably, the immune response is enhanced, increased, stimulated or up-regulated. The bifunctional molecule or pharmaceutical composition may be used to enhance an immune response in a subject in need of treatment. Such as T cell activation. The enhanced immune response may result in the inhibition of binding of PD-L1 and/or PD-L2 to PD-1, thereby reducing the immunosuppressive environment, stimulating the proliferation and/or activation of human T cells and/or IFN γ secretion by human PBMCs.

The present invention provides, inter alia, a method of enhancing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of any of the bifunctional molecules, nucleic acids, vectors, or pharmaceutical compositions comprising the same described herein, such that the immune response in the subject is enhanced.

In some embodiments, the amount of bifunctional molecule described herein is effective in inhibiting PD-1 signaling (e.g., reduces PD-1 signaling by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% as compared to a control). In other embodiments, the amount of bifunctional molecule described herein is effective in activating an immune response (e.g., at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% activation as compared to a control).

In some embodiments, the amount of bifunctional molecule described herein is effective in inhibiting binding of human PD-L1 and/or PD-L2 to human PD-1 (e.g., inhibits binding by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% as compared to a control).

In some embodiments, the amount of bifunctional molecule described herein is sufficient to have antagonist activity of human PD-L1 and/or PD-L2 binding to human PD-1 (e.g., inhibiting binding by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% as compared to a control).

The invention also relates to bifunctional molecules, nucleic acids or vectors encoding the same, or pharmaceutical compositions comprising the same, as described herein, for use in treating a disorder and/or disease in a subject and/or for use as a medicament or vaccine. It also relates to the use of a bifunctional molecule as described herein, a nucleic acid or vector encoding the same, or a pharmaceutical composition comprising the same, for the preparation of a medicament for the treatment of a disorder and/or disease in a subject. Finally, it relates to a method for treating a disease or disorder in a subject, comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition or a bifunctional molecule.

Disclosed herein are methods of treating a patient having a disease and/or disorder, comprising: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of any of the bifunctional molecules, nucleic acids, vectors or pharmaceutical compositions described herein.

The subject in need of treatment may be a human having, at risk of having, or suspected of having a disease associated with a PD-1 mediated signaling pathway. Such patients may be identified by routine medical examination. For example, subjects suitable for such treatment can be identified by examining whether such subjects carry PD-1, PD-L1, and/or PD-L2 positive cells. Preferably, "PD-L1 positive cells" or "PD-L2 positive tumor cells" are intended to refer to a population of tumor cells, wherein PD-L1 or PD-L2, respectively, is expressed in 10% of the tumor cells, preferably at least 20%, 30%, 40% or 50% of the tumor cells.

In one embodiment, the subject in need of treatment is a patient having, suspected of having, or at risk of having a disease, preferably a PD-1, PDL1 and/or PDL2 positive disease, even more preferably a disease wherein PD-1 and/or at least one PD-1 ligand is overexpressed. In such a subject, disruption of the PD-1/PD-L1 and/or PD-1/PD-L2 interaction may enhance the immune response of the subject as a result of administration of the bifunctional molecule or pharmaceutical composition according to the invention. In some embodiments, any of the humanized anti-PD-1 antibodies or pharmaceutical compositions described herein can be used to treat PD-1 positive cells.

-Cancer treatment

It is well known in the art that blocking of PD-1 by antibodies can enhance the immune response against cancer cells in a patient. Thus, in one aspect, the present invention provides a bifunctional molecule or pharmaceutical composition for use in treating a subject suffering from cancer, comprising administering to the individual an effective amount of said bifunctional molecule or pharmaceutical composition, preferably to disrupt or inhibit the PD1/PD-L1 and/or PD1/PD-L2 interaction, and/or to activate the IL7 receptor.

In one embodiment, the subject in need of treatment is a patient having, suspected of having, or at risk of disease, preferably a PD-1 or PD-L1 positive cancer, even more preferably a cancer wherein PD-1 is expressed or overexpressed. In some embodiments, any of the anti-PD-1 antibodies or pharmaceutical compositions described herein can be used to treat PD-1 positive tumor cells. For example, patients suitable for treatment can be identified by investigating whether such patients carry PD-1 positive tumor cells.

In another embodiment, the subject is a patient having, suspected of having, or at risk of developing cancer, preferably a PD-L1 and/or PD-L2 positive cancer. In some embodiments, any of the bifunctional molecules or pharmaceutical compositions described herein can be used to treat a PD-L1 and/or PD-L2 positive tumor. For example, a human patient suitable for treatment can be identified by examining whether such patient carries PD-L1 and/or PD-L2 positive cancer cells.

In other aspects, a bifunctional molecule or pharmaceutical composition is provided for the treatment of cancer, preferably PD-1, PD-L1 and/or PD-L2 positive cancer, even more preferably a cancer wherein PD-1, PD-L1 and/or PD-L2 is overexpressed.

In another embodiment, the invention provides the use of a bifunctional molecule or pharmaceutical composition as disclosed herein for the preparation of a medicament for the treatment of cancer, e.g. for inhibiting the growth of a tumor cell, preferably a PD-1, PD-L1 or PD-L2 positive tumor cell, in a subject.

In one aspect of the disclosure, the cancer to be treated is associated with depleted T cells.

Thus, in one embodiment, the present invention provides a method of treating cancer (e.g. for inhibiting tumor cell growth in a subject) comprising administering to said subject a therapeutically effective amount of a bifunctional molecule or pharmaceutical composition according to the present invention. In particular, the invention relates to the use of bifunctional molecules to treat a subject so as to inhibit the growth of cancer cells.

Any suitable cancer that can be treated using the bifunctional molecules provided herein can be a hematopoietic cancer or a solid cancer. Such cancers include carcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, renal cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, gastric cancer, urinary tract cancer, environmentally induced cancer, and any combination of the cancers. The invention is also useful for treating metastatic cancer, particularly metastatic cancer expressing PD-L1 (Iwai et al (2005) int. Immunol.17: 133-144). In addition, the invention includes refractory or recurrent malignancies.

Preferably, the cancer to be treated or prevented is selected from metastatic or non-metastatic melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, hodgkin's lymphoma, head and neck cancer, urothelial cancer, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic merkel cell cancer, gastric or esophageal cancer, and cervical cancer.

In a particular aspect, the cancer is a hematologic malignancy or solid tumor that overexpresses PD-1 and/or PD-L1. Such cancers may be selected from the following: lymphohematopoietic tumors, angioimmunoblastic T-cell lymphomas, myelodysplastic syndromes, acute myelogenous leukemia.

In a particular aspect, the cancer is a virus-induced or immunodeficiency-associated cancer. Such cancers may be selected from the following: kaposi's sarcoma (e.g., associated with kaposi's sarcoma herpes virus); squamous cell carcinoma of the cervix, anus, penis, and vulva, and oropharyngeal cancer (e.g., associated with human papilloma virus); b-cell non-hodgkin's lymphoma (NHL) including diffuse large B-cell lymphoma, burkitt's lymphoma, plasmablast lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classical hodgkin's lymphoma, and lymphoproliferative disorders (e.g., associated with epstein-barr virus (EBV) and/or kaposi's sarcoma herpes virus); hepatocellular carcinoma (e.g., associated with hepatitis b and/or hepatitis c virus); merkel cell carcinoma (e.g., associated with merkel cell polyoma virus (MPV)); and cancers associated with Human Immunodeficiency Virus (HIV) infection.

Preferred treatments for cancer include cancers that are generally responsive to immunotherapy. Alternatively, the preferred cancer treatment is a cancer that is not responsive to immunotherapy.

Preferably, the bifunctional molecule, nucleic acid, vector, host cell or composition disclosed herein is used to treat a subject suffering from a cancer with a poor prognosis. As used herein, the term "poor prognosis" refers to decreased survival of a subject and/or early cancer progression and/or increased or early cancer recurrence and/or increased risk of metastasis or increased occurrence of metastasis. In particular, poor prognosis is associated with cancers in which a population of Treg cells is present in the tumor or in which the Treg/Teff ratio is high in the tumor (Chraa et al, 2018J Leukoc biol.2018; 1-13.)

By way of example and without wishing to be bound by theory, treatment with anti-cancer antibodies or anti-cancer immunoconjugates or other current anti-cancer therapies that result in cancer cell death will potentiate the immune response mediated by PD-1. Thus, treatment of a hyperproliferative disease (e.g., a cancer tumor) may include a combination, simultaneously or sequentially, or any combination thereof, of a bifunctional molecule with an anti-cancer therapy, which may enhance the anti-tumor immune response of the host. Preferably, the bifunctional molecule may be used in combination with other immunogenic agents, standard cancer therapy or other antibodies.

-Infectious diseases

The bifunctional molecules, nucleic acids, nucleic acid sets, vectors, host cells or pharmaceutical compositions of the present invention are useful for treating patients who have been exposed to a particular toxin or pathogen. Accordingly, one aspect of the present invention provides a method of treating an infectious disease in a subject, comprising administering to said subject a bifunctional molecule according to the present invention or a pharmaceutical composition comprising the same, preferably such that the infectious disease of said subject is treated.

Any suitable infection may be treated using the bifunctional molecules, nucleic acids, nucleic acid sets, vectors, host cells or pharmaceutical compositions provided herein according to the present invention. Some examples of pathogenic viruses that cause infections that can be treated by the methods of the invention include HIV, hepatitis (type A, B, or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus, and arbo encephalitis virus.

In particular, the bifunctional molecule or the pharmaceutical composition according to the invention is used for treating a patient suffering from a chronic viral infection, such infection being caused by a virus selected from the group consisting of: retrovirus, dactylovirus, circovirus, herpesvirus, Varicella Zoster Virus (VZV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), polyoma virus BK, polyoma virus, adeno-associated virus (AAV), herpes simplex type 1 (HSV-1), adenovirus, herpes simplex type 2 (HSV-2), Kaposi's Sarcoma Herpesvirus (KSHV), Hepatitis B Virus (HBV), GB virus C, papilloma virus, Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), Hepatitis D Virus (HDV), human T cell leukemia virus type 1 (HTLV1), xenotropic murine leukemia virus-associated virus (XMLV), rubella virus, German measles, parvovirus B19, measles virus, coxsackie virus.

Some examples of pathogenic bacteria that cause infections that can be treated by the methods of the present invention include chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and streptococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, clostridium botulinum, anthrax, plague, leptospirosis, and lyme disease bacteria.

Some examples of pathogenic fungi that cause infections that can be treated by the methods of the present invention include candida (candida albicans, candida krusei, candida glabrata, candida tropicalis, etc.), cryptococcus neoformans, aspergillus (aspergillus fumigatus, aspergillus niger, etc.), mucorales (mucor, Absidia, rhizopus), sporothrix scherzei, blastomyces dermatitidis, paracoccidioides brasiliensis, coccidioidomycosis immanensis, and histoplasma capsulatum.

Some examples of pathogenic parasites that cause infections that can be treated by the methods of the invention include entamoeba histolytica, large intestine worms, fujiri-grimba, acanthamoeba, giardia lamblia, cryptosporidium, pneumocystis carinii, plasmodium vivax, babesia parvum, trypanosoma brucei, trypanosoma cruzi, doramelis, toxoplasma gondii and brazilian day.

In all of the above methods, the bifunctional molecule may be combined with other forms of immunotherapy such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or any therapy that enhances tumor antigen presentation.

Combination therapy

In particular, the bifunctional molecules according to the present invention can be combined with some other potential strategies to overcome the immune evasion mechanism of drugs in clinical development or already on the market (see Table 1 from Antonia et al, Immuno-interactive combinations: a review of clinical expeditions and future prospects.Clin. cancer Res. of. J. am. Assoc. cancer Res.20, 6258-6268,2014). This combination with the bifunctional molecule according to the invention can be used in particular for:

1-suppression of reverse adaptive immunity (blocking the T cell checkpoint pathway);

2-turn on adaptive immunity (use of agonist molecules (particularly antibodies) to promote T cell costimulatory receptor signaling);

3-improving the function of innate immune cells;

4-activation of the immune system (enhancement of immune cell effector functions), for example by vaccine-based strategies.

Thus, also provided herein is a combination therapy for any disease associated with PD-1 signaling as described herein, using any one of the bifunctional molecules as described herein or a pharmaceutical composition comprising the same, and a suitable second agent. In one aspect, the bifunctional molecule and the second agent may be present in a pharmaceutical composition as described above. Alternatively, as used herein, the term "combination therapy" or "combination therapy" includes administration of these agents (e.g., bifunctional molecules as described herein and additional or second suitable therapeutic agents) in a sequential manner, i.e., wherein each therapeutic agent is administered at a different time, and the therapeutic agents or at least two agents are administered in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each agent may be achieved by any suitable route. The agents may be administered by the same route or by different routes. For example, a first agent (e.g., a bifunctional molecule) can be administered orally, and another therapeutic agent (e.g., an anti-cancer agent, an anti-infective agent, or an immunomodulatory agent) can be administered intravenously. Alternatively, the agents of the selected combination may be administered by intravenous injection, while the other agents of the combination may be administered orally.

In another aspect, the present invention relates to a therapeutic means, in particular a combination product means, comprising as active ingredients: a bifunctional molecule as defined above and a further therapeutic agent, wherein the active ingredients are formulated for separate, sequential or combined therapy, in particular combined or sequential use.

As used herein, unless otherwise specified, the term "sequential" means characterized by a regular order or sequence, e.g., if a dosage regimen comprises administration of a bifunctional molecule and an additional or second agent, a sequential dosage regimen may comprise administration of the bifunctional molecule of the present invention prior to, simultaneously, substantially simultaneously, or after administration of the second agent, but the two agents will be administered in a regular order or sequence. The term "separately" means separating one from another unless otherwise indicated. The term "simultaneously" means, unless otherwise specified, to occur or to be completed simultaneously, i.e. the agents of the invention are administered simultaneously. The term "substantially simultaneously" means that the agents are administered within minutes of each other (e.g., within 15 minutes of each other) and is intended to include combined and continuous administration, but if administration is continuous, it is only separated in time by a short time (e.g., the time required for the physician to administer the two compounds separately).

It is to be understood that any combination as described herein can be used in any order to treat a condition or disease described herein. The combination described herein can be selected based on a variety of factors, including but not limited to, the utility of inhibiting or preventing the progression of the target disease, the utility of alleviating a side effect of another agent of the combination, or the utility of alleviating a symptom associated with the target disease. For example, the combination therapies described herein can reduce any side effects associated with each individual member of the combination.

The present invention also relates to a method for treating a disease in a subject comprising administering to said subject a therapeutically effective amount of a bifunctional molecule or pharmaceutical composition as described herein and a therapeutically effective amount of an additional or second therapeutic agent.

When the bifunctional molecule or pharmaceutical composition described herein is used in conjunction with an additional therapeutic agent, a subtherapeutic dose of the bifunctional molecule, the composition or the second agent, or a subtherapeutic dose of both, can be used to treat a subject, preferably a subject suffering from or at risk of developing a disease or disorder associated with cell signaling mediated by PD-1.

Specific examples of additional or second therapeutic agents are provided on pages 36-43 of WO 2018/053106.

In one aspect, the additional or second therapeutic agent may be selected in a non-exhaustive list comprising: alkylating agents, angiogenesis inhibitors, antimetabolites, antimitotics, antiproliferative agents, antivirals, aurora kinase inhibitors, apoptosis-promoting agents (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T-cell cement) antibodies, antibody drug conjugates, biological response modifiers, Bruton's Tyrosine Kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, Heat Shock Protein (HSP) -90 inhibitors, Histone Deacetylase (HDAC) inhibitors, hormonal therapies, immunological drugs, inhibitors of apoptosis protein Inhibitors (IAPs), intercalating antibiotics, HIV inhibitors of HIV inhibitors, HIV inhibitors of HIV-like, Kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target protein of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate) -ribose polymerase (PARP) inhibitors, platinum-based chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid/retinoid plant alkaloids, small inhibitory ribonucleic acid (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines and the like, epitopes or neo-epitopes from tumor antigens, and combinations of one or more of these agents. For example, the additional therapeutic agent may be selected from the following: chemotherapy, radiation therapy, targeted therapy, anti-angiogenic agents, hypomethylation agents, cancer vaccines, epitopes or neo-epitopes from tumor antigens, bone marrow checkpoint inhibitors, other immunotherapies, and HDAC inhibitors.

In a preferred embodiment, the second therapeutic agent is selected from the following: chemotherapeutic agents, radiation therapy agents, immunotherapeutic agents, cell therapy agents (e.g., CAR-T cells), antibiotics, and probiotics. The immunotherapeutic agent may also be an antibody targeting a tumor antigen, in particular selected from the following: anti-Her 2, anti-EGFR, anti-CD 20, anti-CD 19, anti-CD 52.

In one embodiment, the present invention relates to a combination therapy as defined above, wherein said second therapeutic agent is in particular selected from the group consisting of: therapeutic vaccines, immune checkpoint blockers or activators, particularly adaptive immune cells (T and B lymphocytes) and antibody-drug conjugates. Preferably, suitable agents for use with the anti-hPD-1 antibody or fragment thereof or pharmaceutical composition according to the invention include antibodies that bind to a co-stimulatory receptor (e.g., OX40, CD40, ICOS, CD27, HVEM, or GITR), agents that induce immunogenic cell death (e.g., chemotherapeutic agents, radiotherapeutic agents, anti-angiogenic agents, or agents for targeted therapy), agents that inhibit checkpoint molecules (e.g., CTLA4, LAG3, TIM3, B7H3, B7H4, BTLA, or TIGIT), cancer vaccines, agents that modulate immunosuppressive enzymes (e.g., IDO1 or iNOS), agents that target Treg cells, agents for adoptive cell therapy, or agents that modulate myeloid cells.

In one embodiment, the invention relates to a combination therapy as defined above, wherein the second therapeutic agent is an immune checkpoint blocker or activator of adaptive immune cells (T and B lymphocytes) selected from the group consisting of: anti-CTLA 4, anti-CD 2, anti-CD 28, anti-CD 40, anti-HVEM, anti-BTLA, anti-CD 160, anti-TIGIT, anti-TIM-1/3, anti-LAG-3, anti-2B 4, and anti-OX 40, anti-CD 40 agonists, CD40-L, TLR agonists, anti-ICOS, ICOS-L, and B cell receptor agonists.

In one embodiment, the additional or second therapeutic agent is an antibody targeting a tumor antigen, in particular selected from the group consisting of: anti-Her 2, anti-EGFR, anti-CD 20, anti-CD 19, anti-CD 52.

Combination therapy also relies on the administration of bifunctional molecules in combination with surgery, chemotherapy (e.g., such as docetaxel or dacarbazine), radiation therapy, immunotherapy (e.g., such as antibodies targeting CD40, CTLA-4), gene targeting and modulation, and/or other agents (e.g., immunomodulators, angiogenesis inhibitors), and any combination thereof.

Reagent kit

Any of the antibodies or compositions described herein can be included in the kits provided herein. The present disclosure provides, inter alia, kits for enhancing an immune response and/or treating a disease associated with PD-1 signaling or IL-7 signaling (e.g., cancer and/or infection).

In the context of the present invention, the term "kit" refers to two or more components (one of which corresponds to a bifunctional molecule, nucleic acid molecule, vector or cell according to the present invention) packaged in a container (container/recipient) or otherwise. Thus, a kit may be described as a set of products and/or appliances sufficient to achieve a certain goal, which may be sold as a single unit.

In particular, the kit according to the invention may comprise:

-a bifunctional molecule as defined above,

anti-hPD 1 antibody or antigen-binding fragment thereof linked to IL-7 or a variant thereof

-a nucleic acid molecule or a set of nucleic acid molecules encoding said bifunctional molecule,

-a vector comprising said nucleic acid molecule or group of nucleic acid molecules, and/or

-a cell comprising said vector or nucleic acid molecule or group of nucleic acid molecules.

Thus, in suitable container means, the kit may comprise a pharmaceutical composition and/or a bifunctional molecule and/or a host cell as described herein, and/or a vector encoding a nucleic acid molecule and/or a nucleic acid molecule as described herein, and/or an acid molecule as described herein or a related agent. In some embodiments, means may be provided for obtaining a sample from an individual and/or assaying the sample. In certain embodiments, the kit comprises cells, buffers, cell culture media, vectors, primers, restriction enzymes, salts, and the like. The kit may also comprise means for containing sterile, pharmaceutically acceptable buffers and/or other diluents.

The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a sub-unit dose. In one embodiment, the invention relates to a kit for single dose administration of units as defined above. The kit according to the invention may comprise a first container comprising the dried/lyophilized bifunctional molecule and a second container comprising the aqueous formulation. In certain embodiments of the invention, kits are provided that comprise single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyophile syringes).

The kit of the invention is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed mylar or plastic bottles), and the like. It also relates to packaging for use in conjunction with a particular device such as an inhaler, an intranasal applicator (e.g., nebulizer) or an infusion device such as a micropump. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a bifunctional molecule described herein comprising an anti-hPD-1 antibody linked to IL-7 or a variant thereof, or IL-7 m.

The compositions contained in the kit according to the invention may also be formulated as syringe-compatible compositions. In this case, the container means may itself be a syringe, pipette and/or other such device from which the formulation may be applied to the affected area of the body, and/or even applied to and/or mixed with other components of the kit. The components of the kit may alternatively be provided in the form of a dry powder. When the reagents and/or components are provided in dry powder form, the soluble components can be reconstituted by addition of a suitable solvent. It is envisaged that the solvent may also be provided in another container means and be suitable for administration.

In some embodiments, the kit further comprises an additional agent for treating cancer or an infectious disease, and the additional agent may be used in combination with the bifunctional molecule or other components of the kit of the invention, or may be provided separately in the kit. In particular, the kits described herein may comprise one or more additional therapeutic agents, such as those described in the "combination therapy" described above. The kit may be tailored for a particular cancer of an individual and comprise a corresponding second cancer therapy as described above for the individual.

The instructions related to the use of the bifunctional molecules or pharmaceutical compositions described herein generally comprise information about the dosage, the dosing regimen, the route of administration of the intended treatment, the means for reconstituting the bifunctional molecule and/or the means for diluting the bifunctional molecule according to the invention. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet contained in the kit in the form of a leaflet or instructions). In some embodiments, the kit may comprise instructions for use according to any of the methods described herein. The included instructions may include a description of administering a pharmaceutical composition comprising the bifunctional molecule to enhance an immune response and/or treat a disease as described herein. The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether the individual has a disease associated with PD-1 signaling (e.g., those described herein).

Examples

The following figures and examples are intended to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Example 1: binding of Bicki anti-hPD 1-IL7 molecules to IL7R and PD1

Affinity assessment for CD127(a) or CD132(B) was performed by Biacore on recombinant IL-7 cytokine (rlL7) (Biorad PHP046), IL-7 fused to an Fc domain (IL7-Fc, CHI-HF-22007Adipogen), and Bicki anti-PD 1 antibody fused to IL-7 on its heavy (anti-PDIVH-IL 7, chimeric) or light (anti-PD 1VL-IL7, chimeric) chain. CD127(Sinobiological,10975-H03H-50) was immobilized at 20. mu.g/ml on a CM5 biochip and the indicated proteins were added at serial concentrations (0.34; 1.03; 3.11; 9.3; 28 nM). Two-state reaction models and bivalent models were used to analyze affinity, since IL7-Fc and anti-PD 1-IL7 have a dimeric form. To assess the affinity of IL-7 for CD132, the complex CD127/IL-7 was performed on a biochip, followed by the addition of CD132 receptor (Sinobiological 10555-H08B) at various concentrations (125, 250, 500, 1000et 2000 nM).

The Blitz method was performed using Blitz (Forte Bio; USA; catalog No. C22-2 No. 61010-1). Recombinant hPD1-His (Sino Biologicals, Beijing, China; catalog No. 10377-H08H) was immobilized at 10. mu.g/ml in a Ni-NTA biosensor (Forte Bio; USA; catalog No. 18-0029) for 30 seconds by a histidine tail. Then, the anti-PD 1 antibody was correlated at 20. mu.g/ml for 120 seconds. anti-PD 1 antibody dissociation was performed in kinetic buffer for 120 seconds. Analytical data were generated using the Blitz pro 1.2 software, which calculated the association constant (ka) and dissociation constant (KD) and determined the affinity constant KD (ka/KD).

Table 1: binding of Bicki anti-PD 1-IL7 to CD127 and CD132 receptors: affinity assessment for CD127(a) or CD132(B) was performed by Biacore on recombinant IL-7 cytokine, IL-7 fused to Fc domain (IL7-Fc), and anti-PD 1 antibody fused to IL-7 on its heavy chain (anti-PDIVH-IL 7) or light chain (anti-PD 1VL-IL 7). Steady state affinity model for IL-7 analysis and bivalent model for fusion proteins.

	Blitz analysis
		anti-PD-1 No fusion	1.079nm
anti-PD-1 VH IL-7	1.776nM
		anti-PD-1 VL IL-7	3.225nM
anti-PD-1 VH VL IL-7	1.848nM

Table 2: binding of the Bicki anti-PD 1-IL7 antibody to human recombinant PD-1 protein.

Results

The inventors observed that the fusion of IL-7 to the N-terminal part of the Fc portion (IL7-Fc) reduced the affinity for the CD127 receptor (Table 1), while unexpectedly, the fusion of IL-7 to the C-terminal part of the heavy chain Fc region or the light chain constant domain retained its affinity for CD127 to a similar extent as rIL-7. These data demonstrate that C-terminal fusion of IL7 with an anti-PD 1 antibody (on either the heavy or light chain) will be more effective in the IL-7R activation pathway than conventional IL7-Fc compounds, and will be more effective in the elimination half-life into the patient than IL7 recombinant cytokines for therapeutic use.

Table 2 and figures 1A and B confirm that Bicki anti-PD 1-IL7 molecules (chimeric or humanized) bind to human recombinant PD-1 protein. Humanized forms of anti-PD-1 antibodies bind PD-1 recombinant protein with similar efficacy as the chimeric antibody. However, part a of figure 1A shows that the binding efficacy of Bicki is reduced compared to the anti-PD 1 antibody alone. The inventors have constructed Bicki IL7 molecules with other anti-PD-1 scaffolds (pembrolizumab or nivolumab). Figure 1C shows that these Bicki molecules retain good binding to PD-1.

Furthermore, the bifunctional anti-PD 1/IL-7 molecule allows for accumulation of IL-7 in PD-1+ T cell infiltration and relocation of IL-7 on PD-1+ T cells compared to IL 7-Fc.

Example 2: antagonistic ability of Bicki anti-PD 1-IL7 molecules to block the interaction between PD-1/PD-L1 and PD1-PDL2

PD-L1 was immobilized on a Maxisorp plate and the complex anti-PD 1 antibody + biotinylated recombinant human PD-1 was added. The complexes were generated with a fixed concentration of PD1 (0.6. mu.g/mL) and various concentrations of anti-PD-1 antibody were tested. Chimeric forms of the PD-1 antibody were used in this assay. The revealing was performed using streptavidin peroxidase to detect biotinylated PD1 molecules and the revealing was done colorimetrically at 450nm using TMB substrate. Affinity assessment of PD-1 recombinant proteins preincubated with anti-PD 1 antibody, anti-PD 1VH-IL7 or anti-PD 1VL-IL7 antibody on human PD-L2 recombinant protein was performed by Biacore. Human recombinant PD-L2 was immobilized on a CM5 biochip and complex antibody (200nM) + recombinant human PD-1(100nM) was added. Data are expressed as% relative response to interaction measured by Biacore: 100% ═ PD-1 relative response. In this experiment, a chimeric form of the PD-1 antibody was used.

Results

As shown in figure 2, the Bicki anti-PD 1-IL7 molecule exhibited very good ability to block the interaction of PD1 with PD-L1 and PD-L2. Although anti-PD 1 alone exhibited the highest binding to PD1 compared to the Bicki molecule in the ELISA assay, unexpectedly, inhibition of PD1-PDL1 or PD-1-PDL2 interaction between the antibody and the Bicki format was comparable, confirming that the Bicki anti-PD 1-IL7 molecules described herein can be as effective as the anti-PD 1 antibody.

Example 3: ex vivo IL-7R signaling pathway analysis of human PBMC following stimulation with Bicki anti-PD 1-IL7 molecules

PBMCs isolated from peripheral blood of healthy human volunteers were incubated with recombinant IL-7, Bicki anti-PD 1-IL-7(PD-1VH-IL7/PD1VL-IL7) for 15 minutes. Cells were then fixed, permeabilized and stained with AF 647-labeled anti-pSTAT 5 (clone 47/Stat5(pY 694)). Data were obtained by calculating the MFI pSTAT% pSTAT5+ population and normalizing (100% ═ rIL-757.5nM), which represents the average of 3 different donors in two independent experiments.

Results

After analysis of binding of Bicki anti-PD 1-IL7 molecules to IL7R, IL7R activation was measured by flow cytometry on STAT5 phosphorylation in total human PBMC or on CD4+ and CD8+ T cells.

Figure 3 shows that STAT5 phosphorylation is similarly induced with Bicki anti-PD 1-IL7 molecules fused on either the heavy or light chain. The maximum activity of the Bicki anti-PD 1-IL7 molecule was similar to that of recombinant IL-7 alone, with an EC50 of about 0.7 nM.

Example 4: in vitro and ex vivo analysis of T cell activation and proliferation treated with Bicki anti-PD 1-IL7 molecules

Cell-based assays were performed (Discover' x PD-1Path Hunter Bioassay kit). Jurkat T cells stably expressing an engineered PD-1 receptor fused to a β -gal fragment (ED) and engineered SHP1 fused to a complementary β -gal fragment (EA) were used in the co-culture of Jurkat cells with PD-L1 expressing cells, which resulted in phosphorylation of PD-1 and recruitment of engineered SHP-1, forcing complementation of ED and EA fragments and production of active β -gal enzymes. Upon addition of substrate, β Gal enzyme produces a chemiluminescent signal proportional to activation of PD-1 signaling. The addition of anti-PD-1 antibodies blocked PD-1 signaling, resulting in the loss of bioluminescent signal (RLU). anti-PD-1 antibodies or Bicki anti-PD 1-IL7 molecules were tested at different molar concentrations.

The promega PD-1/PD-L1 bioassay kit was carried out. Two cell lines were used: (1) effector T cells (Jurkat stably expressing PD-1, NFAT-induced luciferase)And (2) activation of target cells (CHO K1 cells stably expressing PDL1 and surface proteins designed to activate homologous TCRs in an antigen-independent manner). When the cells were co-cultured, the PD-L1/PD-1 interaction directly inhibited TCR-mediated activation, thereby blocking NFAT activation and luciferase activity. Addition of anti-PD 1 antibody blocks the inhibitory signal, resulting in NFAT activation and luciferase synthesis. Addition of BioGlo^TMAfter fluorescein, luminescence was quantified, which reflected T cell activation. The series of molar concentrations of anti-PD 1 antibody or Bicki anti-PD 1-IL7 antibody were determined.

Human PBMC cells isolated from peripheral blood of healthy volunteers were stimulated with anti-CD 3/CD28 coated plates (clones OKT3 and CD28.2, 3 μ g/mL, respectively) to induce PD-1 expression. After 20 stimulations, PBMCs were harvested and restimulated on OKT3/PDL1 coated plates (2 and 5. mu.g/mL, respectively) in the presence of recombinant IL7(rIL-7) or an anti-PD 1 antibody fused to IL-7 (on its heavy chain (anti-PD-1 VH IL-7) or light chain (anti-PD-1 VL IL-7)). Chimeric forms of the Bicki anti-PD 1-IL7 antibody were used in this assay at a fixed dose of antibody (5. mu.g/ml) or in multiple doses. On day 5 post-stimulation, T cell proliferation was assessed by 3H thymidine incorporation and secreted IFN- γ was quantified by sandwich ELISA.

Results

To determine the ability of the Bicki anti-PD 1-IL7 molecule to block PD-1 signaling in a cell-based assay, the Discover' x PD-1Path Hunter Bioassay kit was performed. The results are shown in FIG. 4A, showing that Bicki anti-PD 1VH-IL7 is able to inhibit 50% of SHP1 activation at a concentration of 92.1. mu.M. This result is very similar to that obtained with anti-PD 1 alone (80.66 μ M), showing good inhibitory efficacy on PD1/PDL1 interaction, leading to PD1 pathway inhibition. In parallel, inhibition of the inhibitory signal induced by the interaction of PD1 with PDL1 on the surface of T cells was measured using the NFAT bioassay. Figure 4B shows EC50 for each antibody or therapeutic combination tested. In an unexpected manner, the inventors observed that the EC50 of the two Bicki anti-PD 1-IL7 molecules tested was significantly better than either the anti-PD 1 or anti-PD 1+ rIL7 combinations (the average values for Bicki VH-IL7 and VL-IL7 were 0.41 and 0.33nM, while the average values for anti-PD 1 and anti-PD 1+ rIL7 were 3.6 and 6 nM). Unexpectedly, this result highlights that Bicki anti-PD 1-IL7 induces T cell activation on PD-1+ T lymphocytes more efficiently than the combination of anti-PD 1+ IL-7, demonstrating the synergistic effect of the Bicki molecule on PD1+ T cells. Figure 4C shows that these Bicki IL7 molecules constructed with pembrolizumab and nivolumab have similar synergy compared to anti-PD-1 alone, indicating that the present invention can be applied to other anti-PD-1 scaffolds.

In parallel, ex vivo T cell activation and proliferation was tested after treatment with anti-PD 1 antibody, rIL7 or anti-PD 1+ rIL7 or Bicki anti-PD 1-IL7 molecules. The results shown in fig. 5 and 6 indicate that IL7 fused to anti-PD 1 was able to induce IFNg secretion and induce T cell proliferation in a manner comparable to IL7 cytokine alone. However, figure 6B shows that the Bicki molecule has better efficacy in inducing T cell proliferation by providing 20pM of EC50 (compared to IL7 cytokine, 50 pM).

Example 5: integrin expression on cell surface after treatment of Bicki anti-PD 1-IL7 molecule

Human PBMCs were incubated with IL-7(50ng/mL) or anti-PD-1 or anti-PD 1 VH-IL7 (5. mu.g/mL) for 3 days and stained for α 4(BDbiosciences, Rungis, France, Cat. No. 559881), β 7(BDbiosciences, Cat. No. 555945) or LFA-1 integrin (CD11a/CD18) (BDbiosciences, CD11a Cat. No. 555380 and CD18 Cat. No. 557156). FACS was analyzed by LSR and expressed as median fluorescence for each marker. Data represent 3 independent experiments for 3 different donors.

Results

The inventors show in figure 7 that the bicki anti-PD 1-IL7 molecule stimulates the overexpression of some integrins, such as the α 4 and β 7 gut homing integrins and LFA1 integrins (CD11a and CD18), as well as rIL 7. In contrast, IL-2 and IL-5 cytokines did not significantly alter the expression of these integrins (a4, β 7) or at least significantly decreased to the extent of IL-7 and BiCki anti-PD-1 IL-7. These data support the interest of the Bicki anti-PD 1-IL7 molecule for the treatment of PD-1 resistant colorectal cancer, as it demonstrates its ability to promote T cell infiltration into tumor sites by IL-7. LFA-1 mediates T cell trafficking and extravasation in inflamed tissues through binding to its ligand ICAM-1 on endothelial cells. Other studies have shown that IL-7 stimulates the expression of LFA-1 and VLA-4 adhesion molecules, promoting T cell migration to any inflamed tissue. This data supports that anti-PD-1/IL-7 bifunctional molecules can promote T cell infiltration in a variety of cancer subtypes. The lack of T cell infiltration in tumor sites is currently a major obstacle to the efficacy of anti-PD 1.

Example 6: primary (naive), partially depleted and fully depleted T cells treated with Bicki anti-PD 1-IL7 molecules Proliferation and activation of subsets of cells.

Chronic antigen stimulation of T cells leading to exhaustion

Human PBMCs were stimulated repeatedly every 3 days on CD3 CD28 coated plates (3. mu.g/mL OKT3 and 3. mu.g/mL CD28.2 antibody). 24 hours after stimulation, T cells were stained for PD-1, lang 3, and Tim 3 inhibitory receptors to analyze their depletion status after each stimulation. Expression was analyzed by flow cytometry using fluorochrome-labeled antibodies and FACS LSRII. T cells were restimulated 24 hours after each stimulation on CD3/PD-L2 coated plates, proliferative capacity was determined by thymidine 3H incorporation at day 5 after each stimulation, and supernatants were analyzed for IFNg secretion by ELISA. Depleted T cell responses to IL-7 and Bicki anti-PD 1-IL7 molecules were analyzed by STAT5 phosphorylation 48 hours after each stimulation.

T cells were incubated for 15 minutes using sequence dilutions (recombinant IL-7 starting at 29nM to 0.29fM, anti-PD-1 fused to IL-7 (PD-1 VH IL-7/PD-1 VL IL-7) or isotype control fused to IL-7 (B12 VH IL-7). The cells were then fixed, permeabilized and stained with AF 647-labeled anti-pSTAT 5 (clone 47/Stat5(pY694)), the percentage of pSTAT5+ cells was analyzed after treatment with 29nM IL-7 or Bicki anti-PD 1VH-IL7 molecules after each stimulation.

Human PBMC were stimulated repeatedly on CD3 CD28 coated plates (3. mu.g/mL OKT3 and 3. mu.g/mL CD28.2 antibody). T cells were re-stimulated 24 hours after each stimulation on OKT3 coated plates (2. mu.g/mL) in the presence of anti-PD-1, IL-7 or anti-PD-1 fused to IL-7 (anti-PD-1 VH IL7 or anti-PD-1 VL IL-7). H3 incorporation assays were performed on day 5 to determine T cell proliferation. T cells stimulated 3 times were restimulated on anti-CD 3, anti-CD 3+ recombinant PDL1, or anti-CD 3+ recombinant PDL2 coated plates (2 and 5. mu.g/mL, respectively). H3 incorporation assay was performed on day 5.

Results

Using a model of repeated TCR stimulation in vitro, the inventors outlined chronic antigen stimulation of T cells (e.g. in the context of immunogenic tumours) and characterized the ability of T cells to respond to IL-7 following chronic antigen stimulation-induced T cell depletion (fig. 8 and 9). In this model, T cells highly expressed inhibitory receptors (Tim 3, PD1, Lag3) and lost the ability to proliferate and secrete cytokines after stimulation, a key feature of depleting T cells (fig. 8). Contrary to what was previously published in the literature or predicted based on a strong reduction of IL7R expression on depleted T cells, the inventors observed that partially and fully depleted human T cells still responded to IL-7 as shown by pSTAT5 activation (fig. 9), and that IL-7 increased the capacity of T cells to proliferate even when T cells were fully depleted (5 stimuli) (fig. 10). However, as the amount of IL-7 required to activate T cells increased, sensitivity to IL-7 decreased with repeated stimulation (fig. 9B and fig. 10A and B). These data explain the need for high IL-7 concentrations into the tumor microenvironment to activate long-term stimulated depleted T cells. Such high local concentrations of IL-7 can be achieved by increasing the half-life of IL-7 (e.g., by fusion with an antibody or Fc fragment recombinant protein). Another advantage of fusing IL-7 with a monoclonal antibody over the Fc domain (IL7-Fc) is that targeting PD1 with the anti-PD 1 antibody described herein will induce the specific localization of IL-7 cytokines near or directly on the in-tumor PD-1+ depleted T cells, just on cells requiring higher concentrations of IL-7. Furthermore, as PD-1+ T cells accumulate in the tumor microenvironment, the Bicki anti-PD 1-IL7 molecule will result in an increase in local IL-7 concentration where PD-1+ cells accumulate.

Example 7: ex vivo analysis of Treg suppressive activity of effector T cells

CD8+ effector T cells and CD4+ CD25 high CD127 low tregs were isolated from peripheral blood of healthy donors and stained with cell proliferation dye (against CD8+ T cells, with CPDe 450). Treg/CD8+ Teff were then co-cultured on OKT3 coated plates (2. mu.g/mL) at a ratio of 1:1 in the presence or absence of rIL-7(10ng/mL, 0.58nM), anti-PD 1(0.58nM), anti-PD 1+ rIL-7(0.58nM), bicki anti-PD 1VH-IL7(0.58nM) for 5 days. Proliferation of effector T cells was analyzed by cytofluorimetry.

Although anti-PD 1 therapies stimulate T cell effector function, immunosuppressive molecules (TGFB, IDO, IL-10 … …) and regulatory cells (Treg, MDSC, M2 macrophages) create adverse microenvironments that limit the full potential of the therapy. The sensitivity of the intratumoral T reg response to IL-7 treatment was then tested by measuring the proliferation of effector T cells (fig. 11). Although Treg cells express low levels of IL-7R (CD127)), they are still able to stimulate pSTAT5 following IL-7 treatment. In the suppression assay, by co-culturing tregs and T effector cells, the inventors observed fig. 11: treatment with IL-7 or bicki anti-PD 1-IL7 blocked Treg-mediated suppression. The anti-PD 1 antibody failed to inhibit the suppressive activity of tregs on T effector cells. IL7 is known to relieve Treg suppressive function: ( A, etc., j.immunol., 2015). The inventors show in figure 11 that the Bicki anti-PD 1-IL7 molecule abrogates Treg-mediated suppression, resulting in effector T cell proliferation as good as IL7 cytokine. Although Treg cells express low levels of IL-7 receptors, it is well described that IL-7 directly affects tregs and abrogates their suppressive function (Liu W, et al, J Exp Med., 2006, 7/10/2006; Liu W et al; seddidikiki N et al, J Exp Med, 2006, 7/10/2006; codarrri L et al, 2007; Heninger AK et al, J immuonl, 2012, 12/15/2012). Furthermore, figure 11B shows that targeting IL-7 signaling should be more promising than IL-2 and IL-15 signaling, since IL-2 and IL-15 strongly stimulate the proliferation of both tregs relative to IL-7 and Bicki anti-PD-1/IL-7. The inventors have shown in this experiment that the Bicki anti-PD 1-IL7 molecule will bias the T cell effector cells towards T regulatory immune balance by stimulating effector T cell proliferation and survival, while retaining regulatory T cells. Targeted IL-7 signaling compared to IL-2 signalingIt is expected that this would be more promising because IL-2 acts on both Tregs and T effector cells, whereas IL-7 selectively activates T effector cells.

Example 8: efficacy of Bicki anti-PD 1-IL7 molecules in humanized mouse models and on antibodies derived from tumors or ex vivo Ex vivo efficacy of human T cells in human tumor explant culture.

Humanized mouse model:

will 1^e6 human PBMC were injected intraperitoneally into mice. Mice were treated twice weekly with either anti-PD 1 antibody or Bicki anti-PD 1VH-IL7 molecules (5 mg/kg). On day 16 post injection, blood was collected and mice were sacrificed. The percentage of human CD 3T cells was analyzed by flow cytometry in human CD45 cells, human IFN γ was administered in plasma by ELISA, and infiltration of human CD3+ cells in the colon of mice was quantified by immunohistofluorescence. The proximal and distal colon, liver and lung were embedded in TissueIn OCT. Sections were stained for Dapi and human CD 3. For liver and lung, in pixels²/mm²CD3 infiltration was quantified. For the colon, CD3+ infiltrating T cells were counted.

Ex vivo T cell studies of different cancers:

t cells were extracted from renal cancer, metastatic colorectal cancer, hepatocellular carcinoma, schwannoma biopsy tissue and stained for CD3, CD4, CD8, PD-1, CD127 and CD 132. Immunofluorescence was analyzed by FACS LSRII. The CD4+ CD3+ or CD8+ CD3+ populations were analyzed.

Cells were treated with recombinant IL-7 or anti-PD 1VH-IL7 antibody (29nM) for 15 min. Cells were then fixed, permeabilized and stained for pSTAT5 (clone 47/Stat5(pY694)), CD3, CD4 and CD 8. The cells were then washed by centrifugation and resuspended in intact medium with isotype control, anti-PD-1, B12 isotype fused to Il-7 (isotype-VH IL-7) or anti-PD-1 fused to IL-7 (anti-PD-1 VH IL-7) (concentration 5. mu.g/mL). After 48 hours, the supernatant was collected and IFN γ secretion was quantified using MSD technology (Meso scale Discovery) (dose).

Intratumoral FoxP3 Treg staining in CD3+ T cell population. Facs analysis of FoxP3-CD3+ effector T cells versus pSTAT5 in FoxP3-CD3+ Treg cells after treatment with recombinant IL-7 or anti-PD-1 VHIL7(29nM) (15 min incubation). Cells were then fixed, permeabilized and stained for pSTAT5 (clone 47/Stat5(pY694)), CD3 and Foxp 3.

Results

In a humanized mouse model (fig. 12), the inventors observed an increase in the percentage of CD3 positive cells in peripheral blood and an increase in IFNg secretion following treatment with a Bicki anti-PD 1-IL7 molecule, compared to the anti-PD 1 antibody and PBS negative control, demonstrating that the Bicki molecule increases the expansion, survival and activation of human T cells in vivo. Furthermore, this difference was not only associated with high T cell infiltration in the colon, but also in the liver and lung, confirming that the IL-7 portion of the Bicki molecule promotes T cell migration in crinis tissue.

T cell phenotypic analysis of human tumors showed that intratumoral T cells expressed PD-1, IL-7R/γ consensus chains (CD127 and CD132), suggesting that the Bicki anti-PD 1-IL7 molecule may be effective in locally stimulating intratumoral human T cells, contrary to predictions in the literature (figure 13). Indeed, intratumoral T cells responded to IL-7 and Bicki anti-PD 1-IL7 molecules as shown by staining with pSTAT5 (fig. 14), confirming that ex vivo Bicki anti-PD 1-IL7 molecules of human T cells purified from tumors could facilitate treatment of various tumor subtypes. Then, an ex vivo culture model of human tumor explants comprising tumor cells as well as all tumor microenvironments including human immune cells and stromal cells treated with anti-PD 1, isotype VH-IL7, Bicki anti-PD 1-IL7 molecules or IL-7 was used (fig. 15). IFNg secretion was analyzed in the supernatant as a specific marker of T cell activation and a surrogate marker associated with anti-PD 1 response in various clinical trials. The inventors observed that bicki anti-PD-1 IL7 treated human tumor cultures secreted significantly more IFNg, confirming that IL7 acted locally in the tumor bed and increased local T cell activation. Interestingly, cultures of human tumor cells treated with the Bicki anti-PD 1-IL7 molecule secreted more IFNg than either anti-PD 1 alone or IL 7/isoform VHIL7 alone, demonstrating a synergistic effect of combining anti-PD-1 with IL-7 to activate T cells within the tumor (fig. 15B). The percentage of intratumoral T reg (FoxP3+) was also analyzed in patients with cancer (colorectal, schwannoma, renal cancer, and hepatocellular carcinoma). The results presented in figure 16A indicate that Treg cells, which may be the target of Bicki anti-PD 1-IL7 molecules, are present in each cancer tested to disarm the Treg cells. STAT5 activation (pSTAT5) was then analyzed in intratumoral effector cells (FoxP3-) and regulatory T cells (FoxP3+) after treatment with Bicki anti-PD 1-IL7 molecules. The results obtained using cells from colorectal, schwannoma, and pancreatic cancers and shown in figure 16B show that Bicki molecules are able to activate intratumoral effects and modulate the IL-7R pathway in T cells. This result underscores that the Bicki anti-PD 1-IL7 molecule is a twofold sword that biases effector T cell activation into tumors while abrogating Treg suppressive activity.

Example 9 mutation of Fc fusion IL-7 altered binding to IL-7R and pSTAT5 signaling and improved body Internal pharmacokinetics.

To obtain IL-7 mutants, the amino acids involved in the interaction of IL7 with CD127 were replaced with amino acids having similar properties and characteristics. Several mutants were generated, namely Q11E, Y12F, M17L, Q22E, D74E, D74Q, D74N, K81R, W142H, W142F and W142Y.

Disruption of the IL-7 disulfide bond by replacement of cysteine residues with serine residues results in the following substitutions: C2S-C141S + C34S-C129S (mutant named "SS 1"), or C2S-C141S + C47S-C92S (mutant named "SS 2"), or C47S-C92S + C34S-C129S (mutant named "SS 3").

Sample (I)	EC50 ng/mL
		IgG4 G4S3 IL7 WT	18.4
IgG4 G4S3 IL7 Q11E	18.49
		IgG4 G4S3 IL7 Y12F	22.27
IGG4 G4S3 IL7 M17L	20.96
		IGG4 G4S3 IL7 Q22E	17.44
IgG4 G4S3 IL7 D74E	103.94
		IgG4 G4S3 IL7 K81R	20.18
IgG4 IL7 G4S3 W142F	34.86
		IgG4 G4S3 IL7 W142H	136.32
IGG4 G4S3 IL7 W142Y	44.6

Table 3. ED50 assay from fig. 17A, B and C indicates the concentration required to achieve 50% of binding to the CD127 receptor. Each table represents a different experiment and can be compared to the positive control IgG 4G 4S3 IL7 WT.

Sample (I)	Ka(1/Ms)	Kd2(1/s)	KD(M)
				IgG4 Fc G4S3 IL-7 WT	5.76E+06	1.22E-04	4.14E-11
IgG4 Fc G4S3 IL-7 W142H	5.02E+05	2.56E-03	5,68E-08
				IgG4 Fc G4S3 Fc IL-7 SS2	6.11E+05	1.55E-03	7.22E-09
IgG4 Fc G4S3 Fc IL-7 SS3	1962	6.02E-4	1,36E-6

TABLE 4 binding of WT and mutated IL-7 to the CD127 receptor. Evaluation of the affinity of fusion anti-PD-1 IL-7 for CD127 by Biacore. The analysis was performed using a two-state reaction model.

TABLE 5 binding of WT to CD132 receptor compared to mutated IL-7. Affinity assessment of the complex CD127+ IgG fused IL-7 on CD132 was performed by Biacore. The analysis was performed using a steady state reaction model.

Table 6. the ED50 assay from fig. 18A, B and C refers to the concentration required to achieve 50% of the pSTAT5 signal for each anti-PD-1 IL-7 molecule in this assay. Each table represents a different experiment using a different donor, and each table can be compared to the positive control IgG 4G 4S3 IL7 WT.

Sample (I)	Cmax (nM) obtained	Area under the curve (AUG)
			IgG4 G4S3 IL7 WT	13.22	121.4
IgG4 G4S3 IL7D74E	89.19	151.9
			IgG4 G4S3 IL7 W142F	98	Not determined
IgG4 G4S3 IL7 W142H	141	248.2
			IgG4 G4S3 IL7 W142Y	70	Not determined
IgG4 G4S3 SS2	69.9	361.6
			IgG4 G4S3 SS3	140.6	466.5

Table 7 Cmax, area under the curve and half-life determinations from figure 19. Cmax was calculated at a time point of 15 minutes after injection of anti-PD-1 IL 7. AUC was calculated 0 to 144 hours after anti-PD-1 IL-7 injection.

Substitution of one amino acid in the sequence of IL7 did not alter its ability to bind to the PD-1 receptor (fig. 17A, B and C). However, these mutations altered their biological activity as shown by CD127 binding and pSTAT5 signaling in ex vivo T cell assays (fig. 18 and 19 and tables 3 and 6). Mutations D74E and W142H were the most potent mutations that reduced both IL-7 binding to CD127 and pStat5 activation in T lymphocytes (fig. 18A, 18B and 19A, 19B and tables 3 and 7). In another experiment, the effect of disulfide bond disruption was analyzed (fig. 18C). At high concentrations (10 μ g/ml), SS2 or SS3 were able to activate pStat5 in T lymphocytes with no 3log deviation from IL-7 WT (fig. 18C and table 6).

To confirm the binding ability of these mutants, Biacore assays were performed to determine KD (equilibrium dissociation constant between receptor and its antigen, see table 4). Mutants SS2 and W142H had lower affinity for CD127 with KD close to 7 to 57 nM. The SS3 mutant has the lowest affinity for CD127 and the KD is close to 3 μ M. As shown in table 5, the affinity for the CD132 receptor was also assessed. In this experiment, IgG4 alone was used as the baseline KD affinity, since CD127 dimerized with CD132 in the absence of IL-7. IL-7 mutant W142H bound to CD132, but with 5-fold higher affinity than IgG IL-7 WT. As shown in figure 18, this data indicates that mutation W142H reduced binding to CD127 and redirected binding of IL-7 to the CD132 receptor, resulting in loss of activation of pSTAT5 in T cells. In contrast, the inventors observed that the SS2 mutant lost the ability to bind to the CD132 receptor under the conditions tested, indicating that the SS2 mutant preferentially binds CD127 but not the CD132 receptor, resulting in a decrease in pSTAT5 activity in T cells (fig. 19).

To determine the pharmacokinetics/pharmacodynamics of anti-PD-1 IL-7 in vivo, mice were injected intravenously with a dose of IgG-IL-7(34.4 nM/kg). Plasma drug concentrations were analyzed by ELISA specific for human IgG. Figure 19 and table 7 show that the IgG4 IL-7WT molecule has a rapid distribution because the Cmax (maximum concentration 15 minutes after injection) obtained is 30 times lower than the theoretical concentration. All W142Y, F, H mutants tested described better distribution curves with Cmax 5 to 10 fold higher than IL-7WT (FIGS. 19A and 19W) TABLE 7). The W142H mutant exhibited the optimal Cmax. The anti-PD-1 IL-7D 74E mutant also showed a good Cmax. Mutants SS2 and SS3 showed the best PK profile with a Cmax 7 to 13 times higher than IL-7 WT and had a good linear profile. At the same time, the AUC (area under the curve) was determined (table 7 and fig. 20D), from which the extent of drug exposure and its clearance from the body can be understood. These data indicate that AUC increases with IL-7 mutants, which means that IL-7 mutants have improved drug exposure. As shown in fig. 20D, the inventors observed that drug exposure correlated with IL-7 potency of the mutants (as measured by pSTAT5 EC 50). In conclusion, the affinity of IL-7 is related to the pharmacokinetics of the product. Reducing the affinity of IL-7 for its receptors CD127 and CD132Improve the absorption and distribution of the IL-7 bifunctional molecule in vivo.

Example 10: cysteine addition at the C-terminal domain of IgG reduces the flexibility of the IL7 molecule Sex and improving pharmacokinetics in vivo

Cysteine addition at the C-terminal domain of IgG was also tested to create additional disulfide bonds and potentially limit the flexibility of the IL-7 molecule. This mutant was named "C-IL-7". Figure 21 shows that disulfide bond addition in IgG structure reduced pSTAT5 activity of IL-7 compared to anti-PD-1 IL7 WT bifunctional molecule (figure 21A) and increased Cmax (5-fold) in vivo pharmacokinetic analysis (figure 21B).

Example 11: anti-PD-1 IL-7 mutants constructed with IgG1N298A isotype have better affinity with IL-7R Binding, higher pSTAT5 signaling and good in vivo pharmacokinetic profiles

Different isotypes of anti-PD-1 IL-7 bifunctional molecules were tested with IgG4m (S228P) or IgG1m (N298A or N297A, depending on the numbering method). The IgG4 isotype includes the S228P mutation to prevent Fab arm exchange in vivo, while the IgG1 isotype includes the N298A mutation (a mutant named "IgG 4 m" or "IgG 1N 298A") that abrogates the IgG1 isotype from binding to Fc γ R receptors that can reduce nonspecific binding of immune cytokines. anti-PD-1 IL-7 bifunctional molecules were then constructed with 2 different isotypes (mutated IgG1 (called IgG1m) isotype compared to IgG 4S 288P isotype (called IgG4m) in N297A) to determine if isotype structure altered the bioactivity of IL-7 and its pharmacokinetic profile.

Figures 22A and 22B demonstrate that anti-PD-1 IL7 bifunctional molecules constructed with IgG4m or IgG1m isotype have the same binding properties to PD-1 receptor, indicating that the isotype does not alter the conformation of VH and VL and the affinity of anti-PD-1 antibodies to PD-1. However, the inventors observed that, unexpectedly, the IgG1m isotype improved the binding of IL-7D 74, SS2 to CD127 and slightly improved the binding of SS3 to CD127 (fig. 23A, B, C and D), and improved the activation of pSTAT5 to human PBMCs (fig. 24A, B and C). This increase in pSTAT5 signaling was demonstrated in SS2 mutants on another T cell line (Jurkat cells expressing PD-1 and CD127, see fig. 24D), but surprisingly, the IgG1m isotype did not alter the pSTAT5 activity of the anti-PD-1 IL-7 WT bifunctional molecule, indicating that the IgG1m isotype only increased the activity of the IL-7 mutant. To determine the ability of bifunctional molecules comprising anti-PD 1 antibody and IL7 mutant to reactivate TCR-mediated signaling, NFAT bioassay was performed. The results presented in fig. 25A show that the bifunctional molecule activates TCR-mediated signaling (NFAT) better than either anti-PD 1 or anti-PD 1+ rIL7 (as separate compounds), demonstrating the synergistic effect of the bifunctional molecule on PD1+ T cells. The inventors next evaluated the synergistic ability of bifunctional molecules comprising an anti-PD-1 antibody and an IL-7 mutant with mutations D74E, W142H or SS2 constructed with IgG4m compared to IgG1m isotype (fig. 25B, C, D). All mutants tested had a synergistic effect on activating NFAT signaling, the level of activation correlated with their ability to activate pSTAT5 signaling, particularly for bifunctional molecules with IL-7D 74E and IgG4 m.

Pharmacokinetic studies in mice showed that the IgG1 isotype did not alter drug exposure of IL7WT and SS3 molecules and had minimal effect on the W142H molecule (fig. 26A). Taken together, these data indicate that the optimized isotype (IgG1m) is sufficient to enhance the biological activity of the mutant while maintaining good pharmacokinetics of the product in vivo. Other IL-7 mutants were tested using the IgG1m isotype: a combination of the D74N, D74Q, and D74E + W142H mutations. No differences from the anti-PD-1 IL-7D 74E mutant were observed in pSTAT5 activation (FIG. 25B) and pharmacokinetics (FIG. 26B). The double mutant D74E + W142H showed similar characteristics to W124H IgG1, while D74Q showed similar characteristics to the D74E mutant. The inventors also constructed bifunctional molecules with IgG1M isotype + YTE mutations (M252Y/S254T/T256E). This mutation has been described to increase the half-life of the antibody by increasing binding to the FcRn receptor. As shown in figure 23D, YTE mutations did not alter pSTAT5 signaling in bifunctional molecules comprising D74 or W142H mutants.

Example 12: mutation of K444A to the C-terminal lysine residue did not affect pharmacokinetics in vivo

All subclasses of human IgG carry the C-terminal lysine residue of the antibody heavy chain (K444), which is cleaved off in the circulation. This lysis in the blood could potentially compromise the biological activity of the immunocytokine by releasing the linked IL-7 to IgG. To avoid this problem, the K444 amino acid in the IgG domain is replaced with alanine to reduce proteolytic cleavage (a mutation commonly used in antibodies). As shown in FIG. 27, a similar curve was obtained between IgG WT IL-7 and IgG K444A IL-7, indicating that this mutation does not affect the pharmacokinetic profile of the drug.

Example 13 linkers between IgG antibodies do not alter the pharmacokinetics within the variants but improve pSTAT5 signaling Activation

Different linkers between the IgG Fc domain and IL-7m were tested to alter flexibility. Several conditions were tested (e.g., no linker, GGGGS, GGGGSGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS).

For examples 1 and 2, linker (G4S)3 between the C-terminal domain of Fc and the N-terminal domain of IL-7 was used for IgG4m-IL7 and IgG1m-IL-7 construction, respectively. This linker allows a high degree of flexibility and improvement in the IL7 activation signal. To reduce the affinity of IL7 for CD127 and improve pharmacokinetics, different structures were tested using linkers of different lengths (no linker, G4S, (G4S)2 or (G4S) 3). For comparison, IgG1m or IgG4m Fc IL-7 WT was also generated using various linkers.

Pharmacokinetic studies showed that the length of the linker had no effect on the distribution, absorption and elimination of the constructed product of the following tests: anti-PD-1 IL7 WT (FIG. 28A), anti-PD-1 IL-7D 74 (FIG. 28B) and anti-PD-1 IL-7W 142H (FIG. 28C). However, as shown in figure 28D, the length of the linker affected activation of pStat 5. Indeed, anti-PD-1 IL7 constructed with linker (G4S)3 was more effective in activating pSTAT5 signaling than anti-PD-1 IL-7 constructed with (G4S)2 or G4S3 linkers, and even more effective than anti-PD-1 IL-7 constructed without a linker. These data highlight that the use of a (G4S)3 linker can confer flexibility to IL-7 without compromising the in vivo pharmacokinetics of the drug.

Example 14: anti-PD-1 IL-7 mutants allow preferential binding to PD-1+ CD127+ cells relative to PD-1-CD127+ cells + cells

Next, the inventors evaluated the ability of the anti-PD-1 IL-7 bifunctional molecule to target PD-1+ T cells. Jurkat cells expressing CD127+ or co-expressing CD127+ and PD-1+ were stained with 45nM of the following bifunctional molecules: anti-PD-1 IL-7 WT, D74, W142H, SS2 and SS 3. Binding was detected with anti-IgG-PE (Biolegend, clone HP6017) and analyzed by flow cytometry.

As a result:FIG. 29 shows that the anti-PD-1 IL-7 WT and D74 mutants bound PD-1+/CD127+ cells with similar efficacy as PD-1-/CD127+ cells, whereas the anti-PD-1 IL-7 mutants SS2, SS3 bound PD-1+/CD127+ cells with 2 to 3 fold higher efficacy compared to PD-1-/CD127+ cells. The anti-PD-1 IL-7W 142H bifunctional molecule showed moderate effects and bound to PD-1+/CD127+ cells with 1,4 fold higher efficacy. Taken together, these data indicate that IL-7 mutations not only allow better pharmacokinetics of the drug, but also allow IL-7 to preferentially bind to PD-1+ cells, i.e., the drug targets the same cell. This aspect is advantageous for the in vivo bioactivity of the drug, as anti-PD-1 IL-7 concentrates IL-7 on PD-1+ CD127+ depleted T cells into the tumor microenvironment relative to CD127+ naive T cells.

Example 15: the bicki anti-PD-1 IL-7 molecule allows proliferation of CD4 and CD 8T cells and is found in cynomolgus monkeys The preclinical safety is proved.

Cynomolgus monkeys were injected with 6,87nM/Kg (n ═ 2) or 34,35nM (n ═ 1) (equivalent to 1mg/Kg or 5mg/Kg) antibody. Blood analysis was performed until day 15 or 4 hours post injection. Proliferation of CD4/CD 8T or B cells was assessed in blood by flow cytometry using Ki67 marker. After cell fixation and permeabilization, pSTAT5 was analyzed by FACS at multiple time points in CD3+ T cells.

As a result:FIGS. 30A and B show that a single injection of bicki anti-PD-1 IL-7 induced proliferation of CD4 and CD 8T cells, but not B cells. After injection, a rapid activation of pSTAT5 was observed, with a maximum activation time of 1 to 24 hours (as shown in fig. 30C). The drug was well tolerated and safe at this dose, as shown in figure 30D/E/F, with clinical parameters (biochemical and blood cell counts) at or near the normal range following injection of the molecule. These data indicate that the molecule is capable of activating T cells in vivo and exhibits preclinical safety.

Conclusion

The Bicki anti-PD 1-IL7 antibody format with the IL-7 cytokine fused to the C-terminal domain of the Fc portion retained binding to the CD127 receptor, while, unexpectedly, IL7(IL7-Fc) fused to the N-terminal domain of Fc lost its binding capacity. The Bicki format will be more effective in IL-7R activation and elimination half-life in patients. Furthermore, the Bicki anti-PD 1-IL7 antibody format allows IL-7 to accumulate in PD-1+ T cell infiltrates and allows IL-7 to be relocated on PD-1+ T cells compared to IL7-Fc compounds, the use of Bicki having increased T cell activation compared to the combined use of anti-PD 1+ IL7 recombinant proteins. Furthermore, the Bicki anti-PD 1-IL7 molecule is a twolip sword that increases the proliferation and activation of effector T cells by secretion of IFNg cytokines in both in vitro and in vivo models, and that relieves T cell suppression by armed tregs. This has been demonstrated on human T cells isolated from various tumor types and indications, indicating that the Bicki anti-PD 1-IL7 molecule is useful for various tumor subtypes. Tumors resistant to PD-1 therapy exhibit T cell rejection. It is well known that the PD-1 response is correlated with the number of tumor infiltrating T cells. The Bicki anti-PD 1-IL7 molecule increases integrin expression on the cell surface, indicating that the bifunctional molecule of the present invention promotes T cell infiltration into tumors of various cancer subtypes.

Materials and methods

ELISA binding to PD1

For the activity ELISA assay, recombinant hPD1(Sino Biologicals, Beijing, China; catalog No. 10377-H08H) was immobilized on plastic at 0.5. mu.g/ml in carbonate buffer (pH 9.2) and purified antibody was added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; catalog No. 709-.

Affinity measurements using the Biacore method

Affinity assessment for CD127(A) or CD132(B) was performed by Biacore on IgG fused to IL-7 on its heavy chain. CD127(Sinobiological,10975-H03H-50) was immobilized on a CM5 biochip at a concentration of 20. mu.g/ml and the indicated proteins were added at a range of concentrations (0.35; 1.1; 3.3; 10; 30 nM). The affinity was analyzed using a two-state reaction model. To assess the affinity of IL-7 for CD132, CD127 was immobilized on CM5 biochips and each IL-7 construct was injected at a concentration of 30 nM. CD132 receptor (Sinobiological 10555-H08B) was added at various concentrations (e.g., 31.25, 52.5, 125, 250, 500 nM). Analysis was performed using a steady state affinity model.

CD127 binding ELISA

CD127 binding was assessed by a sandwich ELISA method. The antibody backbone-targeted recombinant protein was immobilized, and the antibody fused to IL-7 was then preincubated with CD127 recombinant protein (histidine-tagged, Sino cat. No. 10975-H08H). The revealing was performed using a mixture of an anti-histidine antibody (MBL # D291-6) coupled to biotin and streptavidin coupled to peroxidase (JI 016. sup. 030. sup. 084). Colorimetric determination was performed at 450nm using TMB substrate.

In vivo pSTAT5 analysis

PBMCs isolated from peripheral blood of healthy human volunteers were incubated with recombinant IL-7 or IgG fused IL-7 for 15 minutes. Cells were then fixed, permeabilized and stained with AF 647-labeled anti-pSTAT 5 (clone 47/Stat5(pY 694)). Data were obtained by calculating the MFI pSTAT5 in the CD3+ T cell population.

In vivo pharmacokinetics of IgG fused IL-7

To analyze the pharmacokinetics of IL-7 immunocytokines BalbcRJ mice (female 6-9 weeks old) were injected intraorbitally with a single dose of the molecule. Plasma drug concentrations were determined by ELISA using immobilized anti-human light chain antibody (clone NaM76-5F3), diluted serum (IL-7 containing fusion IgG). Detection was performed using peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; catalog No. 709-.

Cynomolgus monkeys were given 2 doses of Bicki IL-7 intravenously. Blood was collected at various time points post-injection (15/30 min, 1/2/4 h, 1/2/3/6/10/14 days) for analysis of biochemistry and cell counts.

T cell activation assay using a Promega cell-based bioassay

The ability of anti-PD-1 antibodies to restore T cell activation was tested using the Promega PD-1/PD-L1 kit (Cat. No. J1250). Two cell lines were used: (1) effector T cells (Jurkat stably expressing PD-1, NFAT-induced luciferase) and (2) activating target cells (CHO K1 cells stably expressing PDL1 and designed to activate the surface protein of a cognate TCR in an antigen independent manner). When the cells were co-cultured, the PD-L1/PD-1 interaction directly inhibited TCR-mediated activation, thereby blocking NFAT activation and luciferase activity. Addition of anti-PD 1 antibody blocks PD-1 mediated inhibitory signals, resulting in NFAT activation and luciferase synthesis and emission of bioluminescent signals. The experiments were performed as recommended by the manufacturer. Sequence dilutions of the PD-1 antibody were tested. After four hours of co-culture of PD-L1+ target cells, PD-1 effector cells and anti-PD-1 antibody, BioGlo ^TMFluorescein substrate was added to the wells and Tecan was used^TMThe plate was read with a luminometer.

Antibodies and bifunctional molecules

The following antibodies and bifunctional molecules have been used in the different experiments disclosed herein: pembrolizumab (Keytruda, Merck), nivolumab (Opdivo, Bristol-Myers Squibb), and the bifunctional molecules disclosed herein comprise an anti-PD 1 humanized antibody comprising a heavy chain as defined by SEQ ID NO:19, 22 or 24 and a light chain as defined by SEQ ID NO:28, or an anti-PD 1 chimeric antibody comprising a heavy chain as defined by SEQ ID NO:71 and a light chain as defined by SEQ ID NO: 72.