FC-binding fragments comprising the PD-L1 antigen-binding site
阅读说明：本技术 包括pd-l1抗原结合位点的fc结合片段 (FC-binding fragments comprising the PD-L1 antigen-binding site ) 是由 密里班·图纳 弗朗西斯卡·沃尔顿·范·霍克 瑞安·菲勒 穆斯塔法·法鲁迪 弗雷德里克·阿克勒 于 2018-12-19 设计创作，主要内容包括：本申请涉及特异性结合元件,其结合至程序性死亡配体(PD-L1)。该特异性结合元件优选包括位于特异性结合元件的恒定结构域的结构环中的PD-L1抗原结合位点。该特异性结合元件例如在癌症、以及感染性疾病、炎症、与炎症相关联的疾病和病症、及炎性疾病的治疗中获得应用。(The present application relates to a specific binding member that binds to programmed death ligand (PD-L1). The specific binding member preferably includes a PD-L1 antigen binding site located in a structural loop of the constant domain of the specific binding member. The specific binding members find use, for example, in the treatment of cancer, as well as infectious diseases, inflammation, diseases and disorders associated with inflammation, and inflammatory diseases.)
1. A specific binding member that binds to programmed death ligand 1(PD-L1), said specific binding member comprising a PD-L1 antigen binding site located in the CH3 domain of said specific binding member, wherein said PD-L1 binding site comprises:
(i) a first sequence located in the AB structural loop at positions 14-18 of the CH3 domain, wherein the specific binding element comprises an amino acid deletion at position 14, 15, or 16 of the CH3 domain, and wherein the first sequence consists of:
(a) amino acid sequence SGYW (SEQ ID NO: 23), or
(b) SEQ ID NO: 23, wherein
Serine (S) is substituted with amino acid A, E, F, G, H, I, L, P, R, S, T, V or Y, and/or
Glycine (G) is substituted with amino acid A, D, E, F, H, K, L, N, P, R, T, V or Y;
(ii) A second sequence located in the CD structural loop at positions 45.1-78 of the CH3 domain, wherein the second sequence consists of:
(a) the amino acid sequence EPQYWA (SEQ ID NO: 11), or
(b) SEQ ID NO: 11, wherein
Glutamic acid (E) is substituted by amino acid A, G, H, I, L, N, Q, R, S or W, and/or
Proline (P) is substituted by amino acid A, D, E, G, H, N, Q, W or Y, and/or
Glutamine (Q) is substituted by amino acid H or N, and/or
Tyrosine (Y) is substituted by amino acid A, D, H, T or V, and/or
Alanine (a) by amino acid D, E, G, L, R, S or W; and
(iii) a third sequence located in the EF structural loop at positions 92-100 of the CH3 domain, wherein the third sequence consists of:
(a) amino acid sequence SNWRWQMDD (SEQ ID NO: 19), or
(b) SEQ ID NO: 19, wherein
The serine (S) at position 92 is substituted with an amino acid A or G, and/or
Asparagine (N) at position 93 is substituted with amino acid A, E, F, G, H, I, K, L, Q, R, S, T or Y, and/or
Glutamine (Q) at position 97 is substituted with amino acid A, D, E, F, G, H, K, L, N, R, S or V, and/or
Methionine (M) at position 98 by amino acid F, I, L, V, W or Y, and/or
Aspartic acid (D) at position 99 is substituted with amino acid E, A, I, L, R, S, T, V, W, Y or G, and/or
The aspartic acid at position 100 (D) is substituted with amino acid A, E, F, I, K, L, N, R, V, W or Y; and is
Wherein the amino acid at position 101 of the CH3 domain is valine (V), alanine (A), or absent;
wherein the numbering of the amino acid residues is according to the ImmunoGeneTiCs (IMGT) numbering scheme.
2. A specific binding member according to claim 1 wherein the specific binding member comprises a total of up to 5 amino acid substitutions in the first, second and third sequences.
3. A specific binding member according to claim 1 or 2, wherein the methionine (M) at position 98 is substituted by the amino acid L.
4. A specific binding member according to any one of claims 1 to 3, wherein the specific binding member comprises up to two amino acid substitutions in the first sequence and/or up to three amino acid substitutions in the third sequence.
5. A specific binding member according to any one of claims 1 to 4, wherein,
The first sequence consists of:
(a) amino acid sequence SGYW (SEQ ID NO: 23), or
(b) SEQ ID NO: 23, wherein serine (S) is substituted with amino acid E or T;
the second sequence consists of the amino acid sequence EPQYWA (SEQ ID NO: 11); and is
The third sequence consists of:
(a) amino acid sequence SNWRWQMD1D2(SEQ ID NO: 19), or
(b) SEQ ID NO: 19, wherein methionine (M) is substituted with amino acid I, L or V; and is
Aspartic acid (D)1) Optionally substituted with glycine (G); and is
Wherein the amino acid at position 101 of the CH3 domain is valine (V), alanine (A), or absent.
6. A specific binding member according to any one of claims 1 to 5, wherein the first sequence has the sequence shown in SEQ ID NO: 42, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 43, or a sequence set forth in seq id no.
7. A specific binding member according to any one of claims 1 to 5, wherein the first sequence has the sequence shown in SEQ ID NO: 47, said second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 43, or a sequence set forth in seq id no.
8. A specific binding member according to any one of claims 1 to 5, wherein the first sequence has the sequence shown in SEQ ID NO: 47, said second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 78, or a sequence listed in seq id no.
9. A specific binding member according to any one of claims 1 to 5, wherein the first sequence has the sequence shown in SEQ ID NO: 42, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 74 and the residue at position 113 of the CH3 domain of the specific binding member is arginine (R).
10. A specific binding member according to any one of claims 1 to 5, wherein the first sequence has the sequence shown in SEQ ID NO: 47, said second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 70, wherein the residues at positions 84.1, 85.3, 101 and 113 of said CH3 domain of said specific binding member are proline (P), threonine (T), alanine (a) and arginine (R), respectively.
11. A specific binding member according to any one of claims 1 to 5, wherein the first sequence has the sequence shown in SEQ ID NO: 23, said second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 31, or a sequence set forth in seq id no.
12. A specific binding member according to any one of claims 1 to 5, wherein the first sequence has the sequence shown in SEQ ID NO: 23, said second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 19, or a sequence set forth in seq id no.
13. A specific binding member according to any one of claims 1 to 5, wherein the first sequence has the sequence shown in SEQ ID NO: 23, said second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 27, or a sequence set forth in seq id no.
14. A specific binding member according to any one of claims 1 to 5, wherein the first sequence has the sequence shown in SEQ ID NO: 23, said second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 35, or a sequence listed in seq id no.
15. A specific binding member according to any one of claims 6 to 9, wherein the amino acid at position 101 of the CH3 domain is valine (V).
16. A specific binding member according to any one of claims 1 to 15 wherein the CH3 domain is a human IgG1 CH3 domain.
17. A specific binding member according to any one of claims 1 to 16 wherein the amino acid at position 38 of the CH3 domain is a valine or alanine residue.
18. A specific binding member according to any one of claims 1 to 17, wherein the amino acid at position 38 of the CH3 domain is an alanine residue.
19. A specific binding member according to any one of claims 1 to 18, wherein the specific binding member comprises SEQ ID NO: 44. 48, 71, 75, 79, 32, 24, 28, 36, or 39, or a pharmaceutically acceptable salt thereof.
20. A specific binding member according to any one of claims 1 to 19 wherein the specific binding member comprises SEQ ID NO: 44. 48, 71, 75, 79 or 32.
21. A specific binding member according to any one of claims 1 to 20, wherein the specific binding member further comprises a CH2 domain.
22. A specific binding member according to any one of claims 1 to 21 wherein the specific binding member comprises the CH2 domain of human IgG1, IgG2, IgG3 or IgG 4.
23. The specific binding member of any one of claims 1 to 22 wherein the specific binding member comprises the CH2 domain of human IgG 1.
24. The specific binding member according to any one of claims 21 to 23, wherein the CH2 domain has the amino acid sequence of SEQ ID NO: 5. 6 or 82.
25. The specific binding member according to any one of claims 1 to 24, wherein the specific binding member further comprises an immunoglobulin hinge region or portion thereof at the N-terminus of the CH2 domain.
26. A specific binding member according to claim 25 wherein the hinge region or portion thereof is a human IgG1, IgG2, IgG3 or IgG4 hinge region or portion thereof.
27. A specific binding member according to claim 26 wherein the hinge region or portion thereof is a human IgG1 hinge region or portion thereof.
28. The specific binding member according to any one of claims 25 to 27, wherein the hinge region comprises seq id NO: 7, or consists thereof.
29. A specific binding member according to any one of claims 1 to 28 wherein the specific binding member comprises SEQ ID NO: 45. 46, 49, 50, 72, 73, 76, 77, 80, 81, 33, 34, 25, 26, 29, 30, 37, 38, 40, or 41, or a sequence represented by SEQ ID NO: 45. 46, 49, 50, 72, 73, 76, 77, 80, 81, 33, 34, 25, 26, 29, 30, 37, 38, 40, or 41.
30. A specific binding member according to any one of claims 1 to 28 wherein the specific binding member comprises SEQ ID NO: 45. 46, 49, 50, 72, 73, 76, 77, 80, 81, 33 or 34, or a sequence represented by SEQ ID NO: 45. 46, 49, 50, 72, 73, 76, 77, 80, 81, 33 or 34.
31. A specific binding member according to any one of claims 1 to 30, wherein the specific binding member further comprises a second antigen binding site.
32. A specific binding member according to claim 31 wherein the second antigen-binding site is a CDR-based antigen-binding site.
33. A specific binding member according to any one of claims 1 to 32 wherein the specific binding member is an antibody molecule.
34. A specific binding member according to claim 33 wherein the antibody molecule is a human IgG1, IgG2, IgG3 or IgG4 molecule.
35. A specific binding member according to claim 34 wherein the antibody molecule is a human IgG1 molecule.
36. A specific binding member according to any one of claims 32 to 35, wherein the CDR-based antigen binding site of the antibody molecule binds to a checkpoint inhibitor, a co-stimulatory molecule or a tumor associated antigen.
37. A specific binding member according to any one of claims 32 to 36, wherein the CDR-based antigen binding site of the antibody molecule does not bind to OX40, induced T cell co-stimulator (ICOS) or CD 137.
38. The specific binding member according to any one of claims 32 to 37, wherein the CDR-based antigen-binding site of the antibody molecule does not bind to CD27 or glucocorticoid-induced TNFR-related protein (GITR).
39. A specific binding member according to any one of claims 32 to 38, wherein the CDR-based antigen binding site of the antibody molecule does not bind to lymphocyte activation gene 3 (LAG-3).
40. A specific binding member according to any one of claims 32 to 39, wherein the specific binding member or the antibody molecule is conjugated to an immune system modulator, a cytotoxic molecule, a radioisotope or a detectable label.
41. A specific binding member according to claim 40 wherein the immune system modulator or the cytotoxic molecule is a cytokine.
42. A specific binding member according to claim 40, wherein the immune system modulator is a cell surface receptor, or a biologically active fragment thereof; or the immune system modulator is a ligand for a cell surface receptor, or a biologically active fragment of the ligand.
43. A nucleic acid encoding a specific binding member or antibody molecule according to any one of claims 1-42.
44. A vector comprising the nucleic acid of claim 43.
45. A recombinant host cell comprising the nucleic acid of claim 43, or the vector of claim 44.
46. A method of producing a specific binding member or antibody molecule according to any one of claims 1 to 42, the method comprising culturing the recombinant host cell of claim 45 under conditions for production of the specific binding member or antibody molecule.
47. The method of claim 46, further comprising isolating and/or purifying the specific binding member or the antibody molecule.
48. A pharmaceutical composition comprising a specific binding member or antibody molecule according to any one of claims 1-42, and a pharmaceutically acceptable excipient.
49. A specific binding member or antibody molecule according to any one of claims 1 to 42 for use in a method of treating cancer in a patient.
50. A method of treating cancer in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of a specific binding member or antibody molecule according to any one of claims 1-42.
51. A specific binding member or antibody molecule for use, or a method according to claim 49 or 50, wherein the method further comprises administering to the patient a second anti-cancer therapeutic agent.
52. A specific binding member or antibody molecule according to any one of claims 1 to 42 for use in a method of treating an infectious disease.
53. A method of treating an infectious disease in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of a specific binding member or antibody molecule according to any one of claims 1-42.
54. A specific binding member or antibody molecule for use, or a method according to claim 52 or 53, wherein the infectious disease is a chronic infectious disease.
55. A specific binding member or antibody molecule according to any one of claims 1 to 42 for use in a method of treating inflammation, a disease or disorder associated with inflammation, or an inflammatory disease.
56. A method of treating inflammation, a disease or disorder associated with inflammation, or an inflammatory disease in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of a specific binding member or antibody molecule according to any one of claims 1-42.
57. A specific binding member or antibody molecule for use, or a method according to claim 55 or 56, wherein the inflammation, a disease or disorder associated with inflammation, or an inflammatory disease is stroke, stroke-related inflammation, or vascular inflammation.
Technical Field
The present invention relates to a specific binding member that binds to programmed death ligand 1 (PD-L1). The specific binding member preferably includes a PD-L1 antigen binding site located in a structural loop of the constant domain of the specific binding member. The specific binding members of the invention find use, for example, in the treatment of cancer, as well as infectious diseases and inflammation.
Background
Programmed cell death 1 (PD-1) and its ligands PD-L1(CD274, B7-H1) and PD-L2(B7-DC) deliver inhibitory signals that regulate the balance between T cell activation, tolerance and immunopathology. PD-L1 is expressed structurally or transiently on a variety of immune cell types and on certain tumor cells.
PD-L1 is a type I transmembrane protein with two Ig-like domains, a transmembrane domain and a short cytoplasmic domain in the extracellular region. The complete human PD-L1 sequence can be found under the universal protein resource database (UniProt) accession No.
Human PD-L1 with an affinity (K) of 770nMD) Binds to its receptor PD-1. PD-1 is expressed on activated T cells, B cells, and bone marrow cells and modulates the activation or suppression of cellular immune responses. Binding of PD-L1 to PD-1Deliver inhibitory signals, reduce cytokine production and proliferation of T cells. Thus, cellular PD-L1 expression may mediate protection against killing by Cytotoxic T Lymphocytes (CTLs), and cellular PD-L1 expression is a regulatory mechanism that suppresses chronic immune responses during viral infection. Cancer as a chronic and pro-inflammatory (proinflammatory) disease disrupts this immunoprotective pathway by upregulation of PD-L1 expression to evade host immune responses. Under the influence of an active immune response, IFN γ also upregulates the expression of PD-L1.
PD-L1 also mediates immunosuppression by interacting with another protein, B7.1 (also known as CD80), blocking its ability to transmit one of the secondary activation signals on T cells via CD 28.
PD-L1 expression has been shown in a variety of solid tumors (solid tumors). Of the 654 samples examined in one study spanning 19 tumors from different sites, 14% of the samples were positive for PD-L1. The highest frequency of PD-L1 positivity was observed in head and neck (31%), cervix (29%), primary unknown cancer (cancer of unknown origin, CUP; 28%), glioblastoma multiforme (GBM; 25%), bladder cancer (21%), esophageal cancer (20%), Triple Negative (TN) breast cancer (18%) and liver cancer (15%) (Grosso et al, 2013). It has been shown that tumor-associated expression of PD-L1 confers immune resistance and potentially protects tumor cells from T cell-mediated apoptosis.
In murine studies, therapies targeting PD-L1 have shown excellent results. In the B16 murine model of melanoma, treatment with anti-PD-L1 in combination with the GVAX or FVAX vaccination strategy at the end of the study produced significant effects on both survival (30 days for the control group versus 52 days for the PD-L1 treated group) and percentage of tumor-free animals (5%) (Curran et al, 2010). anti-PD-L1 therapy has also been used to study the mechanism of immunosuppression in the P815 murine mammary tumor (mastoma) model. P815 cells injected into mice typically trigger a strong immune response, which leads to their rejection (rejection). When PD-L1 is expressed on P815 cells, these cells evade immune attack, which in turn can be rendered ineffective by administration of anti-PD-L1 antibodies (negate) (Iwai et al, 2002). Clearly, targeting the PD-1/PD-L1 axis (axis) (Herbst et al, 2014) in immunogenic human cancers produces a survival advantage through stimulation of the anti-cancer immune response (Wolchok et al, 2013; Larkin et al, 2015).
Clinical trials have shown the clinical benefit of targeting PD-L1 in patients, leading to the approval of three anti-PD-L1 targeting monoclonal antibodies (anti-PD-L1 targeting monoclonal antibodies) to date.
Atezolizumab (Tecnriq)TM) Is a humanized IgG1 antibody that binds to PD-L1. It is used in clinical trials as monotherapy (monotherapy) and also in combination with other biologic therapies and/or small molecule therapies for the treatment of solid cancers, including colorectal cancer, breast cancer, non-small cell lung cancer, bladder cancer, and renal cell carcinoma.
Abamelumab (Avelumab) (Bavencio)TM) Is a fully human IgG1 antibody that binds to PD-L1 and is being clinically tested in a number of cancers, including bladder, gastric, head and neck, mesothelioma (mesothelioma), non-small cell lung, ovarian, renal, Merkel (Merkel) cell, and metastatic Urothelial (UC).
Durvaluumab (Durvalumab, Imfinzi)TM) Is a human IgG1 antibody that binds to PD-L1 and is being tested in clinical trials for head and neck squamous cell carcinoma (of the head and rock), bladder cancer, pancreatic cancer, alone or in combination with tremelimumab (tremelimumab), and in other solid cancers such as gastric cancer, melanoma, and unresectable hepatocellular carcinoma, in combination with other biomolecules and small molecules.
Duvacizumab has been approved for the treatment of patients with locally advanced or metastatic urothelial cancer and unresectable stage three non-small cell lung cancer (NSCLC).
More anti-PD-L1 antibodies, including BMS-936559(NCT00729664), have been tested in clinical trials, and other antibodies are in preclinical testing.
Infectious diseases show many similarities to oncology (parallell). It is believed that the role of PD-L1 in immune modulation can be exploited to maximize the immune response against pathogens. Immunomodulation in infectious diseases is an emerging field of medicine, and earlier discussions suggest that PD-L1 blockade (blockade) may improve the biological response to infection, in particular helping to counteract T cell failure, manage immune-mediated gaps, and generate long-term immunity (Wykes, m.n. and lewis, s.r., 2018).
Furthermore, recent studies have also shown that the PD-1/PD-L1 pathway can be a therapeutic target (thermitarget) for the treatment of inflammation, diseases and disorders associated with inflammation, inflammatory diseases such as stroke and stroke-related inflammation (Bodhankar et al, 2015), and vascular inflammation (vascularity) or vasculitis (vasculitides), such as medium/large vasculitis, or vasculitis of the central and/or peripheral nervous system (Hid Cadena et al, 2018 and Daxini et al, 2018).
While various anti-PD-L1 therapeutics are being developed, current data show that overall treatment with existing anti-PD-L1 monotherapies results in a response in less than 50% of cancer patients. In clinical tests, the range (spectrum) and severity of adverse reactions reported varied between antibodies. To increase the targeted response rate (ORR), and/or to reduce side effects, the anti-PD-L1 antibody can be combined with other biologies, such as antibodies against other checkpoint modulators; and in combination with small molecule therapies and other immune system activation methods, such as tumor vaccines.
Thus, there remains a need in the art for additional molecules that can target PD-L1 and find use in cancer therapy.
In addition, there remains a need in the art for additional molecules that can target PD-L1 and find use in the treatment of infectious diseases.
Furthermore, there remains a need in the art for additional molecules that can target PD-L1 and find use in the treatment of inflammation, diseases and disorders associated with inflammation, and inflammatory diseases.
Disclosure of Invention
The present inventors have prepared a number of specific binding members which include a non-natural (non-native) binding site for PD-L1 in the CH3 domain of the specific binding member. By nature
The inventors found that specific binding elements identified from an initial affinity maturation program after initial screening of a natural CH3 domain phage library showed increased affinity for PD-L1 and increased blocking (blocking) of the interaction between PD-L1 and its ligand PD-1. However, the specific binding members selected surprisingly have very weak activity in T cell activation assays.
After additional rounds of mutagenesis, screening and selection, the inventors identified specific binding elements that not only have high affinity for PD-L1, but also have excellent activity in T cell activation assays. These specific binding members surprisingly include a CH3 domain having a deletion in the AB structural loop sequence (at
To remove potential manufacturing sequence liability and improve the biophysical properties of selected specific binding elements, the inventors performed further rounds of mutagenesis, screening and selection.
The above method allows the present inventors to identify a large number of specific binding members which comprise a binding site for PD-L1 in their CH3 domain and show excellent binding to PD-L1 and which show or are expected to have high affinity in T cell activation assays. Based on these properties, it is expected that the specific binding members of the present invention will find application in the treatment of human cancer, as well as infectious diseases, inflammation, diseases and conditions associated with inflammation, and inflammatory diseases, through the inhibition of PD-L1.
Specific binding elements have also been shown to have a high affinity for cynomolgus monkey PD-L1, which is comparable to the affinity for human PD-L1, and are expected to be useful for performance assessment of specific binding elements in cynomolgus monkey disease models. The reason for this is that the results obtained are more likely to be predictive of the effect of a specific binding element in human patients than when testing specific binding elements with higher variability in affinity for human and cynomolgus PD-L1 in a cynomolgus monkey model.
Thus, in a first aspect of the invention there is provided a specific binding member that binds to PD-L1 and which comprises a PD-L1 antigen binding site located in a constant domain, such as a CL, CH1, CH2 or CH3 domain, preferably a CH1, CH2 or CH3 domain, most preferably the CH3 domain of the specific binding member.
The PD-L1 binding site of a specific binding member of the invention preferably comprises:
(i) a first sequence located in the AB loop at positions 14-18 of the CH3 domain, wherein the specific binding element comprises an amino acid deletion at
(a) amino acid sequence SGYW (SEQ ID NO: 23), or
(b) SEQ ID NO: 23, wherein
Serine (S) is substituted with amino acid A, E, F, G, H, I, L, P, R, T, V or Y, and/or
Glycine (G) is substituted with amino acid A, D, E, F, H, K, L, N, P, R, T, V or Y;
(ii) a second sequence located in the CD structural loop at positions 45.1-78 of the CH3 domain, wherein the second sequence consists of:
(a) the amino acid sequence EPQYWA (SEQ ID NO: 11), or
(b) SEQ ID NO: 11, wherein
Glutamic acid (E) is substituted by amino acid A, G, H, I, L, N, Q, R, S or W, and/or
Proline (P) is substituted by amino acid A, D, E, G, H, N, Q, W or Y, and/or
Glutamine (Q) is substituted by amino acid H or N, and/or
Tyrosine (Y) is substituted by amino acid A, D, H, T or V, and/or
Alanine (a) by amino acid D, E, G, L, R, S or W; and
(iii) a third sequence located in the EF structural loop at positions 92-100 of the CH3 domain, wherein the third sequence consists of:
(a) amino acid sequence SNWRWQMDD (SEQ ID NO: 19), or
(b) SEQ ID NO: 19, wherein
The serine (S) at
Asparagine (N) at
Glutamine (Q) at
Methionine (M) at
Aspartic acid (D) at
The aspartic acid at position 100 (D) is substituted with amino acid A, E, F, I, K, L, N, R, V, W or Y; and is
Wherein the amino acid at
wherein the numbering of the amino acid residues is according to the numbering scheme of the International immunogenetics database (ImmunoGeneTiCs (IMGT)).
Accordingly, the present invention provides:
[1] a specific binding member that binds to PD-L1, the specific binding member comprising a PD-L1 antigen binding site located in the CH3 domain of the specific binding member, wherein the PD-L1 binding site comprises:
(i) a first sequence located in the AB loop at positions 14-18 of the CH3 domain, wherein the specific binding member comprises an amino acid deletion at
(ii) a second sequence located in the CD structural loop at positions 45.1-78 of said CH3 domain, an
(iii) A third sequence located in the EF loop at positions 92-100 of the CH3 domain.
[2] The specific binding member according to [1], wherein the first sequence consists of the amino acid sequence SGYW (SEQ ID NO: 23) or a variant thereof.
[3] The specific binding member according to [1] or [2], wherein the second sequence consists of the amino acid sequence EPQYWA (SEQ ID NO: 11) or a variant thereof.
[4] The specific binding member according to any one of [1] to [3], wherein the third sequence consists of the amino acid sequence SNWRWQMDD (SEQ ID NO: 19) or a variant thereof.
[5] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid a.
[6] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid E.
[7] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid F.
[8] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid G.
[9] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid H.
[10] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with amino acid I.
[11] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid L.
[12] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid P.
[13] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid R.
[14] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid T.
[15] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid V.
[16] The specific binding member according to any one of [1] to [4], wherein the first sequence consists of SEQ id no: 23 in which serine (S) is substituted with the amino acid Y.
[17] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23, in which glycine (G) is substituted with the amino acid a.
[18] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23, in which glycine (G) is substituted with the amino acid D.
[19] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23 in which glycine (G) is substituted with the amino acid E.
[20] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23 in which glycine (G) is substituted with the amino acid F.
[21] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23 in which glycine (G) is substituted with the amino acid H.
[22] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23 in which glycine (G) is substituted with the amino acid K.
[23] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23 in which glycine (G) is substituted with the amino acid L.
[24] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id no: 23 in which glycine (G) is substituted with the amino acid N.
[25] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23 in which glycine (G) is substituted with the amino acid P.
[26] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23 in which glycine (G) is substituted with the amino acid R.
[27] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23 in which glycine (G) is substituted with the amino acid T.
[28] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23 in which glycine (G) is substituted with the amino acid V.
[29] The specific binding member according to any one of [1] to [16], wherein the first sequence consists of seq id NO: 23 in which glycine (G) is substituted with the amino acid Y.
[30] The specific binding member according to any one of [1] to [29], wherein the second sequence consists of seq id NO: 11 in which glutamic acid (E) is substituted by the amino acid a.
[31] The specific binding member according to any one of [1] to [29], wherein the second sequence consists of seq id NO: 11 in which glutamic acid (E) is substituted by the amino acid G.
[32] The specific binding member according to any one of [1] to [29], wherein the second sequence consists of seq id NO: 11 in which glutamic acid (E) is substituted by the amino acid H.
[33] The specific binding member according to any one of [1] to [29], wherein the second sequence consists of seq id NO: 11 in which glutamic acid (E) is substituted by amino acid I.
[34] The specific binding member according to any one of [1] to [29], wherein the second sequence consists of seq id NO: 11 in which glutamic acid (E) is substituted by the amino acid L.
[35] The specific binding member according to any one of [1] to [29], wherein the second sequence consists of seq id NO: 11 in which glutamic acid (E) is substituted with the amino acid N.
[36] The specific binding member according to any one of [1] to [29], wherein the second sequence consists of seq id NO: 11 in which glutamic acid (E) is substituted by the amino acid Q.
[37] The specific binding member according to any one of [1] to [29], wherein the second sequence consists of seq id NO: 11 in which glutamic acid (E) is substituted by the amino acid R.
[38] The specific binding member according to any one of [1] to [29], wherein the second sequence consists of seq id NO: 11 in which glutamic acid (E) is substituted by the amino acid S.
[39] The specific binding member according to any one of [1] to [29], wherein the second sequence consists of seq id NO: 11 in which glutamic acid (E) is substituted by the amino acid W.
[40] The specific binding member according to any one of [1] to [39], wherein the second sequence consists of SEQ ID NO: 11 in which proline (P) is substituted by amino acid a.
[41] The specific binding member according to any one of [1] to [39], wherein the second sequence consists of SEQ ID NO: 11 in which proline (P) is substituted by amino acid D.
[42] The specific binding member according to any one of [1] to [39], wherein the second sequence consists of SEQ ID NO: 11 in which proline (P) is substituted by amino acid E.
[43] The specific binding member according to any one of [1] to [39], wherein the second sequence consists of SEQ ID NO: 11 in which proline (P) is substituted by the amino acid G.
[44] The specific binding member according to any one of [1] to [39], wherein the second sequence consists of SEQ ID NO: 11 in which proline (P) is substituted by the amino acid H.
[45] The specific binding member according to any one of [1] to [39], wherein the second sequence consists of SEQ ID NO: 11 in which proline (P) is substituted by the amino acid N.
[46] The specific binding member according to any one of [1] to [39], wherein the second sequence consists of SEQ ID NO: 11 in which proline (P) is substituted by the amino acid Q.
[47] The specific binding member according to any one of [1] to [39], wherein the second sequence consists of SEQ ID NO: 11 in which proline (P) is substituted by the amino acid W.
[48] The specific binding member according to any one of [1] to [39], wherein the second sequence consists of SEQ ID NO: 11 in which proline (P) is substituted by amino acid Y.
[49] The specific binding member according to any one of [1] to [48], wherein the second sequence consists of SEQ ID NO: 11 in which glutamine (Q) is substituted with amino acid H.
[50] The specific binding member according to any one of [1] to [48], wherein the second sequence consists of SEQ ID NO: 11 in which glutamine (Q) is substituted with the amino acid N.
[51] The specific binding member according to any one of [1] to [50], wherein the second sequence consists of SEQ ID NO: 11 in which tyrosine (Y) is substituted with the amino acid a.
[52] The specific binding member according to any one of [1] to [50], wherein the second sequence consists of SEQ ID NO: 11 in which tyrosine (Y) is substituted by the amino acid D.
[53] The specific binding member according to any one of [1] to [50], wherein the second sequence consists of SEQ ID NO: 11 in which tyrosine (Y) is substituted with the amino acid H.
[54] The specific binding member according to any one of [1] to [50], wherein the second sequence consists of SEQ ID NO: 11 in which tyrosine (Y) is substituted by the amino acid T.
[55] The specific binding member according to any one of [1] to [50], wherein the second sequence consists of SEQ ID NO: 11 in which tyrosine (Y) is substituted by the amino acid V.
[56] The specific binding member according to any one of [1] to [55], wherein the second sequence consists of SEQ ID NO: 11 in which alanine (a) is substituted with the amino acid D.
[57] The specific binding member according to any one of [1] to [55], wherein the second sequence consists of SEQ ID NO: 11 in which alanine (a) is substituted with the amino acid E.
[58] The specific binding member according to any one of [1] to [55], wherein the second sequence consists of SEQ ID NO: 11 in which alanine (a) is substituted with the amino acid G.
[59] The specific binding member according to any one of [1] to [55], wherein the second sequence consists of SEQ ID NO: 11 in which alanine (a) is substituted with the amino acid L.
[60] The specific binding member according to any one of [1] to [55], wherein the second sequence consists of SEQ ID NO: 11 in which alanine (a) is substituted with the amino acid R.
[61] The specific binding member according to any one of [1] to [55], wherein the second sequence consists of SEQ ID NO: 11 in which alanine (a) is substituted with the amino acid S.
[62] The specific binding member according to any one of [1] to [55], wherein the second sequence consists of SEQ ID NO: 11 in which alanine (a) is substituted with the amino acid W.
[63] The specific binding member according to any one of [1] to [62], wherein the third sequence consists of SEQ ID NO: 19 in which the serine (S) at
[64] The specific binding member according to any one of [1] to [62], wherein the third sequence consists of SEQ ID NO: 19 in which the serine (S) at
[65] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[66] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[67] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[68] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[69] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[70] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[71] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[72] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[73] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[74] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[75] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which asparagine (N) at
[76] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which asparagine (N) at
[77] The specific binding member according to any one of [1] to [64], wherein the third sequence consists of SEQ ID NO: 19 in which the asparagine (N) at
[78] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[79] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[80] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[81] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[82] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[83] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[84] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[85] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[86] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[87] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[88] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[89] The specific binding member according to any one of [1] to [77], wherein the third sequence consists of SEQ ID NO: 19 in which the glutamine (Q) at
[90] The specific binding member according to any one of [1] to [89], wherein the third sequence consists of SEQ ID NO: 19 in which the methionine (M) at
[91] The specific binding member according to any one of [1] to [89], wherein the third sequence consists of SEQ ID NO: 19 in which the methionine (M) at
[92] The specific binding member according to any one of [1] to [89], wherein the third sequence consists of SEQ ID NO: 19 in which the methionine (M) at
[93] The specific binding member according to any one of [1] to [89], wherein the third sequence consists of SEQ ID NO: 19 in which the methionine (M) at
[94] The specific binding member according to any one of [1] to [89], wherein the third sequence consists of SEQ ID NO: 19 in which the methionine (M) at
[95] The specific binding member according to any one of [1] to [89], wherein the third sequence consists of SEQ ID NO: 19 in which the methionine (M) at
[96] The specific binding member according to any one of [1] to [95], wherein the third sequence consists of SEQ ID NO: 19 in which the aspartic acid (D) at
[97] The specific binding member according to any one of [1] to [95], wherein the third sequence consists of SEQ ID NO: 19 in which the aspartic acid (D) at
[98] The specific binding member according to any one of [1] to [95], wherein the third sequence consists of SEQ ID NO: 19 in which aspartic acid (D) at
[99] The specific binding member according to any one of [1] to [95], wherein the third sequence consists of SEQ ID NO: 19 in which the aspartic acid (D) at
[100] The specific binding member according to any one of [1] to [95], wherein the third sequence consists of SEQ ID NO: 19 in which aspartic acid (D) at
[101] The specific binding member according to any one of [1] to [95], wherein the third sequence consists of SEQ ID NO: 19 in which aspartic acid (D) at
[102] The specific binding member according to any one of [1] to [95], wherein the third sequence consists of SEQ ID NO: 19 in which aspartic acid (D) at
[103] The specific binding member according to any one of [1] to [95], wherein the third sequence consists of SEQ ID NO: 19 in which aspartic acid (D) at
[104] The specific binding member according to any one of [1] to [95], wherein the third sequence consists of SEQ ID NO: 19 in which the aspartic acid (D) at
[105] The specific binding member according to any one of [1] to [95], wherein the third sequence consists of SEQ ID NO: 19 in which aspartic acid (D) at
[106] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which the aspartic acid (D) at
[107] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which the aspartic acid (D) at
[108] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which the aspartic acid (D) at
[109] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which the aspartic acid (D) at
[110] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which the aspartic acid (D) at
[111] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which the aspartic acid (D) at
[112] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which the aspartic acid (D) at
[113] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which the aspartic acid (D) at
[114] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which the aspartic acid (D) at
[115] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which the aspartic acid (D) at
[116] The specific binding member according to any one of [1] to [105], wherein the third sequence consists of seq id NO: 19 in which aspartic acid (D) at
[117] The specific binding member according to any one of [1] to [116], wherein the amino acid at
[118] The specific binding member according to any one of [1] to [116], wherein the amino acid at
[119] The specific binding member according to any one of [1] to [118], wherein the amino acid at
[120] The specific binding member according to any one of [1] to [118], wherein the amino acid at
Amino acids may be represented by their one-or three-letter codes, or by their full names. The one and three letter codes, and full names, for each of the 20 standard amino acids are listed below.
The PD-L1 binding site of a specific binding member of the invention preferably comprises: a first sequence, a second sequence, and a third sequence, wherein:
the first sequence consists of:
(a) amino acid sequence SGYW (SEQ ID NO: 23), or
(b) SEQ ID NO: 23, wherein serine (S) is substituted with amino acid E or T;
the second sequence consists of the amino acid sequence EPQYWA (SEQ ID NO: 11); and is
The third sequence consists of:
(a) amino acid sequence SNWRWQMDD (SEQ ID NO: 19), or
(b) SEQ ID NO: 19, wherein the methionine (M) at
Substitution by amino acid I, L or V; and/or
Wherein the amino acid at
In a preferred embodiment, the binding site of the specific binding member of the invention comprises a first sequence, a second sequence and a third sequence of Fcab FS17-33-116, wherein the first sequence has the sequence shown in SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 19, or a sequence set forth in seq id no.
In another preferred embodiment, the binding site of the specific binding member of the invention comprises a first sequence, a second sequence and a third sequence of Fcab FS17-33-288, wherein the first sequence has the sequence shown in SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 27, or a sequence set forth in seq id no.
In another preferred embodiment, the binding site of the specific binding member of the present invention comprises a first sequence, a second sequence and a third sequence of Fcab FS17-33-289 and Fcab FS17-33-334, wherein the first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ id no: 31, or a sequence set forth in seq id no.
In another preferred embodiment, the binding site of the specific binding member of the present invention comprises a first sequence, a second sequence and a third sequence of Fcab FS17-33-296, wherein the first sequence has the sequence shown in SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 35, or a sequence listed in seq id no.
In another preferred embodiment, the binding site of the specific binding member of the present invention comprises a first sequence, a second sequence and a third sequence of Fcab FS17-33-449, wherein the first sequence has the sequence of SEQ ID NO: 42, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 43, or a sequence set forth in seq id no.
In another preferred embodiment, the binding site of the specific binding member of the present invention comprises a first sequence, a second sequence and a third sequence of Fcab FS17-33-451, wherein the first sequence has the sequence shown in SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 43, or a sequence set forth in seq id no.
In addition to the experimental work described above, the present inventors performed a further mutagenesis procedure in order to identify specific binding elements that include a non-natural binding site for PD-L1 in the CH3 domain of the specific binding element that have an increased melting temperature (and are therefore expected to be more stable. This improved stability is expected to be beneficial for the manufacture and storage of specific binding members.
In particular, the inventors have surprisingly found that the reversion of an aspartic acid (D) residue at
Thus, in a preferred embodiment, the specific binding member of the invention comprises a PD-L1 binding site comprising a first sequence, a second sequence and a third sequence as disclosed herein, wherein the third sequence is located in the EF loop, preferably at
(i) leucine (L) at position 84.1 may be substituted with proline (P); and/or
(ii) Serine (S) at position 85.3 may be substituted with threonine (T); and/or
(iii) The amino acid at
Thus, the present invention also provides:
[121] the specific binding member according to any one of [1] to [95] or [106] to [120] above, wherein the third sequence consists of SEQ ID NO: 19, wherein the aspartic acid at position 99 (D) of the CH3 domain is substituted with the amino acid G.
[122] The specific binding member according to any one of [1] to [116] or [119] to [121] above, wherein the amino acid at
[123] The specific binding member according to any one of [1] to [122] above, wherein the amino acid at
[124] The specific binding member according to any one of [1] to [123] above, wherein the amino acid at position 84.1 of the CH3 domain is proline (P).
[125] The specific binding member according to any one of [1] to [124] above, wherein the amino acid at position 85.3 of the CH3 domain is threonine (T).
In a preferred embodiment, the PD-L1 binding site of a specific binding member of the invention comprises a first sequence, a second sequence and a third sequence of FcabFS17-33-488, wherein the first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 70, wherein the residues at positions 84.1, 85.3, 101 and 113 of the CH3 domain of the specific binding member are proline (P), threonine (T), alanine (a) and arginine (R), respectively.
In a further preferred embodiment, the PD-L1 binding site of a specific binding member of the invention comprises a first sequence, a second sequence and a third sequence of Fcab FS17-33-539, wherein the first sequence has the sequence of SEQ ID NO: 42, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 74 and the residue at
In a further preferred embodiment, the specific binding member comprises a first sequence, a second sequence and a third sequence of Fcab FS17-33-548, wherein the first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 78, or a sequence listed in seq id no.
As disclosed herein, the binding site of the specific binding member of the invention may comprise a total of up to 12, up to 11, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or 1 (further) amino acid modification in the first, second and third sequences. In preferred embodiments, the specific binding element may comprise a total of up to 3, up to 2, or 1 (further) amino acid modification in the first, second and third sequences, as disclosed herein. In more preferred embodiments, the specific binding element may comprise a total of up to 2, or 1 (further) amino acid modification in the first, second and third sequences, as disclosed herein. The total number of modifications in the first, second and third sequences refers to the total number of modifications formed in these combined sequences.
As disclosed herein, the binding site of the specific binding member of the invention may comprise up to 2, or 1 (further) amino acid modification in the first sequence. Additionally or alternatively, as disclosed herein, the specific binding element may comprise up to 5, up to 4, up to 3, up to 2, or 1 (further) amino acid modification in the second sequence. Additionally or alternatively, as disclosed herein, a specific binding member of the invention may comprise up to 6, up to 5, up to 4, up to 3, up to 2, or 1 (further) amino acid modification in the third sequence.
In preferred embodiments, the binding site of a specific binding member of the invention may comprise up to 2, or 1 (further) amino acid modification in the first sequence and/or up to 2, or 1 (further) amino acid modification in the third sequence, as disclosed herein. In a more preferred embodiment, the binding element may comprise 1 (further) amino acid modification in the first sequence and/or may comprise 1 (further) amino acid modification in the third sequence, as disclosed herein.
In a preferred embodiment, the binding site of the specific binding member of the invention comprises 1 (further) amino acid modification in the first sequence, the second sequence or the third sequence, as disclosed herein.
The binding site of the specific binding member of the present invention may comprise a third sequence in which the methionine (M) at
In case the specific binding member of the invention comprises 1 or more additional modifications in the first sequence, the amino acids at
In case the specific binding member of the invention comprises 1 or more additional modifications in the second sequence, the amino acid at
Where a specific binding member of the invention includes 1 or more additional modifications in the third sequence, it is preferred that the amino acid at
The modification may be an amino acid substitution, deletion or insertion. Preferably, the modification is a substitution.
In a preferred embodiment, the amino acid at
In an alternative preferred embodiment, the amino acid at
The inventors have shown that certain residues in the AB and EF loops, as well as the framework region, can be converted back to wild-type (WT) residues (see figure 1) with minimal effect on the off-rate (koff) of the specific binding element when bound to PD-L1, with reduced mutational burden of the specific binding element.
Thus, in a preferred embodiment, the binding site of the specific binding member of the invention comprises a first sequence having:
the amino acid T replaces serine (S), and/or
Amino acid K replaces glycine (G).
In another preferred embodiment, the binding site of the specific binding member of the invention comprises a third sequence having:
substitution of the asparagine (N) at
The amino acid N replaces aspartic acid (D) at
The amino acid at
The sequence of the CH3 domain of the specific binding member is not particularly limited, except for the sequence of the PD-L1 antigen binding site. Preferably, the CH3 domain is a human immunoglobulin G domain, such as a human IgG1, IgG2, IgG3 or IgG4 CH3 domain, most preferably a human IgG1 CH3 domain. The sequence of the human IgG1, IgG2, IgG3, or IgG4 CH3 domain is known in the art.
In a preferred embodiment, the specific binding member comprises SEQ ID NO: 24. 28, 32, 36, 39, 44 or 48, more preferably the CH3 domain set forth in SEQ ID NO: 28. 32, 36, 39, 44 or 48, most preferably the domain of CH3 set forth in SEQ ID NO: 32. 44 or 48, or a fragment thereof.
In a further most preferred embodiment, the specific binding member comprises SEQ ID NO: 71. 75 or 79.
Alternatively, the specific binding member may comprise a CH3 domain having an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 24. 28, 32, 36, 39, 44 or 48, preferably SEQ ID NO: 28. 32, 36, 39, 44 or 48, most preferably the sequence set forth in SEQ ID NO: 32. 44 or 48, having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
As a further most preferred embodiment, the specific binding member comprises a CH3 domain having an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 71. 75 or 79 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
The specific binding member may also include a CH2 domain. As in the case of human IgG molecules, the CH2 domain is preferably located at the N-terminus of the CH3 domain. The CH2 domain of the specific binding member is preferably the CH2 domain of human IgG1, IgG2, IgG3 or IgG4, more preferably the CH2 domain of
In a preferred embodiment, the specific binding member comprises: (i) SEQ ID NO: 24. 28, 32, 36, 39, 44, 48, 71, 75 or 79, more preferably SEQ ID NO: 28. 32, 36, 39, 44, 48, 71, 75 or 79, most preferably the CH3 domain set forth in SEQ ID NO: 32. a CH3 domain listed in 44, 48, 71, 75 or 79; and (ii) SEQ ID NO: domain CH2 as set forth in claim 5.
In an alternative preferred embodiment, the specific binding member comprises: (i) SEQ ID NO: 24. 28, 32, 36, 39, 44, 48, 71, 75 or 79, more preferably SEQ ID NO: 28. 32, 36, 39, 44, 48, 71, 75 or 79, most preferably the CH3 domain set forth in SEQ ID NO: 32. a CH3 domain listed in 44, 48, 71, 75 or 79; and (ii) SEQ ID NO: the CH2 domain listed in 6.
Preferably, the specific binding member is in the CH2 domainThe N-terminus comprises an immunoglobulin hinge region (hingereion) or a portion thereof. The immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate (associate) and form a dimer. Preferably, the hinge region or part thereof is a human IgG1, IgG2, IgG3 or IgG4 hinge region or part thereof. More preferably, the hinge region or portion thereof is the IgG1 hinge region or portion thereof. The sequence of the hinge region of human IgG1 is set forth in SEQ ID NO: 69. An appropriately truncated hinge region that may form part of a specific binding member is shown in SEQ ID NO: shown in fig. 7. This hinge region is present in the Fcab molecule tested in the examples, while the full-length hinge region is present in the mimetic mAb 2In a form. Thus, the specific binding member preferably comprises an immunoglobulin hinge region or part thereof at the N-terminus of the CH2 domain, wherein the hinge region has the amino acid sequence set forth in SEQ ID NO: 69 or SEQ ID NO: 7; or wherein the hinge region has an amino acid sequence that is identical to SEQ ID NO: 69 or 7 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. Alternatively, the specific binding member may comprise an immunoglobulin hinge region or portion thereof at the N-terminus of the CH2 domain, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 69, or a fragment thereof, wherein said fragment comprises SEQ id no: 69, at least five, at least six, at least seven, at least eight, at least nine or more, at least ten, at least eleven, at least twelve, at least thirteen or at least fourteen amino acid residues.
In a preferred embodiment, the specific binding member comprises: (i) SEQ ID NO: 24. 28, 32, 36, 39, 44, 48, 71, 75 or 79, more preferably SEQ ID NO: 28. 32, 36, 39, 44, 48, 71, 75 or 79, most preferably the CH3 domain set forth in SEQ ID NO: 32. a CH3 domain listed in 44, 48, 71, 75 or 79; (ii) SEQ ID NO: the CH2 domain listed in 5; and (ii) an immunoglobulin hinge region or portion thereof at the N-terminus of the CH2 domain, wherein the immunoglobulin hinge region has the amino acid sequence of SEQ ID NO: 69.
In an alternative preferred embodiment, the specific binding member comprises: (i) SEQ ID NO: 24. 28, 32, 36, 39, 44, 48, 71, 75 or 79, more preferably SEQ ID NO: 28. 32, 36, 39, 44, 48, 71, 75 or 79, most preferably the CH3 domain set forth in SEQ ID NO: 32. a CH3 domain listed in 44, 48, 71, 75 or 79; (ii) SEQ ID NO: the CH2 domain listed in 6; and (iii) an immunoglobulin hinge region or portion thereof at the N-terminus of the CH2 domain, wherein the immunoglobulin hinge region has the amino acid sequence of SEQ ID NO: 69.
In a preferred embodiment, the specific binding member comprises SEQ ID NO: 25. 26, 29, 30, 33, 34, 37, 38, 40, 41, 45, 46, 49 or 50, or a sequence identical to SEQ ID NO: 25. 26, 29, 33, 34, 30, 37, 38, 40, 41, 45, 46, 49, or 50 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. More preferably, the specific binding member comprises SEQ ID NO: 29. 30, 33, 34, 37, 38, 40, 41, 45, 46, 49 or 50, or a sequence identical to a sequence set forth in SEQ ID NO: 29. 30, 33, 34, 37, 38, 40, 41, 45, 46, 49, or 50, has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. Most preferably, the specific binding member comprises SEQ ID NO: 45. 46, 49 or 50, or a sequence identical to SEQ ID NO: 45. 46, 49 or 50, has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In an alternative most preferred embodiment, the specific binding member comprises SEQ ID NO: 33 or 34, or a sequence identical to SEQ ID NO: 33 or 34, has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
In a further most preferred embodiment, the specific binding member comprises SEQ ID NO: 72. 73, 76, 77, 80 or 81, or a sequence identical to SEQ ID NO: 72. 73, 76, 77, 80 or 81, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
In recent years, monoclonal antibody-based cancer immunotherapy, such as anti-CTLA-4 or anti-PD-1/PD-L1 antibodies, has had a major impact in the treatment of cancer patients. However, despite the significant clinical efficacy of these monotherapies in a large number of malignancies, only a small subset of cancer patients respond to these treatments. Recently, combined inhibition of PD-1 and CTLA-4 in melanoma and non-small cell lung cancer has highlighted the potential to further enhance the clinical benefit of such monotherapies by combining PD-1 and CTLA-4 with other agents with complementary mechanisms of action. Furthermore, the inventors have surprisingly found that when specific binding members of the invention are prepared which comprise CDR-based antigen binding sites for CTLA-4 from ipilimumab (ipilimumab) in addition to the PD-L1 antigen binding site located in the CH3 domain of the specific binding member and which are tested in the staphylococcal enterotoxin B assay (SEB assay), the specific binding members exhibit greater T cell activation compared to (i) specific binding members comprising the same PD-L1 antigen binding site in the CH3 domain and a CDR-based antigen binding site for an unrelated antigen, (ii) ipilimumab, or a combination of (i) and (ii). This suggests that incorporating an anti-PD-L1 antigen-binding site into the CH3 domain, and a CDR-based antigen-binding site for a second antigen into the same specific binding member, can both result in a synergistic increase in specific binding member activity, as compared to the combined use of two separate molecules that include the same antigen-binding site.
Thus, in addition to the PD-L1 antigen binding site in the CH3 domain of the specific binding element, the specific binding element may also include one or more additional antigen binding sites to create a bispecific or multispecific molecule. Preferably, the specific binding member comprises a CDR-based antigen binding site. CDR-based antigen binding sites are present in naturally-occurring (naturally-occuring) immunoglobulin molecules, and their structures are known in the art. Where the specific binding member includes a CDR-based antigen binding site, the specific binding member is preferably an antibody molecule. The antibody molecule is not particularly limited so long as it includes the CH3 domain and CDR-based antigen binding site defined herein. In a preferred embodiment, the antibody molecule is a human immunoglobulin G molecule, such as a human IgG1, IgG2, IgG3 or IgG4 molecule, more preferably a human IgG1 molecule. The sequence of human immunoglobulin G molecules is known in the art, and as disclosed herein, the introduction of the CH3 domain or CH3 domain sequence into such molecules would not present any difficulty to the skilled artisan.
In a preferred embodiment, the specific binding member of the present invention comprises: (i) SEQ ID NO: 45. 46, 49, 50, 72, 73, 76, 77, 80, 81, 33, 34, 25, 26, 29, 30, 37, 38, 40 or 41, preferably SEQ id no: 45. 46, 49, 50, 72, 73, 76, 77, 80, 81, 33, or 34; and (ii) a CDR-based antigen binding site.
The CDR-based antigen binding site can bind an antigen, wherein binding of the antigen is expected to be beneficial in cancer therapy.
In one embodiment, where the specific binding member comprises a CDR-based antigen binding site, the CDR-based antigen binding site may bind a non-redundant and complementary checkpoint inhibitor (checkpoint inhibitor), such as CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, CD73, CSF-1R, kir. B7-H3, B7-H4, 2B4, NKG2A, CD47, SIRPI, BTLA, CCR4, CD200R, or TGFG.
Inhibition of the PD-1/PD-L1 axis and stimulation of co-stimulatory molecules (costimulatory molecules) represent complementary strategies for enhancing immune responses in human patients. Reversal of T cell depletion by checkpoint blockade may allow these cells to be activated more efficiently and to develop full antitumor activity. Thus, in another embodiment, a specific binding member of the invention may comprise a CDR-based antigen-binding site that binds to and is an agonist of a costimulatory molecule expressed by a T cell, such as OX40, ICOS, CD40, HVEM, NKG2D or
In a further embodiment, a specific binding member of the invention may comprise a CDR-based antigen binding site that binds to a Tumor Associated Antigen (TAA). Such specific binding members are expected to lead to tumor specific T cell responses by local immune activation. Examples of TAAs are c-Met, B7-H3, B7-H4, EGFR, HER-2, EPCAM, CEACAM, FAP, VEGF, MSLN, GPC3, CD38, CD19 and
As detailed above, infectious diseases show many similarities to oncology. The role of PD-L1 in immune regulation can be exploited to maximize the immune response against pathogens. Immunomodulation in the context of infectious disease treatment is an emerging field of medicine, and earlier discussions suggest that PD-L1 blockade may improve the biological response to infection, in particular helping to counteract T cell failure, manage immune-mediated gaps, and generate long-term immunity (Wykes and lewis, 2018).
In some infectious diseases, excessive pro-inflammatory responses and suboptimal antigen-specific T cell activity are responsible for severe tissue damage (Rao M et al, 2017). Without wishing to be bound by theory, it is believed that the use of specific binding members of the invention, including CDR-based antigen binding sites, may find application in the treatment of these diseases by localizing beneficial immunomodulatory activity to the pathogen environment.
Alternatively, the use of specific binding members of the invention comprising CDR-based antigen binding sites that bind to immune cell targets either agonistically (agonism) or antagonistically (antasonism) may result in increased specificity and activity of T cells.
Thus, in one embodiment, where the specific binding member comprises a CDR-based antigen binding site, the CDR-based antigen binding site may bind to an immune cell target such as PD-1, PD-L2, CTLA-4, LAG-3, TIGIT, TIM3, OX40, CD40, ICOS, CD28 or
Alternatively, where the specific binding member includes a CDR-based antigen binding site, the CDR-based antigen binding site may bind to a pathogenic target, i.e., an antigen expressed by a human pathogen. The pathogen may be a virus, bacterium, fungus or parasite. Preferably, the pathogen is a virus, bacterium or fungus. More preferably, the pathogen is a virus or a bacterium. Most preferably, the pathogen is a virus. Examples of viral antigens include the proteins p24, gp120 and gp41 expressed by Human Immunodeficiency Virus (HIV), the hepatitis b surface antigen (HBsAg) expressed by Hepatitis B Virus (HBV), and hemagglutinin and neuraminidase expressed by influenza virus. Examples of bacterial antigens include Rv1733, Rv2389 and Rv2435n expressed by Mycobacterium tuberculosis (Mycobacterium tuberculosis).
In some embodiments, the CDR-based antigen binding site of a specific binding member of the invention may not bind to OX 40. Additionally or alternatively, the CDR-based antigen-binding site of the specific binding member of the invention may not bind to CD 137. Additionally or alternatively, the CDR-based antigen-binding site of the specific binding member of the invention may not bind to CD 27. Additionally or alternatively, the CDR-based antigen-binding site of the specific binding member of the invention may not bind to glucocorticoid-induced TNFR-related protein (GITR). Additionally or alternatively, the CDR-based antigen binding site of a specific binding member of the invention may not bind to lymphocyte activation gene 3 (LAG-3). Additionally or alternatively, the CDR-based antigen-binding site of the specific binding member of the invention may not bind to an inducible T-cell costimulator (ICOS). For example, the CDR-based antigen-binding site of a specific binding member of the invention may not bind to ICOS: (i) the PD-L1 binding site of a specific binding member includes: a first sequence located in the AB loop at positions 14-18 of the CH3 domain, wherein the specific binding element comprises an amino acid deletion at
The specific binding member may be further conjugated to an immune system modulator (immune system modulator), cytotoxic molecule, radioisotope or detectable label. The immune system modulator may be a cytotoxic molecule (cytokine), such as a cytokine.
The invention also provides a nucleic acid encoding a specific binding member or antibody molecule of the invention, and a vector comprising such a nucleic acid.
Also provided is a recombinant host cell comprising a nucleic acid or vector of the invention. Such recombinant host cells may be used to produce the specific binding members of the invention. Thus, also provided is a method of producing a specific binding member or antibody molecule of the invention, said method comprising culturing said recombinant host cell under conditions for production of said specific binding member or antibody molecule. The method may further comprise the step of isolating and/or purifying the specific binding member or antibody molecule.
The specific binding members and antibodies of the invention are expected to find use in therapeutic applications, particularly in human therapy, such as cancer therapy, the treatment of infectious diseases, inflammation, diseases and disorders associated with inflammation, and the treatment of inflammatory diseases. Thus, also provided is a pharmaceutical composition comprising a specific binding member or antibody molecule according to the invention, and a pharmaceutically acceptable excipient.
The invention also provides a specific binding member or antibody molecule of the invention for use in a method of treatment. Also provided is a method of treating a patient, wherein the method comprises administering to the patient a therapeutically effective amount of a specific binding member or antibody molecule according to the invention. Further provided is the use of a specific binding member or antibody molecule according to the invention in the manufacture of a medicament. As mentioned herein, the patient is preferably a human patient.
The invention also provides a specific binding member or antibody molecule of the invention for use in a method of treating cancer in a patient. Also provided is a method of treating cancer in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of a specific binding member or antibody molecule according to the invention. Further provided is the use of a specific binding member or antibody molecule according to the invention in the manufacture of a medicament for the treatment of cancer in a patient. The treatment may also include administering a second anti-cancer agent and/or therapy to the patient, such as an anti-tumor vaccine and/or chemotherapeutic agent. The second anti-cancer agent and/or therapy may be administered to the patient simultaneously, separately or sequentially with the specific binding member or antibody molecule of the invention.
The invention also provides a specific binding member or antibody molecule of the invention for use in a method of treating an infectious disease in a patient. Also provided is a method of treating an infectious disease in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of a specific binding member or antibody molecule according to the invention. Further provided is the use of a specific binding member or antibody molecule according to the invention in the manufacture of a medicament for the treatment of an infectious disease in a patient. The treatment may also include administering to the patient a second agent and/or therapy for treating the infectious disease. The second agent and/or therapy may be administered to the patient simultaneously, separately or sequentially with the specific binding member or antibody molecule of the invention.
The invention also provides a specific binding member or antibody molecule of the invention for use in a method of treating inflammation, a disease or disorder associated with inflammation, or an inflammatory disease in a patient. Also provided is a method of treating inflammation, a disease or disorder associated with inflammation, or an inflammatory disease in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of a specific binding member or antibody molecule according to the invention. Further provided is the use of a specific binding member or antibody molecule according to the invention in the manufacture of a medicament for the treatment of inflammation, a disease or disorder associated with inflammation, or an inflammatory disease in a patient. The treatment may also include administering to the patient a second agent and/or therapy for treating inflammation, a disease or disorder associated with inflammation, or an inflammatory disease. The second agent and/or therapy may be administered to the patient simultaneously, separately or sequentially with the specific binding member or antibody molecule of the invention.
Drawings
FIG. 1A shows the sequence of AB, CD and EF loops at frame positions 38, 84.1, 85.3 and 113 of the CH3 domain of Fcab FS17-33, FS17-33-37, FS17-33-116, FS17-33-288, FS17-33-289, FS17-33-296, FS17-33-334, FS17-33-449, FS17-33-451, FS17-33-488, FS17-33-539 and FS17-33-548, as well as the wild-type (WT) Fcab. Numbering of residues according to the IMGT numbering system is shown. Fig. 1B and C show the identity of the antibody residue numbering system including the IMGT, sequential numbering (IMGT exon numbering), EU numbering and Kabat numbering system for the CH3 domain residues of the IgG1 CH3 domain, and the wild-type IgG1 CH3 domain amino acid sequence. FIG. 1D shows an alignment (alignment) of the sequences of the CH3 domain of Fcab FS17-33, FS17-33-37, FS17-33-116, FS17-33-288, FS17-33-289, FS17-33-296, FS17-33-334, FS17-33-449, FS17-33-451, FS17-33-488, FS17-33-539 and FS17-33-548, and the wild-type (WT) Fcab.
FIG. 2 shows two Fcabs (A and B) and a mAb2(C) Size Exclusion Chromatography (SEC) profile of (a). The parent Fcab FS17-33 shows a single shoulder peak (A). Fcab FS17-33-116 shows split peaks (B). SEC analysis shows mAb2FS17-33-116/40420(C) has a small amount of aggregation, which is a typical feature of the parent 4420 mAb. Unlike Fcab, there is no distinct split or shoulder.
FIG. 3A shows that anti-PD-L1 Fcab, FS17-33-116AA inhibited human PD-L1 activity, resulting in the release of IL-2 in the DO 11.10T cell activation assay. This release was comparable to the positive control anti-PD-L1 mAb, yw243.55.S1 (S1). anti-PD-L1 Fcab, FS17-33-37AA, and wild-type (WT) Fcab did not show significant T cell activation in this assay. FIG. 3B shows anti-PD-L1 Fcab, FS17-33-288AA, FS17-33-289AA, and FS17-33-296AA, all showing IL-2 release in the DO 11.10T cell activation assay, which is comparable to the IL-2 release of the positive control mAb, S1. As indicated by "AA" in the clone name, all fcabs tested contained a LALA mutation in the CH2 domain.
FIG. 4A shows Fcab and mock mAb2Forms of anti-PD-L1 Fcab, FS17-33-288AA, FS17-33-289AA and FS17-33-296AA, and FS17-33-334AA inhibited human PD-L1, resulting in the release of IL-2 in the DO 11.10T cell activation assay. Fcab to mock mAb2The conversion of form results in a partial reduction of IL-2 release in this assay. FIG. 4B shows a mimetic mAb 2Forms of anti-PD-L1 Fcab, FS17-33-449 and FS17-33-451 are also capable of inhibiting PD-L1 and releasing IL-2 in the DO 11.10T cell activation assay.
Fig. 5 shows a representative plot (plot) from the SEB assay. Fcab and mock mAb compared to IgG1 isotype control mAb HelD1.32Both show an increased IFN γ release from T cells (a and B). The activity of Fcab was close to or comparable to that of the anti-PD-L1 positive control mAb, yw243.55.s70 (S70). Mimetic mAbs2Form Fcab showed a slight decrease in IFN γ release in this experiment. Fig. 5C shows that the presence of LALA mutation (AA) in Fcab did not interfere with Fcab activity in SEB assay.
Fig. 6A and B show representative plots from SEB trials. Mimicry of mAbs compared to ipilimumab (anti-CTLA-4)2And PD-L1/CTLA-4mAb2Both show an increased IFN γ release by T cells.
Detailed Description
The present invention relates to specific binding members that bind to PD-L1. In particular, the specific binding member of the present invention comprises a PD-L1 antigen binding site located in the constant domain of the specific binding member. Unless the context requires otherwise, the term "PD-L1" may refer to human PD-L1 and/or cynomolgus monkey PD-L1. Preferably, the term "PD-L1" refers to human PD-L1.
The term "specific binding member" describes an immunoglobulin or fragment thereof comprising a constant domain, preferably a CH3 domain, that includes the PD-L1 antigen binding site. Preferably, the specific binding member comprises a CH2 and CH3 domain, wherein the CH2 or CH3 domain, preferably the CH3 domain, comprises the PD-L1 antigen binding site. In a preferred embodiment, the specific binding member further comprises an immunoglobulin hinge region or portion thereof at the N-terminus of the CH2 domain. Such molecules are also referred to herein as antigen binding Fc fragments, or FcabsTM. The specific binding member may be partially or completely synthetically produced.
Thus, as used herein, the term "specific binding member" includes fragments so long as the fragment includes the PD-L1 antigen binding site located in a constant domain of the specific binding member, such as a CL, CH1, CH2, or CH3 domain, preferably a CH1, CH2, or CH3 domain, and most preferably a CH3 domain. Thus, as used herein, the term "specific binding member" corresponds to a "specific binding member or fragment thereof" unless the context requires otherwise.
In a preferred embodiment, the specific binding member is an antibody molecule. The term "antibody molecule" encompasses fragments of antibody molecules as long as such fragments comprise a constant domain, such as a CH1, CH2 or CH3 domain, preferably a CH3 domain, which comprises the PD-L1 antigen binding site. The antibody molecule may be human or humanized (humanized). The antibody molecule is preferably a monoclonal antibody molecule. Examples of antibody molecules are immunoglobulin isotypes (immunoglobulin isotypes), such as immunoglobulin G and isotypes subclasses thereof, such as IgG1, IgG2, IgG3 and IgG4, and fragments thereof.
Monoclonal and other antibodies can be employed and techniques of recombinant DNA technology (technology) used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve the introduction of CDRs or variable regions into different immunoglobulins. The introduction of the CDRs of one immunoglobulin into another is described, for example, in EP-A-184187, GB2188638A and EP-A-239400. Similar techniques can be employed for related constant domain sequences by introducing the PD-L1 antigen binding site into another constant domain, such as a different CH3 domain, or CH1 or CH2 domain.
Since antibodies can be modified in many ways, the term "specific binding member" should be interpreted to cover antibody fragments, derivatives, functional equivalents (functional equivalents) and homologues (homologue) of antibodies, whether natural or wholly or partially synthetic. An example of an antibody fragment comprising the CH3 domain is the Fc domain of an antibody. An example of an antibody fragment that includes both a CDR sequence and a CH3 domain is a minibody (minibody), which includes a single chain antibody fragment (scFv) linked to a CH3 domain (Hu et al, 1996).
The specific binding member of the present invention binds to PD-L1. Binding in this context may refer to specific binding. The term "specific" may refer to a situation in which a specific binding member will not exhibit any significant binding to a molecule other than its specific binding partner, here PD-L1. The term "specific" is also useful where the specific binding member is specific for a particular epitope (epitope) carried by multiple antigens, such as an epitope on PD-L1, in which case the specific binding member will be capable of binding to the various antigens carrying the epitope. The specific binding member may not bind to programmed death ligand 1(PD-1) and CD80, or may not exhibit any significant binding to programmed death ligand 1(PD-1) and CD80, such as human and murine CD80, and human and murine PD-1.
The specific binding member of the present invention preferably comprises a PD-L1 antigen binding site. The PD-L1 antigen binding site is located in the constant domain of the specific binding member, such as the CH1, CH2, CH3 or CH4 domain. Preferably, the PD-L1 antigen binding site is located in the CH3 domain of the specific binding member.
The PD-L1 binding site preferably includes:
(i) a first sequence located in the AB structural loop, wherein the specific binding element comprises an amino acid deletion at
(a) amino acid sequence SGYW (SEQ ID NO: 23), or
(b) SEQ ID NO: 23, wherein
Serine (S) is substituted with amino acid A, E, F, G, H, I, L, P, R, T, V or Y, and/or
Glycine (G) is substituted with amino acid A, D, E, F, H, K, L, N, P, R, T, V or Y;
(ii) a second sequence located in a CD structural loop, wherein the second sequence consists of:
(a) the amino acid sequence EPQYWA (SEQ ID NO: 11), or
(b) SEQ ID NO: 11, wherein
Glutamic acid (E) is substituted by amino acid A, G, H, I, L, N, Q, R, S or W, and-
Or
Proline (P) is substituted by amino acid A, D, E, G, H, N, Q, W or Y, and/or
Glutamine (Q) is substituted by amino acid H or N, and/or
Tyrosine (Y) is substituted by amino acid A, D, H, T or V, and/or
Alanine (a) by amino acid D, E, G, L, R, S or W; and
(iii) a third sequence located in an EF structural loop, wherein the third sequence consists of:
(a) amino acid sequence SNWRWQMD1D2(SEQ ID NO: 19) or
(b) SEQ ID NO: 19, wherein
Serine (S) is substituted by amino acid A or G, and/or
Asparagine (N) is substituted with amino acid A, E, F, G, H, I, K, L, Q, R, S, T or Y, and/or
Glutamine (Q) is substituted with amino acid A, D, E, F, G, H, K, L, N, R, S or V, and/or
Methionine (M) is substituted by amino acid H, S, F, I, L, V, W or Y, and/or
Aspartic acid (D)1) By amino acid E, A, I, L, R, S, T, V, W, Y or G, and/or
Aspartic acid (D)2) Substitution by amino acid A, E, F, I, K, L, N, R, V, W or Y; and is
Wherein the amino acid at
In an alternative preferred embodiment, the PD-L1 binding site includes:
(i) a first sequence located in the AB structural loop, wherein the specific binding element comprises an amino acid deletion at
(a) Amino acid sequence SGYW (SEQ ID NO: 23), or
(b) SEQ ID NO: 23, wherein
Serine (S) is substituted with amino acid A, E, F, G, H, I, L, P, R, T, V or Y, and/or
Glycine (G) is substituted with amino acid A, D, E, F, H, K, L, N, P, R, T, V or Y;
(ii) a second sequence located in a CD structural loop, wherein the second sequence consists of:
(a) the amino acid sequence EPQYWA (SEQ ID NO: 11), or
(b) SEQ ID NO: 11, wherein
Glutamic acid (E) is substituted by amino acid A, G, H, I, L, N, Q, R, S or W, and/or
Proline (P) is substituted by amino acid A, D, E, G, H, N, Q, W or Y, and/or
Glutamine (Q) is substituted by amino acid H or N, and/or
Tyrosine (Y) is substituted by amino acid A, D, H, T or V, and/or
Alanine (a) by amino acid D, E, G, L, R, S or W; and
(iii) a third sequence located in an EF structural loop, wherein the third sequence consists of:
(a) amino acid sequence SNWRWQMD1D2(SEQ ID NO: 19) or
(b) SEQ ID NO: 19, wherein
Serine (S) is substituted by amino acid A or G, and/or
Asparagine (N) is substituted with amino acid A, E, F, G, H, I, K, L, Q, R, S, T or Y, and/or
Glutamine (Q) is substituted with amino acid A, D, E, F, G, H, K, L, N, R, S or V, and/or
Methionine (M) is substituted by amino acid H, S, F, I, L, V, W or Y, and/or
Aspartic acid (D)1) By amino acid E, A, I, L, R, S, T, V, W or Y, and/or
Aspartic acid (D)2) Substitution by amino acid A, E, F, I, K, L, N, R, V, W or Y; and is
Wherein the amino acid at
The specific binding member of the present invention may also include an arginine (R) at
(i) the amino acid at position 84.1 of the CH3 domain may be proline (P); and/or
(ii) The amino acid at position 85.3 of the CH3 domain may be threonine (T); and/or
(iii) The amino acid at
Unless otherwise indicated, amino acid residues herein are numbered according to the international immunogenetic database (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al, 2005.
Preferably, the first sequence is located at residues 14-18 of the CH3 domain, the second sequence is located at positions 45.1-78 of the CH3 domain, and/or the third sequence is located at positions 92-100 or 92-101 of the CH3 domain, wherein the amino acid residues are numbered according to the international immunogenetics database (IMGT) numbering scheme.
As an alternative to IMGT numbering, the positions of residues comprising the modified CH3 domain described herein may alternatively be indicated according to a consecutive numbering, also referred to as SEQ ID NO: 4, IMGT exon numbering, EU numbering, or Kabat numbering of residues in the wild-type IgG1 CH3 domain sequence set forth in seq id no.
The identity between IMGT numbering, sequential numbering (IMGT exon numbering), EU numbering and Kabat numbering (concordance) of residues of the WT IgG1 CH3 domain (SEQ ID NO: 4) is shown in FIGS. 1B and C.
Shown in fig. 1A, relative to SEQ ID NO: the wild-type CH3 domain sequence set forth in 4, identity between IMGT numbering and consecutive numbering of modified residue positions in the CH3 domain of Fcab FS17-33, FS17-33-37, FS17-33-116, FS17-33-288, FS17-33-289, FS17-33-296, FS17-33-334, FS17-33-449, FS17-33-451, FS17-33-488, FS17-33-539, and FS 17-33-548.
Thus, for example, as shown in fig. 1A, where the application relates to modification of the AB structural loop at positions 14-18, the CD structural loop at positions 45.1-78, and/or the EF structural loop at positions 92-100 of the CH3 domain, where the residue numbering is according to the IMGT numbering scheme, the amino acid sequence according to SEQ ID NO: 4 are positions 18-22, 46-51 and 73-81, respectively, of the CH3 domain.
In a preferred embodiment, the PD-L1 binding site includes a first sequence located in the AB loop, a second sequence located in the CD loop, and a third sequence located in the EF loop,
wherein the first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 19, or a sequence set forth in seq id no;
the first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 27, or a sequence set forth in seq id no;
the first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 31, or a sequence set forth in seq id no;
the first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 35;
the first sequence has the sequence of SEQ ID NO: 42, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 43;
the first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 43;
The first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 78; or
The first sequence has the sequence of SEQ ID NO: 42, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 74 and the residue at
Most preferably, the PD-L1 binding site includes a first sequence located in the AB loop, a second sequence located in the CD loop, and a third sequence located in the EF loop, wherein
The first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 31, or a sequence set forth in seq id no;
the first sequence has the sequence of SEQ ID NO: 42, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 43; or
The first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 43, or a sequence set forth in seq id no.
In an alternative most preferred embodiment, the PD-L1 binding site of a specific binding member of the invention may include a first sequence located in the AB loop, a second sequence located in the CD loop, and a third sequence located in the EF loop,
wherein the first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 78; or
The first sequence has the sequence of SEQ ID NO: 42, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 74 and the residue at
The first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 70, wherein the residues at positions 84.1, 85.3, 101 and 113 of the CH3 domain of the specific binding member are proline (P), threonine (T), alanine (a) and arginine (R), respectively.
The first, second and third sequences are preferably located in the structural loops of the constant domains of the specific binding members. For example, the introduction of sequences into the structural loop regions of antibody constant domains to create new antigen binding sites is described in WO2006/072620 and WO 2009/132876.
The structural loops of the antibody constant domain include the AB, CD and EF loops. In the CH3 domain, the AB, CD, and EF loops are located at residues 11-18, 43-78, and 92-101 of the CH3 domain. Preferably, the first sequence is located at residues 14-18 of the CH3 domain, the second sequence is located at positions 45.1-78 of the CH3 domain, and/or the third sequence is located at positions 92-100 or 92-101 of the CH3 domain.
Specific binding members of the invention may also include alanine and valine residues, preferably an alanine residue, at
the first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 31, or a sequence set forth in seq id no;
the first sequence has the sequence of SEQ ID NO: 42, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 43;
the first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 43;
The first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 78;
the first sequence has the sequence of SEQ ID NO: 42, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 74 and the residue at
The first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 70, wherein the residues at positions 84.1, 85.3, 101 and 113 of the CH3 domain of the specific binding member are proline (P), threonine (T), alanine (a) and arginine (R), respectively;
an alanine residue at
Similarly, a specific binding member comprising a first sequence, a second sequence and a third sequence, wherein
The first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 19, or a sequence set forth in seq id no;
The first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 27, or a sequence set forth in seq id no;
the first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 31, or a sequence set forth in seq id no; or
The first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: the sequence set forth in 35, wherein,
a valine residue at
Additionally or alternatively, a specific binding member of the invention may also comprise a valine residue or an amino acid deletion at
The first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 19, or a sequence set forth in seq id no;
the first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 27, or a sequence set forth in seq id no;
The first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 31, or a sequence set forth in seq id no;
the first sequence has the sequence of SEQ ID NO: 23, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 35;
a deletion of the amino acid at
Alternatively, a specific binding member of the invention may also comprise an alanine residue (a) at
The first sequence has the sequence of SEQ ID NO: 47, the second sequence having the sequence set forth in SEQ ID NO: 11, and the third sequence has the sequence set forth in SEQ ID NO: 70, wherein the residues at positions 84.1, 85.3 and 113 of the CH3 domain of the specific binding member are proline (P), threonine (T) and arginine (R), respectively, may comprise the alanine residue (a) at
In a preferred embodiment, the specific binding member of the invention comprises a CH3 domain, the CH3 domain comprising, having or consisting of SEQ ID NO: 24. 28, 32, 36, 39, 44, 48, 71, 75 or 79, preferably having the sequence set forth in SEQ ID NO: 28. 32, 36, 39, 44, 48, 75 or 79, or a pharmaceutically acceptable salt thereof.
A specific binding member of the invention may comprise a CH3 domain, the CH3 domain comprising, having or consisting of seq id NO: 24. 28, 32, 36, 39, 44, 48, 71, 75 or 79, wherein the sequence set forth in SEQ ID NO: 24. the lysine residue (K) immediately adjacent (immedate) the C-terminus of the sequence shown in 28, 32, 36, 39, 44, 48, 71, 75 or 79 has been deleted.
Furthermore, the specific binding member of the invention may comprise the CH2 domain of an immunoglobulin G molecule, such as the CH2 domain of an IgG1, IgG2, IgG3 or IgG4 molecule. Preferably, the specific binding member of the present invention comprises the CH2 domain of an IgG1 molecule. The CH2 domain may have the amino acid sequence of SEQ ID NO: 6.
The CH2 domain of the specific binding element may comprise one or more mutations that reduce or abolish (abrogate) binding of the CH2 domain to one or more Fc γ receptors such as Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIII and/or to complement (complement). The present inventors postulated that reducing or abolishing binding to Fc γ receptors would reduce or eliminate antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by antibody molecules. Similarly, reducing or abolishing binding to complement is expected to reduce or eliminate Complement Dependent Cytotoxicity (CDC) mediated by the antibody molecule. Mutations that reduce or abolish the binding of the CH2 domain to one or more Fc γ receptors and/or complement are known in the art (Wang et al, 2018). These mutations include the "LALA mutations" described in Bruhns et al, 2009 and Hezareh et al, 2001, which involve the substitution of alanine for leucine residues at positions 1.3 and 1.2 of IMGT of the CH2 domain (L1.3A and L1.2A). Alternatively, it is also known that IgG1 effector function is reduced by generating aglycosylated antibodies by mutation of the conserved N-linked glycosylation site by mutating asparagine (N) at position 84.4 of IMGT of CH2 domain to alanine, glycine or glutamine (N84.4A, N84.4G or N84.4Q) (Wang et al, 2018). As a further alternative, it is known to reduce complement activation (C1q binding) and ADCC (Idusogene et al, 2000; Klein et al, 2016) by mutating the proline at position 114 of the IMGT of the CH2 domain to alanine or glycine (P114A or P114G). These mutations can also be combined to generate antibody molecules with further reduced or no ADCC or CDC activity.
Thus, a specific binding member may comprise a CH2 domain, wherein the CH2 domain comprises:
(i) alanine residues at positions 1.3 and 1.2; and/or
(ii) Alanine or glycine at position 114; and/or
(iii) Alanine, glutamine or glycine at position 84.4;
wherein the numbering of the amino acid residues is according to the IMGT numbering scheme.
In a preferred embodiment, the specific binding member comprises a CH2 domain, wherein the CH2 domain comprises:
(i) an alanine residue at position 1.3; and
(ii) an alanine residue at position 1.2;
wherein the numbering of the amino acid residues is according to the IMGT numbering scheme.
For example, the CH2 domain may have the sequence of SEQ ID NO: 5, or a sequence listed in seq id no.
In an alternative preferred embodiment, the antibody molecule comprises a CH2 domain, wherein the CH2 domain comprises:
(i) an alanine residue at position 1.3;
(ii) an alanine residue at position 1.2; and
(iii) alanine at position 114;
wherein the numbering of the amino acid residues is according to the IMGT numbering scheme.
For example, the CH2 domain may have the sequence of SEQ ID NO: 82, or a sequence listed in seq id no.
In a preferred embodiment, the specific binding member may include one or more additional antigen binding sites that bind one or more additional antigens in addition to the PD-L1 antigen binding site located in the constant domain of the specific binding member. The one or more other antigen binding sites preferably specifically bind their cognate antigens.
The one or more additional antigen binding sites may bind PD-L1 or another antigen. Thus, the specific binding member may be a multispecific molecule, such as a bispecific, trispecific or tetraspecific molecule, preferably a bispecific molecule. In a preferred embodiment, the specific binding member is capable of binding to PD-L1, and one or more other antigens simultaneously.
Antibody molecules are known to have modular structures comprising discrete domains that can be combined in a number of different ways to create multispecific antibody formats, such as bispecific, trispecific, or tetraspecific antibody formats. Exemplary multispecific antibody formats are described, for example, in Spiess et al (2015) and Kontermann (2012). Specific binding members of the invention may be utilized in such a multispecific antibody format. This has the following additional advantages: antigen binding sites are introduced into such multispecific antibody formats by the presence of antigen binding sites in the constant domains of specific binding elements, such as the CH3 domain.
For example, a specific binding member of the invention may be a heterodimeric antibody molecule, such as a heterodimeric whole immunoglobulin molecule or a fragment thereof. In this case, a portion of the antibody molecule will have one or more sequences as described herein. For example, where the specific binding element of the invention is a bispecific heterodimeric antibody molecule, the specific binding element can comprise a heavy chain comprising a CH3 domain as described herein paired with a heavy chain that binds an antigen other than PD-L1. Techniques for making heterodimeric antibodies are known in the art and include the knob-to-hole (KIHs) process, which involves building the CH3 domain of an antibody molecule to create a "knob" or "hole" to promote chain heterodimerization. Alternatively, heterodimeric antibodies can be prepared by introducing charge pairs into the antibody molecule to avoid homodimerization of the CH3 domain by electrostatic repulsion and direct heterodimerization by electrostatic attraction. Examples of heterodimeric antibody formats include CrossMab, mAb-Fv, SEED-body, and KIH IgG.
Alternatively, the multispecific specific binding element of the present invention may comprise an intact immunoglobulin molecule or fragment thereof, and one or more additional antigen binding portions. For example, the antigen binding portion may be an Fv, an scFv, or a single domain antibody, and may be fused to an intact immunoglobulin molecule or a fragment thereof. Examples of multispecific antibody molecules comprising additional antigen-binding moieties fused to an intact immunoglobulin molecule include DVD-IgG, DVI-IgG, scFv4-IgG, IgG-scFv and scFv-IgG molecules (Spiess et al, 2015; FIG. 1). For example, examples of multispecific antibody molecules comprising an additional antigen-binding moiety fused to an immunoglobulin fragment comprising a CH3 domain (Spiess et al, 2015; FIG. 1) include scDiabody-CH3, Diabody-CH3, and scFv-CH3 KIH.
Other suitable multispecific formats will be apparent to the skilled person.
In a preferred embodiment, a specific binding member according to the invention comprises a second antigen-binding site, wherein the second antigen-binding site is preferably a CDR-based antigen-binding site. The term "CDR-based antigen binding site" refers to an antigen binding site of a variable region of a specific binding member composed of six CDR residues.
The second antigen binding site is preferably specific for a checkpoint inhibitor, a co-stimulatory molecule or a tumor associated antigen.
Determination of antibody molecules directed against a given antigen, such as a tumor-associated antigen, and the CDR sequences of such antibody molecules is well within the skill of the artisan, and many suitable techniques are known in the art. In addition, antibodies comprising CDR sequences directed against various immune system modulators are known in the art. Thus, the skilled person would have no difficulty in preparing a specific binding member that includes a CDR-based antigen binding site for a second antigen in addition to the PD-L1 binding site described herein.
In certain embodiments, a specific binding member of the invention does not comprise a CDR-based antigen binding site.
Specific binding members of the invention may also include variants of the structural loops, CH3 domain, CH2 domain, CH2 and CH3 domain, CDRs, VH domain, VL domain, light or heavy chain sequences disclosed herein. Suitable variants may be obtained by sequence alteration, or mutation, and screening methods. In a preferred embodiment, a specific binding element comprising one or more variant sequences retains one or more functional properties of the parent specific binding element, such as binding specificity and/or binding affinity for PD-L1. For example, a specific binding member comprising one or more variant sequences, preferably binds to PD-L1 having the same affinity as or a higher affinity than the (parent) specific binding member. The parent specific binding member is a specific binding member that does not comprise amino acid substitutions, deletions and/or insertions that have been incorporated into the variant specific binding member.
For example, a specific binding member of the invention may comprise a loop, CH3 domain, CH2 387domain, CH2 and CH3 domain, CDR, VH domain, VL domain, light chain or heavy chain sequence having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% to a loop, CH3 domain, CH2 domain, VH domain, VL domain, light chain or heavy chain sequence disclosed herein.
In a preferred embodiment, the specific binding member of the invention comprises a CH3 domain sequence that is identical to the sequence of SEQ ID NO: 24. 28, 32, 36, 39, 44, 48, 71, 75 or 79, preferably SEQ ID NO: 28. 32, 36, 39, 44, 48, 71, 75 or 79, most preferably SEQ ID NO: 32. the CH3 domain sequences listed in 44, 48, 71, 75, or 79 have at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity. In particular, the specific binding member of the invention comprises a CH3 domain that is homologous to the CH3 domain of SEQ ID NO: the CH3 domain sequences listed in 24 have at least 96% sequence identity.
In a further preferred embodiment, the specific binding member of the invention comprises a CH2 domain sequence that is identical to the sequence of SEQ ID NO: the CH2 domain sequences listed in 5 or 6 have at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.
In another preferred embodiment, the specific binding member of the invention comprises or consists of a sequence that is identical to the sequence of SEQ ID NO: 25. 26, 29, 30, 33, 34, 37, 38, 40, 41, 45, 46, 49, 50, 72, 73, 76, 77, 80, or 81, most preferably SEQ ID NO: 45. 46, 49, 50, 72, 73, 76, 77, 80, or 81 has at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity. In an alternative most preferred embodiment, the specific binding member of the invention comprises, or consists of, a sequence that is substantially identical to the sequence of SEQ ID NO: 33 or 34, has at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.
Tables 9A, B and C show that, in the case of the molecules prepared in the examples, certain residues in the structural loops could not be modified without an increase in the off-rate of Fcab when bound to PD-L1, compared to the off-rate of the parent molecule.
Thus, in the following cases:
(i) the specific binding member comprises a CH3 domain sequence consisting of the sequence of the CH3 domain and the sequence of SEQ ID NO: 24. 28, 32, 36, 39, 44, 48, 71, 75 or 79; or
(ii) The specific binding member comprises or consists of a sequence consisting of a sequence substantially identical to the sequence set forth in any one of the preceding paragraphs or elsewhere herein as set forth in SEQ ID NO: 25. 26, 29, 30, 33, 34, 37, 38, 40, 41, 45, 46, 49, 50, 72, 73, 76, 77, 80 or 81,
preferably the amino acid deletion at
preferably the amino acid at
Preferably the amino acid at
Sequence identity is usually defined with reference to the algorithm GAP (Wisconsin GCG software package, Accelerys corporation, san diego, usa). GAP uses the neiderian (Needleman) and Wunsch (Wunsch) algorithms to align two complete sequences, maximizing the number of matches and minimizing the number of GAPs (GAPs). Typically, using the default parameter (default parameter), the gap creation penalty (gap creation penalty) is equal to 12 and the gap extension penalty (gap extension penalty) is equal to 4. The use of GAP may be preferred, but other algorithms may be used, such as BLAST (which uses the method of Altschul et al, 1990), FASTA (which uses the method of Pearson and Lipman, 1988), or the Smith-Waterman algorithm (Smith and Waterman,1981), typically with default parameters, or the TBLASTN program of Altschul et al, 1990, described above. In particular, the psi-Blast algorithm can be used (Altschul et al, 1997).
Specific binding members of the invention may also include: having one or more amino acid sequence alterations (additions, deletions, substitutions and/or insertions of amino acid residues), preferably 20 or less alterations, 15 or less alterations, 10 or less alterations, 5 or less alterations, 4 or less alterations, 3 or less alterations, 2 or less alterations, or 1 altered structural loop, a CH3 domain, a CH2 domain, a CH2 and CH3 domain, a CDR, a VH domain, a VL domain, a light chain or heavy chain sequence as compared to the structural loop, CH3 domain, CH2 domain, CH2 and CH3 domain, CDR, VH domain, VL domain, light chain or heavy chain sequence disclosed herein. In particular, the alteration may be formed in one or more framework regions of the specific binding member.
In a preferred embodiment, the specific binding member of the invention may comprise a CH3 domain sequence that is substantially identical to the sequence of SEQ ID NO: 24. 28, 32, 36, 39, 44, 48, 71, 75 or 79 has one or more amino acid sequence alterations (additions, deletions, substitutions and/or insertions of amino acid residues) as compared to the CH3 domain sequence, preferably 20 or less alterations, 15 or less alterations, 10 or less alterations, 5 or less alterations, 4 or less alterations, 3 or less alterations, 2 or less alterations, or 1 alteration.
In a further preferred embodiment, the specific binding member of the invention comprises a CH2 domain sequence that is identical to the sequence of SEQ ID NO: the CH2 domain sequences listed in 5 or 6 have one or more amino acid sequence alterations (additions, deletions, substitutions, and/or insertions of amino acid residues) as compared to the sequence, preferably 20 or less alterations, 15 or less alterations, 10 or less alterations, 5 or less alterations, 4 or less alterations, 3 or less alterations, 2 or less alterations, or 1 alteration.
In a further preferred embodiment, the specific binding member of the invention comprises or consists of a sequence that is identical to the sequence of SEQ ID NO: 25. 26, 29, 30, 33, 34, 37, 38, 40, 41, 45, 46, 49, 50, 72, 73, 76, 77, 80, or 81 has one or more amino acid sequence alterations (additions, deletions, substitutions, and/or insertions of amino acid residues) as compared to the sequence listed, preferably 40 or fewer alterations, 30 or fewer alterations, 20 or fewer alterations, 15 or fewer alterations, 10 or fewer alterations, 5 or fewer alterations, 4 or fewer alterations, 3 or fewer alterations, 2 or fewer alterations, or 1 alteration.
Also contemplated is a specific binding member that competes with, or binds to the same epitope on PD-L1 as the specific binding member of the invention for binding to PD-L1, wherein the specific binding member preferably comprises a PD-L1 antigen binding site in the CH3 domain of the specific binding member. Methods for determining the competition of two antibodies for an antigen are known in the art. For example, surface plasmon resonance, such as a biomacromolecule interaction analyzer (Biacore), can be used to determine competition of two antibodies for binding to an antigen. Methods for mapping epitopes bound by antibodies are also known in the art.
Specific binding members of the invention may bind to human PD-L1 and/or cynomolgus monkey PD-L1. Preferably, the specific binding member of the present invention binds to human PD-L1.
The specific binding member of the present invention is preferably capable of binding to PD-L1 expressed on the surface of a cell. The cell is preferably a cancer cell.
Where the specific binding member includes a second antigen-binding site, such as a CDR-based antigen-binding site, specific for a second antigen, the specific binding member is preferably capable of binding to both PD-L1 and the second antigen. Preferably, the specific binding member is capable of binding to PD-L1 and the second antigen simultaneously, wherein PD-L1 and the second antigen are expressed on the surface of a single cell, or on the surface of two separate cells.
The specific binding members of the invention preferably have an affinity (K) of 5nM, 4nM, 3nM or 2nMD) Or greater affinity to monomeric PD-L1, preferably human monomeric PD-L1. Preferably, the specific binding members of the present invention have an affinity (K) of 2nMD) Or greater affinity to PD-L1, preferably human PD-L1.
The specific binding members of the invention preferably have an affinity (K) of 20nM, 19nM, 18nM, 17nM or 16nMD) Or greater affinity to PD-L1, preferably human PD-L1, expressed on the cell surface. Preferably, a specific binding member of the invention has an affinity (K) of 16nMD) Or greater affinity to PD-L1, preferably human PD-L1, expressed on the cell surface.
For example, the binding affinity of a specific binding member for a cognate antigen, such as PD-L1, can be determined by Surface Plasmon Resonance (SPR), such as Biacore. The binding affinity of a specific binding member to a cognate antigen expressed on the cell surface, such as PD-L1, can be determined by flow cytometry (flow cytometry).
Fcab has a smaller binding interface than monoclonal antibodies because the binding site of Fcab forms a relatively compact antibody fragment with two binding sites located proximally. In contrast, the Fab arms (arm) of a typical mAb are separated by a flexible hinge region. The two antigen-binding sites of Fcab are also spatially close to each other compared to the antigen-binding sites of typical mabs. Based on this smaller binding interface and reduced flexibility of the two binding sites, it was surprising that anti-PD-L1 Fcab was able to bind to PD-L1 and inhibit PD-L1 with similar affinity and potency (potency) to a monoclonal antibody specific for PD-L1.
When the specific binding member is in the form of an intact immunoglobulin molecule, it is also referred to herein as a mAb2The specific binding member may have an EC of 9nM or less, 8nM or less, 7nM or less, 6nM or less, 5nM or less, 4nM or less, 3nM or less, or 2nM or less, preferably 2nM or less, in a T cell activation assay, such as the DO11.10 assay50。
When the specific binding member is in the form of an Fcab consisting of a CH3 domain, a CH2 domain and a truncated immunoglobulin hinge region located at the N-terminus of the CH2 domain, the specific binding member may have an EC of 2nM or less, 1nM or less, or 0.5nM or less, preferably 0.5nM or less, in a T cell activation assay, such as the DO11.10 assay50。
The DO11.10 assay is an IL-2 release assay based on T-and B-lymphocyte hybridoma cell lines and can be used for functional screening of specific binding members of the invention. IL-2 release is a marker of T cell activation. The DO11.10 assay may be a DO11.10 assay as described in example 6 herein.
For example, a DO11.10 assay may involve, for example, preparing a dilution of the specific binding member of interest in an experimental medium, such as intact DO11.10 medium without a purotoxin 5B cell hybridoma cells per ml 1: 1 mix, the B cell hybridoma cells transduced with a lentiviral vector (lentiviral vector) containing human PD-L1 to overexpress human PD-L1 (referred to as "B cell hybridoma cells") in experimental media in the presence of 2.46 μ M OVA peptide (e.g., 100 μ L of B cell hybridoma cells/specific binding element) per well, e.g., in a 96 round bottom plate, and can be at 37 ℃, 5% CO2Specific binding members were incubated for 1 hour at the
In the case of DO 11.10T cell activation assays, DO 11.10T cell hybridoma cells transduced with an empty lentiviral vector can be prepared using the lentiviral transduction methodology, for example to generate DO11.10 cells containing an empty lentiviral vector pLVX using the Lenti-X HTX encapsulation system. Lenti-X expression vector (pLVX) can be co-transfected into a Lenti-X293T cell line along with a Lenti-X HTX encapsulation mixture to generate virus. Do11.10 cell lines can be transduced using lentivirus particles produced in the Lenti-X HTX encapsulation system.
In the case of the DO 11.10T cell activation assay, B cell hybridoma cells, such as LK 35.2B cell hybridoma cells (ATCC, HB-98), transduced with a lentiviral vector containing human PD-L1 to overexpress human PD-L1 can be prepared using a lentiviral transduction methodology, for example using the Lenti-X HTX encapsulation system. A Lenti-X expression vector (pLVX) containing human PD-L1 cDNA was co-transfected into a Lenti-X293T cell line along with a Lenti-X HTX encapsulation mixture to generate virus. B cell hybridoma cell lines can be transduced with lentiviral vectors generated with a Lenti-X HTX encapsulation system.
When the specific binding member is in the form of an intact immunoglobulin molecule, it is also referred to herein as a mAb 2The specific binding member may have a molecular weight of 5nM or less in a Staphylococcal Enterotoxin B (SEB) assayEC of 4nM or less, 3nM or less, or 2nM or less, preferably 2nM or less50。
When the specific binding member is in the form of an Fcab consisting of a CH3 domain, a CH2 domain, and a truncated immunoglobulin hinge region located at the N-terminus of the CH2 domain, the specific binding member may have an EC of 5nM or less, 4nM or less, 3nM or less, 2nM or less, 1nM or less, 0.5nM or less, 0.3nM or less, or 0.2nM or less, preferably 0.2nM or less, in a staphylococcal enterotoxin b (seb) assay50。
Staphylococcal enterotoxin B is a superantigen and binds to MHC class II molecules on Antigen Presenting Cells (APCs), as well as to the v β chain of the T Cell Receptor (TCR), causing non-specific activation of T cells and cytokine release. The presence of an antigen-specific TCR for T cell activation is not required. SEB assay uses stimulated human Peripheral Blood Mononuclear Cells (PBMCs) with physiological levels of checkpoint inhibitors, and SEB assay can be used to confirm that T cell activation is enhanced by specific binding elements in the human system.
The SEB test may be an SEB test as described in example 7 herein.
For example, SEB trials may include:
(i) for example, CD4+ T cell isolation kits (e.g., from Miltenyi Biotechnology Ltd, 130-rotech), 200-02-50. mu.g) of 1.0 × 10 in RPMI 6The required amount of CD4+ T cells per cell/ml was transferred to a T75 flask (e.g., from Greiner Bio-one, 690195) at a 3:1 magnetic bead to cell ratio and + 5% CO at 37 ℃ + 5%2After 3 days of incubation, the cells can be gently resuspended and counted, the cell density can be maintained at 0.8-1 × 10 by adding fresh medium (e.g., RPMI-10% FBS + penicillin streptomycin solution 1X +50IU/ml rhuIL2)6Between cells/ml on
(ii) (ii) isolating the uncontacted monocytes from human PBMCs, e.g.using a human Pan (Pan) monocyte isolation kit (such as from America whirlwind Biotechnology, Inc., 130-096-537), e.g.using the method described in (i). Human Mo-DC differentiation media (such as from Gentiana and whirlpool biotechnology, Inc., 130-; and/or
(iii) For example, dilutions of specific binding members of interest are prepared in AIM medium (such as from Gibco, 12055-one 091.) for example MoiDC prepared as described in (ii) can be mixed with T cells from the same donor at a ratio of 1:10 (5ml of 2 × 10) 5Individual cells/ml of iDC can be mixed with 5ml of 2 × 106Individual cells/ml of T cell combination). 20 μ l of SEB (e.g.from Sigma, S4881) at 0.1 μ g/ml can be added to 10ml of cells. In a round bottom 96 well plate, 100. mu.l of the cell/SEB mixture can be added to 100. mu.l of the specific binding member dilution, giving 10 with 0.1ng/ml SEB per 200. mu.l of medium (e.g., AIM medium) per well4iDC cells and 105Proportion of individual T cells. The cells can be cultured at 37 deg.C and 5% CO2The following incubations were carried out for 4 days. An ELISA, such as a human IFN γ ELISA kit (e.g., from R) can be used&D system, PDIF50), supernatants were tested for IFN γ. The assay can be performed using a supernatant diluted 1:30 by PBA (DPBS, 2% BSA, such as from sigma, a 7906-100G). Can convert human IFN gammaIs plotted against the log concentration of the specific binding member. The resulting curve can be fitted using a log (agonist) response equation, for example in GraphPadPrism software.
The specific binding member may have an EC of 30nM or less, 20nM or less, 15nM or less, 14nM or less, 13nM or less, 12nM or less, or 11nM or less, preferably 11nM or less 50Blocking the interaction between PD-L1 and PD-1, preferably human PD-L1 and human PD-1.
When the specific binding member is in the form of an intact immunoglobulin molecule, it is also referred to herein as a mAb2The specific binding member may have an EC of 50nM or less, 45nM or less, 40nM or less, or 30nM or less, preferably 30nM or less50Blocking the interaction between PD-L1 and CD80, preferably human PD-L1 and
When the specific binding member is in the form of an Fcab consisting of a CH3 domain, a CH2 domain and a truncated immunoglobulin hinge region located at the N-terminus of the CH2 domain, the specific binding member may be capable of an EC of 20nM or less, 15nM or less, 14nM or less, 13nM or less, 12nM or less, 11nM or less, 10nM or less, preferably 10nM or less50Blocking the interaction between PD-L1 and CD80, preferably human PD-L1 and
Methods for testing the ability of specific binding members of the invention to block the interaction between human PD-L1, and human PD-1 or human CD80 are known in the art and described herein. For example, blockade of the interaction between human PD-L1, and human PD-1 or human CD80 can be tested using cell-based receptor binding assays. Biotinylated human PD-L1 at 2. mu.g/mL can be incubated for 1 hour with Fcab at a titration concentration ranging from 400nM to 3 pM. The mixture can be incubated with cells overexpressing human PD-1 or human CD80, such as HEK293 cells for an additional hour. The level of bound biotinylated human PD-L1 on the cells can be detected using streptavidin 647 and the level of fluorescence can be measured using FACS, whereby the detected level of fluorescence is indicative of the level of PD-L1 and PD-1 or human CD80 expressed on the cells.
The specific binding member may have a temperature of above 60 ℃ and 61 DEG CA melting temperature of 62 ℃ or higher, 63 ℃ or higher, 64 ℃ or higher, 65 ℃ or higher, 66 ℃ or higher, 67 ℃ or higher, or 68 ℃ or higher, preferably 65 ℃ or higher, 66 ℃ or higher, 67 ℃ or higher, or 68 ℃ or higher, more preferably 66 ℃ or higher, 67 ℃ or higher, or 68 ℃ or higher. When melting temperature is measured, the specific binding member may be in the form of an intact immunoglobulin molecule, also referred to herein as a mAb2。
The melting temperature of the specific binding members of the invention can be measured by known methods. For example, the melting temperature of the specific binding member of the present invention can be measured by Differential Scanning Calorimetry (DSC) or Differential Scanning Fluorescence (DSF).
Aggregation of therapeutic antibodies may lead to loss of drug efficacy and may cause unwanted immunogenicity. Therefore, for this purpose, antibodies that show little or no aggregation are preferred. The specific binding member may exhibit no more than 5%, 4% or 3%, preferably 3% aggregation when in aqueous solution. For example, Size Exclusion Chromatography (SEC) can be used to determine aggregation of specific binding members in aqueous solution.
Specific binding members of the invention may be conjugated to a bioactive agent, a therapeutic agent or a detectable label. In this case, the specific binding member may be referred to as a conjugate. Such conjugates find use in the treatment of diseases and conditions as described herein.
For example, the specific binding member may be conjugated to an immune system modulator, a cytotoxic molecule, a radioisotope, or a detectable label. The immune system modulator or cytotoxic molecule may be a cytokine. The immune system modulator may also be a cell surface receptor or a biologically active fragment thereof, e.g., a fragment comprising or consisting of the ligand binding domain of the receptor. Alternatively, the immune system modulator may be a ligand, e.g. a peptide ligand, of a cell surface receptor, or a biologically active fragment of the ligand, e.g. a fragment comprising or consisting of a receptor binding domain of the ligand. The detectable label may be a radioisotope, e.g., a non-therapeutic radioisotope.
The specific binding member may be conjugated to the bioactive agent, therapeutic agent or detectable label by a peptide bond or linker (linker), i.e. within a fusion polypeptide (fusion polypeptide) comprising the bioactive agent, therapeutic agent or detectable label and the specific binding member or polypeptide chain component thereof (polypeptide chain component). Other means for conjugation include chemical conjugation, particularly cross-linking using bifunctional REAGENTS (e.g., using DOUBLE-REAGENTS) TMGuidelines for Selection of crosslinking Reagents (Cross-linking Reagents Selection Guide), Pierce).
Thus, the specific binding member and the biologically active agent, therapeutic agent or detectable label may be directly linked to each other, e.g. by any suitable chemical bond or by a linker, e.g. a peptide linker.
The peptide linker may be short (2-20, preferably 2-15 amino acid residue extensions). Suitable examples of peptide linker sequences are known in the art. One or more different linkers may be used. The linker may be about 5 amino acids in length.
For example, the chemical bond may be a covalent bond or an ionic bond. Examples of covalent bonds include peptide bonds (amide bonds) and disulfide bonds. For example, the specific binding member and the bioactive agent, therapeutic agent or detectable label (diagnostic agent) may be covalently linked, for example, by a peptide bond (amide bond). The biologically active agent, therapeutic agent or detectable label may be directly linked to the C-terminus or N-terminus of the specific binding member by a covalent bond, such as a peptide bond (amide bond). In particular embodiments, the bioactive agent, therapeutic agent, or detectable label is directly linked to the N-terminus of the specific binding member via a peptide bond (amide bond). Thus, the specific binding member, as well as the bioactive agent, therapeutic agent or detectable label (diagnostic agent), may be produced (secreted) as a single chain polypeptide.
The invention also provides isolated nucleic acids encoding the antibody molecules of the invention. The skilled person will have no difficulty in preparing such nucleic acids using methods well known in the art. Isolated nucleic acids may be used to express a specific binding member of the invention, for example by expression in a bacterial, yeast, insect or mammalian host cell. Preferred host cells are mammalian cells, such as chinese hamster ovary Cells (CHO), human embryonic kidney cells (HEK), or myeloma cells (NS 0). The nucleic acid will generally be provided in the form of a recombinant vector for expression.
In vitro host cells comprising such nucleic acids and vectors are part of the invention, as are the use of such in vitro host cells for expressing a specific binding member of the invention, which may subsequently be purified from cell culture (culture) and optionally formulated into a pharmaceutical composition. Thus, the invention further provides a method of producing a specific binding member of the invention, the method comprising culturing a recombinant host cell of the invention under conditions for production of the specific binding member. Methods for culturing suitable host cells as described above are well known in the art. The method may further comprise isolating and/or purifying the specific binding member. The method may further comprise formulating the specific binding member into a pharmaceutical composition, optionally together with a pharmaceutically acceptable excipient or other substance as described below.
PD-L1 is known to be expressed on many cancer cells, as well as on cells of the immune system.
Thus, the specific binding members of the present invention may be used in methods of treating cancer in a patient. The patient is preferably a human patient.
The cells of the cancer to be treated with the specific binding members of the invention may, for example, express PD-L1 on their cell surface. In one embodiment, the cells of the cancer to be treated may have been determined to express PD-L1, for example, on their cell surface. Methods for determining antigen expression on the surface of cells are known in the art and include, for example, flow cytometry.
The treatment of various types of cancer with anti-PD-L1 or anti-PD-1 antibodies has been studied in clinical trials and showed satisfactory results. These cancers include solid tumors such as ovarian cancer, prostate cancer, colorectal cancer, fibrosarcoma, renal cell carcinoma, melanoma (advanced metastatic melanoma), pancreatic cancer, breast cancer, glioblastoma multiforme, lung cancer (such as non-small cell lung cancer and small cell lung cancer), head and neck cancer (such as head and neck squamous cell carcinoma), gastric cancer (stomachic cancer) (gastric cancer), bladder cancer, cervical cancer, uterine cancer (endometrial cancer, uterine cervical cancer), vulval cancer, testicular cancer, penile cancer, esophageal cancer, hepatocellular carcinoma, nasopharyngeal carcinoma, merkel cell carcinoma, mesothelioma, DNA mismatch repair deficient colorectal cancer, DNA mismatch repair deficient endometrial cancer, thyroid cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma, follicular lymphoma, indolent non-Hodgkin's lymphoma, melanoma, and melanoma) Mantle cell lymphoma), leukemias (such as chronic lymphocytic leukemia, myeloid leukemia, acute lymphoblastic (lymphoblastic) leukemia, or chronic lymphoblastic leukemia), multiple myeloma, and peripheral T-cell lymphoma. Thus, the specific binding members of the present invention may find application in the treatment of these cancers. The tumors of these cancers are known or predicted to contain immune cells expressing P-L1, such as Tumor Infiltrating Lymphocytes (TILs).
In particular, the use of anti-PD-L1 antibody for the treatment of melanoma, colorectal cancer, breast cancer, bladder cancer, renal cell carcinoma, gastric cancer, head and neck cancer (such as head and neck squamous cell carcinoma), mesothelioma, lung cancer (such as non-small cell lung cancer), ovarian cancer, mercker cell carcinoma, pancreatic cancer, melanoma, and hepatocellular carcinoma has been studied in clinical trials and shown to be satisfactory. Thus, the cancer to be treated using the antibody molecule of the invention may be melanoma, colorectal cancer, breast cancer, bladder cancer, renal cell carcinoma, bladder cancer, gastric cancer, head and neck cancer (such as head and neck squamous cell carcinoma), mesothelioma, lung cancer (such as non-small cell lung cancer), ovarian cancer, mercker cell carcinoma, pancreatic cancer, melanoma, or hepatocellular carcinoma.
Where the application relates to a particular type of cancer, such as breast cancer, this involves malignant transformation of the relevant tissue, in this case breast tissue. Cancers derived from malignant transformation of different tissues, e.g. ovarian tissue, may cause metastatic lesions in other locations of the body, such as the breast, but are not therefore the breast cancer referred to herein, but ovarian cancer.
The cancer may be primary (primary) or secondary (secondary) cancer. Thus, the specific binding members of the present invention may be used in a method of treating cancer in a patient, wherein the cancer is a primary tumor and/or tumor metastasis (tumor metastasis).
It is also contemplated that the specific binding members of the present invention find use in the treatment of infectious diseases, such as viral, bacterial, fungal and/or parasitic infections. Preferably, the infectious disease is a viral, bacterial or fungal disease; more preferably viral or bacterial diseases; viral diseases are most preferred. The infectious disease may be chronic or acute, but is preferably chronic.
Examples of viral diseases that can be treated using specific binding members according to the invention include: human Immunodeficiency Virus (HIV) infection, influenza virus infection, enterovirus infection, Hepatitis B Virus (HBV) infection, Hepatitis C Virus (HCV) infection, Hepatitis A Virus (HAV) infection, Hepatitis D Virus (HDV) infection, and Hepatitis E Virus (HEV) infection, Respiratory Syncytial Virus (RSV) infection, herpes virus infections such as Epstein-barr virus (Epstein-Barrvirus), herpes simplex virus 1(HSV-1), herpes simplex virus 2(HSV-2), Cytomegalovirus (CMV), and papilloma virus infections.
Examples of bacterial diseases that can be treated with the specific binding members of the present invention include: mycobacterium tuberculosis infection, gram-negative bacteria (such as Acinetobacter (Acinetobacter), klebsiella (klebsiella), Enterobacter (Enterobacter)), gram-positive bacteria (gram-positive bacteria) (such as Clostridium difficile (Clostridium difficile), staphylococcus aureus (staphylococcus aureus)), and Listeria (Listeria), for example, Listeria monocytogenes (Listeria).
Examples of fungal diseases that can be treated with the specific binding members of the present invention include: aspergillus (Aspergillus) and Candida (Candidia) infections.
Examples of parasitic diseases that can be treated with the specific binding members of the present invention include: malaria infection, toxoplasma infection, and Leishmania (Leishmania) infection.
Specific binding members of the invention are also expected to find application in inflammation, diseases and disorders associated with inflammation, and inflammatory diseases, such as stroke, stroke-related inflammation, and vascular or vasculitis, e.g., medium/large vessel vasculitis, or vasculitis of the central and/or peripheral nervous system.
The specific binding members of the present invention are designed for use in methods of treatment of patients, preferably human patients. The specific binding member will generally be administered in the form of a pharmaceutical composition which may include at least one component other than the specific binding member, such as a pharmaceutically acceptable excipient. For example, the pharmaceutical composition of the present invention may include, in addition to the active ingredient, a pharmaceutically acceptable excipient, carrier (carrier), buffer, stabilizer, or other material known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be by injection, for example, intravenous or subcutaneous injection. The specific binding member may be administered intravenously or subcutaneously.
Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. Physiological saline solution, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included.
For intravenous injection or injection at the site of affliction, the specific binding member or the pharmaceutical composition comprising the specific binding member is preferably in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art are fully enabled to prepare suitable solutions using, for example, isotonic vehicles (isotonics vehicles) such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed as desired. Many methods of preparing pharmaceutical formulations are known to those skilled in the art. See, for example, Robinoned, Sustainated and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978.
Depending on the condition to be treated, the composition comprising the specific binding member according to the invention may be administered alone or in combination with other treatments, simultaneously with one or more other therapeutic agents, sequentially or as a combined preparation with one or more other therapeutic agents. For example, a specific binding member of the invention may be administered in combination with an existing therapeutic agent for the disease to be treated, e.g., cancer as described above. For example, a specific binding member of the invention may be administered to a patient in combination with a second anti-cancer therapy, such as chemotherapy, anti-tumor vaccination (also known as cancer vaccination), radiation therapy, immunotherapy, oncolytic virus (oncocytic virus), Chimeric Antigen Receptor (CAR) T cell therapy, or hormonal therapy.
It is envisaged that the specific binding members of the invention may be used as adjuvants in anticancer therapy such as chemotherapy, anti-tumour vaccination or radiotherapy. Without wishing to be bound by theory, it is believed that administration of a specific binding member to a patient as part of chemotherapy, anti-tumor vaccination, or radiation therapy will trigger a greater immune response against the cancer-associated antigen PD-L1 than would be achieved with chemotherapy, anti-tumor vaccination, or radiation therapy alone.
Accordingly, a method of treating cancer in a patient may comprise: a therapeutically effective amount of a specific binding member according to the invention is administered to a patient in combination with a chemotherapeutic agent, an anti-tumor vaccine, a radionuclide, an immunotherapeutic agent, an oncolytic virus, CAR-T cells or an agent for hormone therapy. The chemotherapeutic, anti-tumor vaccine, radionuclide, immunotherapeutic agent, oncolytic virus, CAR-T cell or agent for hormone therapy is preferably a chemotherapeutic, anti-tumor vaccine, radionuclide, immunotherapeutic agent, oncolytic virus, CAR-T cell or agent for hormone therapy for the cancer in question, i.e. a chemotherapeutic, anti-tumor vaccine, radionuclide, immunotherapeutic agent, oncolytic virus, CAR-T cell or agent for hormone therapy that has shown efficacy in treating the cancer in question. The choice of suitable chemotherapeutic agents, anti-tumor vaccines, radionuclides, immunotherapeutics, oncolytic viruses, CAR-T cells, or agents for hormone therapy that have been shown to be effective against the cancer in question is well within the capabilities of the skilled physician.
For example, where the method comprises administering to a patient a therapeutically effective amount of a specific binding member according to the invention in combination with a chemotherapeutic agent, the chemotherapeutic agent may be selected from the group consisting of: taxanes (taxanes), cytotoxic antibiotics, tyrosine kinase inhibitors, poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors, B _ RAF enzyme inhibitors, alkylating agents (alkylating agents), platinum analogs, nucleoside analogs, thalidomide derivatives, antineoplastic chemotherapeutic agents, and others. Taxanes include docetaxel, paclitaxel, and albumin-bound paclitaxel; cytotoxic antibiotics include actinomycin (actinomycin), bleomycin (bleomycin), anthracyclines (anthracyclines), doxorubicin (doxorubicin) and valrubicin (valrubicin); tyrosine kinase inhibitors include sunitinib (sunitinib), erlotinib (erlotinib), gefitinib (gefitinib), axitinib (axitinib), PLX3397, imatinib (imatinib), cobitinib (cobeminib), and trametinib (trametinib); PARP inhibitors include piraparib (piraparib); B-Raf enzyme inhibitors include Vemurafenib (vemurafenib) and dabrafenib (dabrafenib); alkylating agents include dacarbazine (dacarbazine), cyclophosphamide (cyclophosphamide), temozolomide (temozolomide); platinum analogs include platinum (carboplatin), cisplatin (cissplatin) and oxaliplatin (oxaliplatin); nucleoside analogs include gemcitabine (gemcitabine) and azacitidine (azacitidine); antineoplastic agents include fludarabine (fludarabine). Other chemotherapeutic agents suitable for use in the present invention include methotrexate (methotrexate), denafinil (defactinib), entinostat (enterostat), pemetrexed (pemetrexed), capecitabine (capecitabine), eribulin (eribulin), irinotecan (irinotecan), fluorouracil (fluorouracil) and vinblastine (vinblastine).
Vaccination strategies for the treatment of cancer have been implemented clinically and are discussed in detail in the scientific literature (such as Rosenberg, s.2000). This is primarily directed to strategies to suggest that the immune system responds to various cellular markers expressed by autologous or heterologous cancer cells, whether or not granulocyte-macrophage colony stimulating factor (GM-CSF) is used, by using these cells as a method of immunization. GM-CSF elicits a strong response in antigen presentation and works particularly well when employed with the strategy described.
Where the method comprises administering to a patient a therapeutically effective amount of a specific binding member according to the invention in combination with an immunotherapeutic agent, the immunotherapeutic agent may be selected from the group consisting of: antibodies that bind to checkpoint inhibitors, co-stimulatory molecules or soluble factors, such as antibodies that bind to CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, CD73, CSF-1R, KIR, OX40, CD40, HVEM, TGFB, IL-10, CSF-1. Alternatively, the immunotherapeutic agent may be one or more cytokines or cytokine-based therapies selected from the group consisting of: IL-2; prodrugs conjugated with IL2, GM-CSF, IL-7, IL-12, IL-9, IL-15, IL-18, IL-21; and type I interferon (type I interferon).
Administration may be in a "therapeutically effective amount," which is an amount sufficient to show benefit to the patient. Such benefit can be at least an improvement in at least one symptom. Thus, "treatment" of a particular disease refers to the amelioration of at least one symptom. The actual amount administered, the rate and timing of administration (time-course) will depend on the nature and severity of the disease being treated, the particular patient being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the type of specific binding member, the method of administration, the regimen of administration, and other factors known to the physician. Prescription of treatment, e.g., determination of dosage, etc., is within the responsibility of general practitioners and other physicians, and may depend on the severity of the symptoms and/or progression of the disease being treated. Appropriate dosages of specific binding members are well known in the art (Ledermann et al, 1991; and Bagshawe et al, 1991). Specific dosages as indicated herein, or as indicated in the Physician's manual (2003), may be used, as appropriate for the specific binding member to be administered. A therapeutically effective amount or suitable dose of a specific binding member may be determined by comparing its in vitro and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other test animals to humans are known. The exact dosage will depend on many factors, including the size and location of the area to be treated, and the exact nature of the specific binding member. Treatment may be repeated at daily, twice weekly, weekly or monthly intervals, at the discretion of the physician. Treatment may be given before and/or after surgery and may be applied or applied directly at the anatomical site of the surgical treatment.
Further aspects and embodiments of the invention will be apparent to those skilled in the art in view of this disclosure, including the following experimental illustrations.
As used herein, "and/or" is considered a specific disclosure of each of two specified features or components, with or without the other. For example, "a and/or B" is considered a specific disclosure of each of (i) a, (ii) B, and (iii) a and B, as if each were set forth separately herein.
Unless the context indicates otherwise, the description and definition of the features described above is not limited to any particular aspect or embodiment of the invention, and applies equally to all aspects and embodiments described.
Certain aspects and embodiments of the present invention will now be illustrated by way of example and with reference to the drawings described above.
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