阅读说明：本技术 高效表达的egfr和pd-l1双特异性结合蛋白 (High-expression EGFR and PD-L1 bispecific binding protein ) 是由 吴辰冰 宫世勇 于 2019-07-08 设计创作，主要内容包括：本文公开了同时结合EGFR和PD-L1的双特异性Fabs-In-Tandem免疫球蛋白(FIT-Ig)结合蛋白。这样的双特异性EGFR/PD-L1 FIT-Ig结合蛋白被有效表达并且可用于阻断EGFR信号传导、阻断PD-L1信号传导以及治疗癌症。(Bispecific Fabs-In-Tandem immunoglobulin (FIT-Ig) binding proteins that bind EGFR and PD-L1 simultaneously are disclosed herein. Such bispecific EGFR/PD-L1FIT-Ig binding proteins are efficiently expressed and are useful for blocking EGFR signaling, blocking PD-L1 signaling, and treating cancer.)

1. A Fabs-In-Tandem immunoglobulin (FIT-Ig) binding protein that binds EGFR and PD-L1 and comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein:

the first polypeptide chain comprises, from amino-terminus to carboxy-terminus, a VL_EGFR-CL-VH_PD-L1-CH1-Fc, wherein VL_EGFRIs an antibody light chain variable domain of a first parent antibody that binds EGFR, CL is an antibody light chain constant domain, VH_PD-L1Is an antibody heavy chain variable domain of a second parent antibody that binds PD-L1, CH1 is a first constant region of an antibody heavy chain, and Fc is an antibody Fc region; wherein CL is directly fused to VH_PD-L1Wherein no artificial linker is interposed between the variable domain and the constant domain, and wherein:

VL_EGFRcomprises the amino acid sequence of SEQ ID NO: 1 of the amino acid residues 1 to 107,

VH_PD-L1comprises the amino acid sequence of SEQ ID NO: 1 amino acid residue 215-331;

the second polypeptide chain comprises, from amino-terminus to carboxy-terminus, a VH_EGFR-CH1, wherein VH_EGFRIs an antibody heavy chain variable domain of said first parent antibody that binds EGFR, wherein CH1 is the first constant domain of an antibody heavy chain, wherein in VH_EGFRAnd CH1, and wherein:

VL_EGFRcomprises the amino acid sequence of SEQ ID NO: 2, 1-119 of amino acid residues of (2),

the third polypeptide chain comprises, from amino-terminus to carboxy-terminus, a VL_PD-L1-CL wherein VL_PD-L1The light chain of said second parent antibody that binds PD-L1 mayA variable domain, wherein CL is an antibody light chain constant domain, wherein at VL_PD-L1And CL with no manual joint interposed therebetween, and wherein:

VL_PD-L1comprises the amino acid sequence of SEQ ID NO: 3, amino acid residues 1-107.

2. The FIT-Ig binding protein of claim 1, wherein said binding protein is a six polypeptide chain binding protein comprising two of said first polypeptide chain, two of said second polypeptide chain, and two of said third polypeptide chain, wherein said polypeptide chains are joined to form four Fab binding units, wherein two Fab binding units bind EGFR and two Fab binding units bind PD-L1.

3. The FIT-Ig-binding protein of claim 1 or claim 2, wherein the antibody CL domain in the first and third polypeptide chains is derived from a human IgG1 antibody.

4. The FIT-Ig-binding protein of claim 1 or claim 2, wherein the antibody CL domain in the first and third polypeptide chains comprises SEQ ID NO: 1 amino acid residue 108-214.

5. The FIT-Ig binding protein of claim 1 or claim 2, wherein the antibody CH1 domain present in the first and second polypeptide chains is derived from a human IgG1 antibody.

6. The FIT-Ig-binding protein according to claim 1 or claim 2, wherein the antibody CH1 domain present in the first and second polypeptide chains comprises SEQ ID NO: 1, amino acid residue 332-434.

7. The FIT-Ig-binding protein of claim 1 or claim 2, wherein the antibody Fc present in the first polypeptide chain is derived from a human IgG1 antibody.

8. The FIT-Ig-binding protein of claim 1 or claim 2, wherein the antibody Fc present in the first polypeptide chain comprises SEQ ID NO: 1 amino acid residue 435-661.

9. The FIT-Ig binding protein of claim 1 or claim 2, wherein:

said first polypeptide chain comprises a sequence according to SEQ ID NO: 1;

said second polypeptide chain comprises a sequence according to SEQ ID NO: 2; and

said third polypeptide chain comprises a sequence according to SEQ ID NO: 3.

10. The EGFR/PD-L1FIT-Ig binding protein of claim 1, wherein the FIT-Ig binding protein binds EGFR and PD-L1 simultaneously.

11. The EGFR/PD-L1FIT-Ig binding protein of claim 1, wherein the FIT-Ig binding protein has a human glycosylation pattern.

12. A composition comprising the FIT-Ig-binding protein according to claim 1, wherein the composition comprises less than or equal to 0.1% FIT-Ig-binding protein aggregates.

13. A pharmaceutical composition comprising the EGFR/PD-L1FIT-Ig binding protein of claim 1 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, further comprising one or more additional therapeutically active compounds.

15. The pharmaceutical composition according to claim 14, wherein the one or more additional therapeutically active compounds are selected from the group consisting of: cytotoxic metal-containing anticancer compounds, cytotoxic radioisotope-based anticancer compounds, antibiotics, antiviral compounds, sedatives, stimulants, local anesthetics, anti-inflammatory steroids, analgesics, antihistamines, non-steroidal anti-inflammatory drugs, and combinations thereof.

16. The pharmaceutical composition of claim 15, wherein the anti-inflammatory steroid is a natural anti-inflammatory steroid, a synthetic anti-inflammatory steroid, or a combination thereof.

17. The pharmaceutical composition of claim 15, wherein the analgesic is selected from the group consisting of: acetylsalicylic acid, acetaminophen, naproxen, ibuprofen, COX-2 inhibitors, morphine, oxycodone, and combinations thereof.

18. The pharmaceutical composition of claim 15, wherein the non-steroidal anti-inflammatory drug is selected from the group consisting of: acetylsalicylic acid, ibuprofen, naproxen, COX-2 inhibitors, and combinations thereof.

19. A composition for releasing crystallized EGFR/PD-L1FIT-Ig binding protein, comprising crystallized EGFR/PD-L1FIT-Ig binding protein, an excipient component, and at least one polymeric carrier.

20. The composition of claim 19, wherein the excipient ingredient is selected from the group consisting of: albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-beta-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

21. The composition of claim 19, wherein the polymeric carrier is a polymer selected from one or more of the following: poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutyrate), poly (caprolactone), poly (dioxanone); polyethylene glycol, poly (hydroxypropyl) methacrylamide, poly [ (organo) phosphazene ], poly (orthoester), polyvinyl alcohol, poly (vinyl pyrrolidone), maleic anhydride/alkyl vinyl ether copolymers, pluronic polyols, albumin, alginates, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosaminoglycans, sulfated polysaccharides, blends thereof, and copolymers thereof.

22. An isolated nucleic acid molecule encoding one or more of:

comprising a sequence according to SEQ ID NO: 1;

comprising a sequence according to SEQ ID NO: 2;

comprising a sequence according to SEQ ID NO: 3.

23. A vector comprising one or more isolated nucleic acid molecules according to claim 22.

24. The vector of claim 23, wherein the vector is an expression vector and the one or more isolated nucleic acids are operably linked to transcription and translation sequences that allow expression of the encoded one or more polypeptide chains.

25. The vector of claim 24 selected from the group consisting of pcDNA, pcdna3.1, pTT3, pEFBOS, pBV, pJV, pcdna3.1 TOPO, pEF6 TOPO, and pBJ.

26. An isolated host cell comprising one or more vectors according to claim 24.

27. An isolated host cell comprising one or more expression vectors, wherein the one or more vectors encode three polypeptide chains that form the EGFR/PD-L1FIT-Ig binding protein of claim 1.

28. The isolated host cell of claim 27, wherein the host cell is an isolated prokaryotic host cell.

29. The isolated host cell of claim 27, wherein the host cell is an isolated eukaryotic host cell.

30. The isolated eukaryotic host cell of claim 29, wherein the isolated eukaryotic host cell is an isolated mammalian host cell.

31. The isolated mammalian host cell according to claim 30, wherein the mammalian host cell is selected from the group consisting of: chinese Hamster Ovary (CHO) cells, COS cells, Vero cells, SP2/0 cells, NS/0 myeloma cells, human embryonic kidney (HEK293) cells, mouse kidney (BHK) cells, HeLa cells, human B cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells, and MDCK cells.

32. A method of producing EGFR/PD-L1FIT-Ig binding protein, comprising culturing the isolated mammalian host cell of claim 30 under conditions sufficient for production of EGFR/PD-L1FIT-Ig binding protein.

33. The method according to claim 32, wherein the mammalian host cell is a HEK293 cell.

34. The method of claim 33, wherein the FIT-Ig binding protein is expressed at a level greater than 10 mg/L.

35. An EGFR/PD-L1FIT-Ig binding protein produced according to the method of claim 32.

36. A method of inhibiting EGFR signaling in a cell, the method comprising contacting a cell expressing EGFR with the EGFR/PD-L1FIT-Ig binding protein of claim 1.

37. A method of inhibiting PD-L1 signaling in a cell, the method comprising contacting a cell expressing PD-L1 with the EGFR/PD-L1FIT-Ig binding protein of claim 1.

38. A method of treating cancer in a human subject, the method comprising administering to the subject the EGFR/PD-L1FIT-Ig binding protein of claim 1.

39. The method of claim 38, wherein the cancer is an epithelial cancer.

40. The method of claim 38, wherein the cancer is selected from the group consisting of: melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, and lymphoma.

41. The method of claim 40, wherein the melanoma is metastatic malignant melanoma.

42. The method of claim 40, wherein the renal cancer is clear cell renal cell carcinoma.

43. The method of claim 40, wherein the prostate cancer is hormone refractory prostate adenocarcinoma.

44. The method of claim 40, wherein the lung cancer is non-small cell lung cancer.

45. Use of the EGFR/PD-L1FIT-Ig binding protein of claim 1 in the manufacture of a medicament for the treatment of cancer.

46. The use of claim 45, wherein the cancer is an epithelial cancer.

47. The use of claim 45, wherein the cancer is selected from the group consisting of: melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, and lymphoma.

48. The use of claim 47, wherein the melanoma is metastatic malignant melanoma.

49. The use of claim 47, wherein the renal cancer is clear cell renal cell carcinoma.

50. The use of claim 47, wherein the prostate cancer is hormone refractory prostate adenocarcinoma.

51. The method of claim 47, wherein the lung cancer is non-small cell lung cancer.

Technical Field

The present invention relates to novel engineered bispecific binding proteins that recognize Epidermal Growth Factor Receptor (EGFR) and programmed death ligand 1 (PD-L1). The bispecific binding protein can be used for treating cancer.

Background

Programmed death ligand 1(PD-L1) is a type I transmembrane glycoprotein of approximately 40 kilodaltons (kD) in size. In humans, PD-L1 is expressed on a variety of immune cell types, including activated and inactive/depleted T cells, naive and activated B cells, myeloid Dendritic Cells (DCs), monocytes, mast cells and other Antigen Presenting Cells (APCs). It is also expressed on non-immune cells, including pancreatic islets of langerhans, Kupffer cells of the liver, vascular endothelium and selected epithelia, such as airway epithelium and renal tubule epithelium, the expression of which is enhanced during the onset of inflammation. PD-L1 is also present at elevated levels in a number of malignancies, including but not limited to breast, colon, colorectal, lung, kidney (including renal cell carcinoma), gastric, bladder, non-small cell lung (NSCLC), Hepatocellular (HCC), pancreatic and melanoma. It has also been shown that by stimulating IFN-gamma, the expression of PD-L1 on the cell surface can be upregulated.

PD-L1(CD274, B7-H1) binds to programmed cell death protein 1(PD-1, CD279), which is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. PD-1 is typically expressed in immune cells, such as T cells, B cells, monocytes, and Natural Killer (NK) cells. Both PD-L1 and PD-L2(CD273, B7-DC) are cell surface glycoprotein ligands of PD-1. Binding of PD-1 to PD-L1 or PD-L2 initiates signals that inhibit T cell activation and cytokine secretion. This down-regulation of T cell activation in turn leads to reduced T cell proliferation, IL-2 secretion, IFN- γ secretion, and secretion of other growth factors and cytokines. Freeman et al, J.Exp.Med.,192:1027-1034 (2000); latchman et al, nat. Immunol.,2:261-8 (2001); carter et al, Eur.J.Immunol.,32:634-43 (2002); ohigashi et al, Clin. cancer Res.,11:2947-53 (2005). It is believed that signaling through the PD-1/PD-L1 interaction plays a critical, non-redundant role within the immune system through negative regulation of T cell responses. This regulation is involved in the development of T cells in the thymus, regulation of chronic inflammatory responses, and maintenance of peripheral tolerance and immune privilege. The key properties of these functions are exemplified in PD-1 deficient mice, which exhibit an autoimmune phenotype. PD-1 deficiency in C57BL/6 mice results in chronic progressive lupus-like glomerulonephritis and arthritis. In Balb/c mice, PD-1 deficiency leads to severe cardiomyopathy due to the presence of heart tissue-specific autoreactive antibodies.

PD-L1 has been proposed to play a role in tumor immunity by increasing apoptosis of antigen-specific T cell clones. Dong et al, nat. Med.,8:793-800 (2002). In addition, it has been shown that PD-L1 may be involved in intestinal mucositis, inhibiting PD-L1 from inhibiting wasting diseases associated with colitis. Kanai et al, J.Immunol.,171:4156-63 (2003). In general, inhibition of PD-L1 signaling has been proposed as a means of enhancing T cell immunity for the treatment of cancer (e.g., tumor immunity) and infections, including acute and chronic (e.g., persistent) infections.

Since PD-L1, PD-L2, and PD-1 are involved in down-regulating immune responses, including suppressing anti-tumor immune responses, they are referred to as "immune checkpoint" proteins. Pardol, nat. Rev. cancer,12:252-264 (2012). Clinical studies using immune checkpoint inhibitors (e.g., antibodies targeting PD-1, PD-L1, or CTLA-4) have achieved promising results, however, it has been observed that only a fraction of patients initially respond to current inhibitors, and increasing clinical evidence suggests that a substantial proportion of initial responders eventually relapse, becoming fatal drug-resistant disease after months or years. Syn et al, The Lancet Oncology,18(12) e 731-e 741 (2017).

Epidermal Growth Factor Receptor (EGFR) is a transmembrane glycoprotein and is a member of the ErbB superfamily of receptor tyrosine kinases. EGFR has been shown to play an important role in a complex signaling cascade that promotes the development, survival and metastasis of epithelial cancers. EGFR signaling is triggered when EGFR binds to its cognate ligand, Epidermal Growth Factor (EGF), and then forms a dimer with another EGFR (homodimerization) or another receptor tyrosine kinase (heterodimerization). Thereafter, dimers are internalized to degrade or accumulate in the nucleus where EGFR can regulate transcription of multiple genes involved in cancer transformation. Therefore, EGFR has been considered as an attractive therapeutic target for anti-tumor therapy. Approved EGFR-targeted anti-cancer therapies include the monoclonal antibodies cetuximab (a human murine chimeric anti-EGFR monoclonal antibody) and panitumumab (a human anti-EGFR monoclonal antibody). Many small molecule tyrosine kinase inhibitors that inhibit EGFR and other tyrosine kinase receptors have been approved for use as anti-cancer therapies, including gefitinib, erlotinib, lapatinib, and canertinib. These approved drugs have been used alone or in various combinations for the treatment of various cancers. For an overview of targeting EGFR in anti-cancer therapy, see seshacharyuu et al, Expert opin.

PD-L1 and EGFR are involved in the regulation of different signaling pathways, both of which are known to contribute to the initiation, growth, maintenance and spread of cancer cells in humans. However, in certain cancer cells, activation of EGFR has been shown to up-regulate PD-L1 expression, indicating that there is some degree of "cross-talk" between these two pathways (Chen et al, j.thorac. oncol.,10(6): 910-.

Disclosure of Invention

The present invention meets the above needs by providing an engineered bispecific protein that binds EGFR and PD-L1. In particular, the invention provides bispecific, multivalent binding proteins that bind human EGFR and human PD-L1. Preferred bispecific binding proteins of the invention are "Fabs-In-Tandem immunoglobulin" (FIT-Ig) binding proteins that bind EGFR and PD-L1. As shown herein, this "EGFR/PD-L1" FIT-Ig binding protein according to the present invention is produced in mammalian cell culture in significantly high yield and does not exhibit significant aggregate formation. Low productivity and significant aggregate formation are issues that preclude preclinical and clinical-stage assessments of previously prepared FIT-Ig-binding proteins against EGFR and PD-L1, which are necessary to determine whether such binding proteins are useful as therapeutic anticancer drugs.

In one embodiment, the invention provides an EGFR/PD-L1FIT-Ig binding protein that binds EGFR and PD-L1, comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein

The first polypeptide chain ("heavy chain") comprises, from amino-terminus to carboxy-terminus, a VL_EGFR-CL-VH_PD-L1-CH1-Fc, wherein VL_EGFRIs an antibody light chain variable domain of a first parent antibody that binds EGFR, CL is an antibody light chain constant domain, VH_PD-L1Is an antibody heavy chain variable domain of a second parent antibody that binds PD-L1, CH1 is the first constant region of the antibody heavy chain, and Fc is the antibody Fc region (comprising the hinge-CH 2-CH 3); wherein CL is directly fused to VH_PD-L1Wherein no artificial linker is interposed between the variable domain and the constant domain, and wherein:

VL_EGFRcomprises the amino acid sequence of SEQ ID NO: 1 of the amino acid residues 1 to 107,

VH_PD-L1comprises the amino acid sequence of SEQ ID NO: 1 amino acid residue 215-331;

the second polypeptide chain ("first light chain") comprises, from amino-terminus to carboxy-terminus, a VH_EGFR-CH1, wherein VH_EGFRIs an antibody heavy chain variable domain of said first parent antibody that binds EGFR, wherein CH1 is the constant domain of the first antibody heavy chain, wherein in VH_EGFRAnd CH1, and wherein:

VH_EGFRcomprises the amino acid sequence of SEQ ID NO: 2, amino acid residues 1-119;

a third polypeptide chain ("second light chain") comprising, from amino-terminus to carboxy-terminus, a VL_PD-L1-CL wherein VL_PD-L1Is a light chain variable domain of said second parent antibody that binds PD-L1, wherein CL is an antibody light chain constant domain, wherein at VL_PD-L1And CL with no manual joint interposed therebetween, and wherein:

VL_PD-L1comprises the amino acid sequence of SEQ ID NO: 3, amino acid residues 1-107.

In a preferred embodiment, the above EGFR/PD-L1FIT-Ig binding protein is a six polypeptide chain FIT-Ig binding protein comprising two of the above first polypeptide chains, two of the above second polypeptide chains, and two of the above third polypeptide chains, wherein the polypeptide chains bind to form four Fab binding units, wherein two Fab binding units bind to EGFR and two Fab binding units bind to PD-L1.

Preferably, the CL domain present in one or more polypeptide chains (e.g., the first and third polypeptide chains described above) of the FIT-Ig binding protein of the present invention is a human CL kappa domain (hC κ). Preferably, the CL domain of the FIT-Ig binding protein of the invention is derived from the human IgG1(hIgG1) antibody. The preferred hIgG1 CL kappa domain present in one or more polypeptide chains of the EGFR/PD-L1FIT-Ig binding protein of the present invention comprises the amino acid sequence of SEQ ID NO: 1 amino acid residue 108-214.

Preferably, the CH1 domain present in one or more polypeptide chains of the FIT-Ig binding protein of the invention (e.g., in the first and second polypeptide chains described above) is derived from a human IgG1 antibody. A preferred hIgG1 CH1 domain present in one or more polypeptide chains of the EGFR/PD-L1FIT-Ig binding protein of the present invention comprises SEQ ID NO: 1, amino acid residue 332-434.

Preferably, the Fc present in the polypeptide chain of the first polypeptide chain (or "heavy chain") of the FIT-Ig binding protein of the present invention comprises an antibody Fc region comprising the hinge-CH 2-CH3 domain. Preferably, the Fc is derived from a human IgG1 antibody. The preferred hIgG1 Fc region present in the first polypeptide chain of the EGFR/PD-L1FIT-Ig binding protein of this invention comprises the amino acid sequence of SEQ ID NO: 1 amino acid residue 435-661.

The invention also provides an EGFR/PD-L1FIT-Ig binding protein that binds EGFR and PD-L1, comprising:

a first polypeptide chain comprising a sequence according to SEQ ID NO: 1;

a second polypeptide chain comprising a sequence according to SEQ ID NO: 2; and

a third polypeptide chain comprising a sequence according to SEQ ID NO: 3;

wherein EGFR/PD-L1FIT-Ig comprises a Fab binding unit of EGFR and a Fab binding unit of PD-L1.

In a preferred embodiment, the EGFR/PD-L1FIT-Ig binding protein described above is a hexapolypeptide chain FIT-Ig binding protein comprising two polypeptide chains comprising a sequence according to SEQ ID NO: 1, two polypeptide chains comprising a sequence of amino acid residues according to SEQ ID NO: 2, and two polypeptide chains comprising a sequence of amino acid residues according to SEQ ID NO: 3, wherein the polypeptide chains bind to form four Fab binding units, two of which bind to EGFR and two of which bind to PD-L1.

Preferably, the EGFR/PD-L1FIT-Ig binding protein described herein binds both EGFR and PD-L1. In another embodiment, the EGFR/PD-L1FIT-Ig binding protein of the invention binds to two EGFR proteins and two PD-L1 proteins. In a more preferred embodiment, the EGFR/PD-L1FIT-Ig binding protein described herein binds both EGFR protein and both PD-L1 protein simultaneously.

In one embodiment, the EGFR/PD-L1FIT-Ig binding protein according to the present invention binds EGFR and PD-L1, wherein the affinity for EGFR and PD-L1 is substantially the same (i.e., the same or within 30%) as the affinity of each parent antibody from which each EGFR and PD-L1 antigen binding site of the FIT-Ig binding protein is derived, to EGFR and PD-L1.

In one embodiment, the EGFR/PD-L1FIT-Ig binding protein of the present invention binds EGFR and has an association rate constant (k) for human EGFR_on) Is at least 1 × 10⁵M^-1s^-1More preferably at least 2X 10⁵M^-1s^-1As determined by bio-layer interferometry. In a further embodiment, the EGFR/PD according to the inventionK of-L1 FIT-Ig binding protein to human EGFR_onK to human EGFR over parent antibody_onApproximately 40% lower, the anti-EGFR specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody.

In one embodiment, the EGFR/PD-L1FIT-Ig binding protein of the invention binds human EGFR and has an off-rate constant (k) for human EGFR_off) Less than 1.1X 10^-4sec^-1As determined by bio-layer interferometry. In a further embodiment, the k of the EGFR/PD-L1FIT-Ig binding protein of the present invention to human EGFR_offK to human EGFR over parent antibody_offAbout 50% lower, the anti-EGFR specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody.

In one embodiment, the EGFR/PD-L1FIT-Ig binding protein of the present invention binds human EGFR and has a dissociation constant (KD) for human EGFR of less than 1X 10^-9M, preferably less than 7X 10^-10M, more preferably less than 6X 10^-10M, and still more preferably less than or equal to 5X 10^-10M, as determined by biolayer interferometry. In a further embodiment, the K of the EGFR/PD-L1FIT-Ig binding protein of the present invention to human EGFR_DK to human EGFR with parent antibodies_DSubstantially identical (i.e., identical or within 25%), the anti-EGFR specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody.

In one embodiment, the EGFR/PD-L1FIT-Ig binding protein of the present invention binds to PD-L1 and has an association rate constant (k) to human PD-L1_on) Is at least 5 x 10⁵M^-1s^-1More preferably at least 7X 10⁵M^-1s^-1Even more preferably at least 8X 10⁵M^-1s^-1As determined by bio-layer interferometry. In a further embodiment, the EGFR/PD-L1FIT-Ig binding protein according to the invention is k to human PD-L1_onK to parent antibody to human PD-L1_onThe anti-PD-L1 specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody, which is the same or approximately within 90% thereof.

In one embodiment of the process of the present invention,the EGFR/PD-L1FIT-Ig binding protein of the present invention binds to human PD-L1 and has an off-rate constant (k) for human PD-L1_off) Less than 2 x 10^-2sec^-1More preferably less than 1.5X 10^-2sec^-1As determined by bio-layer interferometry. In a further embodiment, the EGFR/PD-L1FIT-Ig binding protein of the present invention is k to human PD-L1_offK to human PD-L1 over the parent antibody_offAbout 20% higher, the anti-PD-L1 specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody.

In one embodiment, the EGFR/PD-L1FIT-Ig binding protein of the present invention binds to human PD-L1 and has a dissociation constant (k) for PD-L1_D) Less than 2 x 10^-8M, more preferably less than 1.7X 10^-8M, as determined by biolayer interferometry. In a further embodiment, the K of the EGFR/PD-L1FIT-Ig binding protein of the present invention to PD-L1_DK to PD-L1 of parent antibody_DSubstantially identical (i.e., identical or within 30%), the anti-PD-L1 specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody.

In one embodiment, the EGFR/PD-L1FIT-Ig binding protein according to the present invention is expressed at a level greater than 10mg/L in mammalian cell culture.

Fully assembled hexapolypeptide chain EGFR/PD-L1FIT-Ig binding protein "monomers" can be purified from cell culture media using protein a affinity chromatography. Potential aggregates of a solution or suspension of EGFR/PD-L1FIT-Ig binding protein that has been purified using protein a affinity chromatography can be further analyzed using Size Exclusion Chromatography (SEC), where the protein aggregates are detected as molecular species having a molecular weight greater than that of the six-chain EGFR/PD-L1FIT-Ig binding protein monomer, which has a molecular weight of about 240,000 daltons. In one embodiment, the invention provides a composition (e.g., solution or suspension) comprising an EGFR/PD-L1FIT-Ig binding protein described herein that has been purified using protein a affinity chromatography (preferably, a chromatography column) and has less than or equal to 0.1% (≦ 0.1%) FIT-Ig protein aggregates.

In another embodiment, the EGFR/PD-L1FIT-Ig binding protein described herein is glycosylated. Preferably, the glycosylation is a human glycosylation pattern.

In one embodiment, the EGFR/PD-L1FIT-Ig binding protein described herein inhibits or blocks EGFR signaling or PD-L1 signaling. Preferably, the EGFR/PD-L1FIT-Ig binding protein of the present invention inhibits or blocks EGFR signaling and PD-L1 signaling.

In one embodiment, the EGFR/PD-L1FIT-Ig binding protein described herein inhibits the growth or survival of cancer cells.

The present invention also provides one or more isolated nucleic acids encoding one or more polypeptide chains of the EGFR/PD-L1FIT-Ig binding protein described above.

In a preferred embodiment, the isolated nucleic acid molecule of the invention encodes a first polypeptide chain (heavy chain), a second polypeptide chain (first light chain), or a third polypeptide chain (second light chain) of an EGFR/PD-L1FIT-Ig binding protein, wherein:

the first polypeptide chain (heavy chain) comprises a sequence according to SEQ ID NO: 1;

the second polypeptide chain (first light chain) comprises a sequence according to SEQ ID NO: 2; and

the third polypeptide chain (second light chain) comprises a sequence according to SEQ ID NO: 3.

In one embodiment, the present invention provides an expression vector comprising one or more of the isolated nucleic acid molecules described above encoding one or more polypeptide chains of the EGFR/PD-L1FIT-Ig binding protein, wherein the one or more isolated nucleic acid molecules are operably linked to suitable transcriptional and/or translational sequences required for expression of the one or more encoded polypeptide chains of the EGFR/PD-L1FIT-Ig binding protein in a host cell compatible with the expression vector. Preferably, a single expression vector comprises a single nucleic acid encoding only one of the three component polypeptide chains of the FIT-Ig binding protein described herein, such that three separate expression vectors (each encoding and expressing only one of the three component polypeptides) must be present in a host cell to produce the FIT-Ig binding protein described herein.

Preferred vectors for cloning and expressing Nucleic Acids described herein include, but are not limited to, pcDNA, pcDNA3.1, pTT (Durocher et al Nucleic Acids Res.,30(2e9):1-9(2002)), pTT3 (pTT with other multiple cloning sites), pEFBOS (Mizushima and Nagata, Nucleic Acids Res.,18(17):5322(1990)), pBV, pJV, pcDNA3.1 TOPO, pEF6 TOPO, and pBJ.

The vector of the present invention may be an autonomously replicating vector or may be a vector which is incorporated into the genome of a host cell.

In another embodiment, the present invention provides an isolated host cell comprising one or more of the vectors described above. Such an isolated host cell may be an isolated prokaryotic cell or an isolated eukaryotic cell.

In one embodiment of the invention, the isolated prokaryotic host cell comprising one or more of the vectors described herein is a bacterial host cell. The bacterial host cell may be a gram-positive, gram-negative or gram-variable bacterial cell. Preferably, the bacterial host cell comprising one or more of the vectors described herein is a gram-negative bacterium. Even more preferably, the bacterial host cell comprising one or more of the vectors described herein is an escherichia coli cell.

In one embodiment of the invention, the isolated host cell comprising one or more of the vectors described herein is a eukaryotic host cell. Examples of isolated eukaryotic host cells that may comprise one or more vectors described herein include, but are not limited to, mammalian host cells, insect host cells, plant host cells, fungal host cells, eukaryotic algal host cells, nematode host cells, protozoan host cells, and fish host cells.

An isolated fungal host cell that may comprise one or more of the vectors described herein is selected from the group consisting of: aspergillus (Aspergillus), Neurospora (Neurospora), Saccharomyces cerevisiae (Saccharomyces), Pichia pastoris (Pichia), Hansenula (Hansenula), Schizosaccharomyces (Schizosaccharomyces), Kluyveromyces (Kluyveromyces), Yarrowia (Yarrowia), and Candida (Candida). Preferred fungal host cells are Saccharomyces cerevisiae host cells. More preferably, the s.cerevisiae host cell is a s.cerevisiae (Saccharomyces cerevisiae) cell. The insect cell used as a host cell according to the present invention is an insect Sf9 cell.

In a preferred embodiment, the host cell according to the invention is an isolated mammalian host cell comprising one or more expression vectors as described herein, wherein said mammalian host cell expresses three polypeptide chains encoded on the one or more expression vectors, and wherein the polypeptide chains bind to form a FIT-Ig binding protein comprising two Fab binding units binding to EGFR and two Fab binding units binding to PD-L1. Particularly preferred are mammalian host cells selected from the group consisting of: chinese Hamster Ovary (CHO) cells, COS cells, Vero cells, SP2/0 cells, NS/0 myeloma cells, human embryonic kidney (HEK293) cells, mouse kidney (BHK) cells, HeLa cells, human B cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells, and MDCK cells.

More preferably, the isolated mammalian host cell according to the present invention comprises three expression vectors, wherein each expression vector encodes and expresses one of the three component polypeptide chains of the FIT-Ig-binding protein described herein, and wherein the three expressed polypeptide chains are joined to form a FIT-Ig-binding protein comprising two Fab binding units that bind EGFR and two Fab binding units that bind PD-L1.

The invention also provides a method of producing the EGFR/PD-L1FIT-Ig binding protein described herein, the method comprising culturing an isolated host cell comprising one or more expression vectors described herein under conditions sufficient for production of the EGFR/PD-L1FIT-Ig binding protein.

Another aspect of the invention is an EGFR/PD-L1FIT-Ig binding protein produced by a method comprising culturing an isolated host cell comprising one or more expression vectors described herein under conditions sufficient for the production of the EGFR/PD-L1FIT-Ig binding protein.

The EGFR/PD-L1FIT-Ig binding protein described herein may be conjugated with another compound, e.g., in a manner similar to other conjugated antibodies, along or at one or both strands first (heavy)) The carboxy-terminal conjugation of the CH3 domain of the Fc region of the polypeptide chain. Such compounds that may be conjugated to the EGFR/PD-L1FIT-Ig binding protein include, but are not limited to, imaging agents and therapeutic agents. Preferred imaging agents that can be conjugated to EGFR/PD-L1FIT-Ig binding protein include, but are not limited to: radiolabels, enzymes, fluorescent labels, luminescent labels, bioluminescent labels, magnetic labels, biotin, streptavidin and avidin. Radiolabels that may be conjugated to the EGFR/PD-L1FIT-Ig binding protein described herein include, but are not limited to³H、¹⁴C、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹³¹I、¹⁷⁷Lu、¹⁶⁶Ho and¹⁵³sm. Preferred therapeutic compounds that may be conjugated to the EGFR/PD-L1FIT-Ig binding protein described herein include, but are not limited to, antibiotics, antivirals, small molecule receptor tyrosine kinase inhibitors, and cytokines.

In another embodiment, the EGFR/PD-L1FIT-Ig binding protein described herein may be a crystallized EGFR/PD-L1FIT-Ig binding protein that retains the binding affinity of the amorphous EGFR/PD-L1FIT-Ig binding protein to EGFR and PD-L1. This crystallized EGFR/PD-L1FIT-Ig binding protein also provides carrier-free controlled release of EGFR/PD-L1FIT-Ig binding protein when administered to an individual. The crystalline EGFR/PD-L1FIT-Ig binding protein of the invention may also exhibit a greater in vivo half-life when administered to an individual compared to the non-crystalline form. The crystallized binding proteins of the present invention may be produced according to methods known in the art, as disclosed in international publication No. WO 02/072636(Shenoy et al), which is incorporated herein by reference.

One embodiment of the present invention provides a composition for releasing crystallized EGFR/PD-L1 FIT-Ig-binding protein, wherein the composition comprises the crystallized EGFR/PD-L1 FIT-Ig-binding protein described herein, an excipient component, and at least one polymeric carrier. Preferably, the excipient ingredient is selected from: albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-beta-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol. Preferably, the polymeric carrier is a polymer selected from one or more of the following: poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (beta-hydroxybutyrate), poly (caprolactone), poly (dioxanone); polyethylene glycol, poly (hydroxypropyl) methacrylamide, poly [ (organo) phosphazene ], poly (orthoester), polyvinyl alcohol, poly (vinyl pyrrolidone), maleic anhydride/alkyl vinyl ether copolymers, pluronic polyols, albumin, alginates, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosaminoglycans, sulfated polysaccharides, blends thereof, and copolymers thereof.

The pharmaceutical compositions of the invention comprise the EGFR/PD-L1FIT-Ig binding protein described herein and one or more pharmaceutically acceptable components, such as a pharmaceutically acceptable carrier (vehicle, buffer), pharmaceutically acceptable excipients, and/or other pharmaceutically acceptable ingredients.

Preferred pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the present invention include, but are not limited to: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and combinations thereof.

The pharmaceutical composition of the present invention may further comprise an isotonic agent. In the pharmaceutical compositions of the invention, useful preferred isotonic agents are selected from: sugars, polyols (e.g., mannitol or sorbitol), sodium chloride, and combinations thereof.

Pharmaceutical compositions comprising the EGFR/PD-L1 FTI-Ig binding protein described herein may further comprise one or more additional therapeutically active compounds (therapeutic agents). Examples of such other therapeutic agents that may be incorporated into the pharmaceutical compositions of the present invention include, but are not limited to, anti-cancer agents other than the EGFR/PD-L1 FTI-Ig binding proteins described herein (e.g., cytotoxic metal-containing anti-cancer compounds or cytotoxic radioisotope-based anti-cancer compounds and combinations thereof), antibiotics, anti-viral compounds, sedatives, stimulants, local anesthetics, anti-inflammatory steroids (e.g., natural or synthetic anti-inflammatory steroids and combinations thereof), analgesics (e.g., acetylsalicylic acid, acetaminophen, naproxen, ibuprofen, COX-2 inhibitors, morphine, oxycodone and combinations thereof), antihistamines, non-steroidal anti-inflammatory drugs ("acetylsalicylic acid", NSAIDs, naproxen, COX-2 inhibitors and combinations thereof), and combinations thereof.

In another embodiment, the pharmaceutical composition of the invention comprises an EGFR/PD-L1FIT-Ig binding protein as described herein, a pharmaceutically acceptable carrier, and an adjuvant, wherein the adjuvant provides general stimulation of the immune system of a patient.

In one embodiment, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject EGFR/PD-L1FIT-Ig binding protein as described herein.

The invention also provides a method of inhibiting or blocking EGFR signaling in a cell, the method comprising contacting a cell expressing EGFR with an EGFR/PD-L1FIT-Ig binding protein as described herein.

In another embodiment, the invention provides a method of inhibiting or blocking PD-L1 signaling in a cell, the method comprising contacting a cell expressing PD-L1 with an EGFR/PD-L1FIT-Ig binding protein as described herein.

In one embodiment, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising EGFR/PD-L1FIT-Ig binding protein as described herein.

In another embodiment, the present invention provides a method for treating cancer in a cancer patient, wherein EGFR/PD-L1FIT-Ig binding protein as described herein is administered to a subject, and wherein the cancer is a cancer that is generally responsive to immunotherapy. In another embodiment, the cancer is a cancer not associated with immunotherapy. In another embodiment, the cancer is a refractory or relapsed malignancy.

Preferably, the cancer treated using the method according to the invention is epithelial cancer.

In another embodiment, the cancer treated using the method according to the invention is selected from: melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell renal cell carcinoma, "CCRCC"), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.

In another embodiment, the invention provides a method for treating a human subject suffering from a disease in which EGFR and/or PD-L1 activity is detrimental, the method comprising administering to the subject an EGFR/PD-L1FIT-Ig binding protein of the invention such that the activity mediated by PD-L1/PD1 binding and/or EGFR/EGF binding in the subject is reduced or blocked.

In one embodiment, the invention provides a method of detecting EGFR and/or PD-L1 in a sample, wherein the sample contains or is suspected of containing EGFR or PD-L1 or cells expressing EGFR or PD-L1, wherein the sample is contacted with a FIT-Ig binding protein as described herein. For example, the EGFR/PD-L1FIT-Ig binding protein of the invention can be used to detect EGFR or PD-L1 or both in conventional immunoassays, such as enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA) or tissue immunohistochemistry, using the FIT-Ig binding protein instead of an anti-EGFR antibody or an anti-PD-L1 antibody. The invention provides a method for detecting EGFR or PD-L1 in a biological sample, the method comprising contacting the biological sample with an EGFR/PD-L1FIT-Ig binding protein of the invention, and detecting whether binding to a target antigen (EGFR or PD-L1) occurs, thereby detecting the presence or absence of the target in the biological sample. The FIT-Ig binding protein may be labeled, directly or indirectly, with a detectable substance to facilitate detection of bound or unbound FIT-Ig binding protein. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescent materialsElemental, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; examples of suitable radioactive materials include³H、¹⁴C、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I、¹⁷⁷Lu、¹⁶⁶Ho or¹⁵³Sm。

Drawings

FIG. 1 is a diagram illustrating the general procedure for constructing three expression vectors for expressing the three types of polypeptide chains of each FIT-Ig binding protein described in example 1.

As shown in FIG. 1, to express the first polypeptide chain of the FIT-Ig binding protein ("heavy chain"), VL encoding the first polypeptide chain was synthesized_A-CL-VH_BDNA molecules of the fragments ("DNA synthesis"). The DNA molecule is then inserted ("inserted") into the Multiple Cloning Site (MCS) of the pcdna3.1 expression vector so that the inserted DNA molecule is located downstream of the vector's strong Cytomegalovirus (CMV) enhancer promoter, also downstream of and in-frame with the DNA fragment encoding the amino terminal Signal Peptide (SP), and upstream and in-frame with the inserted DNA molecule encoding the antibody CH1 domain, the CH1 domain is linked to the antibody Fc region (designated "h-CH 2-CH 3") which comprises the native contiguous hinge-CH 2-CH3 domain.

As shown in FIG. 1, to express the second polypeptide chain of the FIT-Ig binding protein ("light chain #1), the antibody VH was synthesized_AThe DNA fragment of the domain, which is then inserted into the Multiple Cloning Site (MCS) of the pcdna3.1 expression vector, such that the inserted DNA molecule is located downstream of the vector's strong CMV enhancer promoter, also downstream of and in frame with the DNA fragment encoding the amino terminal Signal Peptide (SP), and upstream and in frame with the inserted DNA molecule encoding the antibody CH1 domain.

For expression of the third polypeptide chain ("light chain #2), the synthesis encoded the antibody VL_BA DNA fragment of the domain which is then inserted into the Multiple Cloning Site (MCS) of the pcDNA3.1 expression vector such that the inserted DNA molecule is located downstream of the strong CMV enhancer promoter of the vector, also located in the DN encoding the amino terminal Signal Peptide (SP)The a fragment is downstream of and in reading frame with the inserted DNA molecule encoding the antibody CL domain.

For more details, see example 1.

FIG. 2 shows a size exclusion chromatography elution profile of a FIT-Ig1 sample previously purified by protein A affinity chromatography. The elution profile is complex, indicating a significant proportion of FIT-Ig1 aggregates. FIT-Ig1 six-chain monomer accounts for less than 30% of the purified protein. For detailed information, see example 1.7.

FIG. 3 shows a size exclusion chromatography elution profile of a FIT-Ig2 sample previously purified by protein A affinity chromatography. The elution profile shows the main peak of FIT-Ig2 six-chain monomer and several peaks indicating aggregates. The table below the figure provides the results of the analysis of several peaks. FIT-Ig2 six-chain monomer accounts for less than 75% of the purified protein. For detailed information, see example 1.7.

FIG. 4 shows a size exclusion chromatography elution profile of a FIT-Ig3 sample previously purified by protein A affinity chromatography. The elution profile is complex, indicating a significant proportion of aggregates. FIT-Ig3 six-chain monomer accounts for less than 40% of the purified protein. For detailed information, see example 1.7.

FIG. 5 shows a size exclusion chromatography elution profile of a previously purified FIT-Ig4 sample by protein A affinity chromatography. The elution profile shows the main peak of FIT-Ig4 six-chain monomer and several peaks indicating low proportions of aggregates. The table below the figure provides the results of the analysis of several peaks. FIT-Ig4 six-chain monomer constitutes about 91% of the purified protein. For detailed information, see example 1.7.

FIG. 6 shows a size exclusion chromatography elution profile of a previously purified FIT-Ig5 sample by protein A affinity chromatography. The elution profile shows the main peak of FIT-Ig5 six-chain monomer and a small peak indicating very low proportions of aggregates. The table below the figure provides the results of the analysis of several peaks. FIT-Ig5 six-chain monomer accounts for more than 98% of the purified protein. For detailed information, see example 1.7.

FIG. 7 shows a size exclusion chromatography elution profile of a FIT-Ig6 sample previously purified by protein A affinity chromatography. The elution profile shows the main peak and almost undetectable peak of FIT-Ig6 six-chain monomer, which may indicate that aggregates, if any, are present in very low proportions. The table below the figure provides the results of the analysis of several peaks. FIT-Ig6 six-chain monomer surprisingly constituted 99.9% of the purified protein. For detailed information, see example 1.7.

FIG. 8 shows a graph of the serum concentration of FIT-Ig6 in three male Sprague-Dawley rats as a function of time. FIT-Ig6 was administered to each rat in an intravenous dose of 5mg/kg body weight. For detailed information, see example 3.

Detailed Description

The Fabs-In-Tandem immunoglobulin ("FIT-Ig") binding protein format has been shown to be highly adaptable to provide bispecific, multivalent binding proteins against multiple different target antigen pairs or against different epitopes on the same antigen. See, for example, international publication nos. WO2015/103072 a1 and WO 2017/136820 a 2. In a preferred form, the FIT-Ig binding protein comprises four Fab binding units (instead of two of the native IgG antibodies), wherein each of the two Fab units binds to a first antigen (or epitope) and each of the other two Fab units binds to a second antigen (or epitope). Although the successful use of the FIT-Ig format has resulted in a number of bispecific binding proteins that bind to a target antigen pair relevant to therapy, FIT-Ig binding proteins that bind PD-L1 and EGFR have not previously been produced in suitable quantities and qualities for routine preclinical-stage evaluation as candidate anticancer therapeutics. For example, as shown herein, many of the FIT-Ig-binding proteins that bind PD-L1 and EGFR are not expressed at sufficiently high (i.e., greater than 10mg/L) levels in standard mammalian cell cultures to provide sufficient amounts of the proteins needed to support preclinical evaluations, including, for example, standard chemical, manufacturing and control ("CMC") stage evaluations. Another problem is that the previously produced FIT-Ig binding proteins that bind PD-L1 and EGFR have shown extensive aggregate formation, which greatly reduces the amount of functional hexapeptide chains (the "monomers" of EGFR and PD-L1 binding proteins) necessary for drug development. Unacceptable levels of FIT-Ig aggregates were evident in fractions of FIT-Ig binding proteins eluted from protein A affinity chromatography. Cost analysis showed that no existing FIT-Ig binding protein construct was available in sufficient quantity and quality to enable preclinical stage anticancer evaluation.

The present invention is based on the discovery of an EGFR/PD-L1FIT-Ig binding protein that is expressed at sufficiently high levels in mammalian cell culture and that does not have significant levels of aggregate formation, thereby enabling preclinical and clinical evaluation as a therapeutic anti-cancer drug.

The FIT-Ig binding proteins described herein contain two or more antigen binding sites and are typically tetravalent (four antigen binding sites) proteins. Preferred FIT-Ig binding proteins according to the invention bind to EGFR and PD-L1 and are therefore bispecific. Illustratively, in a FIT-Ig binding protein, two first (heavy) polypeptide chains (each having the general structure "V-C-V-C-Fc," where "V" is an antibody variable domain and C is an antibody constant domain) and four "light" polypeptide chains (each having the general structure "V-C") associate to form a hexamer with four Fab binding units (VH-CH1 paired with VL-CL, sometimes referred to as VH-CH1:: VL-CL). Each Fab binding unit comprises an antigen binding site comprising a heavy chain Variable (VH) domain and a light chain Variable (VL) domain, each antigen binding site having a total of six CDRs. Thus, each half of the FIT-Ig binding protein comprises one heavy polypeptide chain and two light polypeptide chains, and complementary immunoglobulin pairings of the VH-CH1 and VL-CL elements of these three chains result in two Fab binding units, arranged in tandem. In the present invention, the immunoglobulin domains of the Fab binding units are fused directly in the heavy chain polypeptide without the use of artificial interdomain linkers. That is, the N-terminal V-C element of each heavy polypeptide chain is fused at its C-terminus directly to the N-terminus of another V-C element, which in turn is linked to the C-terminal antibody Fc region. In the bispecific FIT-Ig binding protein, the tandem Fab binding units will react with different antigens.

A description of the design, expression and characterization of the FIT-Ig binding protein is provided in International publication No. WO 2015/103072. Preferred examples of such FIT-Ig molecules described herein include one heavy polypeptide chain and two different light polypeptide chains. Heavy chain comprises a knotStructure formula VL_A-CL-VH_B-CH1-Fc, wherein CL is directly connected to VH_BThe fusion, or heavy chain, comprising the structural formula VH_B-CH1-VL_A-CL-Fc in which CH1 is directly linked to VL_AFusion of wherein VL_AIs a variable light chain domain, VH, from a parent antibody that binds antigen A_BIs a variable heavy chain domain from a parent antibody that binds antigen B, CL is the human IgG1 light chain kappa constant domain, CH1 is the first human IgG1 antibody heavy chain constant domain, and Fc is an immunoglobulin Fc region (e.g., the C-terminal hinge-CH 2-CH3 portion of the human IgG1 antibody heavy chain). The two light polypeptide chains of FIT-Ig each have the formula VH_A-CH1 and VL_B-CL. In the embodiment of bispecific FIT-Ig, antigen A and antigen B are different antigens, or different epitopes of the same antigen. In the present invention, one of A and B is human EGFR, and the other is human PD-L1. Most preferably, in the present invention, A is human EGFR and B is human PD-L1.

Using the above protocol, each V domain of each polypeptide chain of the FIT-Ig binding protein may be named with the antigen (or epitope) specificity of the antigen binding site from which it is derived. For example, using this "antigen-specific" nomenclature scheme, the structure of the first polypeptide chain (polypeptide chain #1) of the FIT-Ig6 binding protein described in example 1.6 and Table 6 can be designated "VL_EGFR-CL-VH_PD-L1-CH1-Fc, wherein "VL_EGFR"indicates that the VL domain is derived from the EGFR-specific antigen-binding site of an anti-EGFR parent antibody," VH_PD-L1By "is meant that the VH domain is derived from the PD-L1 specific antigen binding site of the anti-PD-L1 parent antibody. The structure of the second polypeptide chain (polypeptide chain #2) of FIT-Ig6 can be named "VH_EGFR-CH1 ", wherein VH_EGFRIndicates that the VH domain is derived from the EGFR-specific antigen-binding site of the anti-EGFR parent antibody. Similarly, the third polypeptide chain of FIT-Ig6 (polypeptide chain #3) may be designated "VL_PD-L1-CL' in which "VL_PD-L1"means that the VL domain is derived from the PD-L1 specific antigen binding site of an anti-PD-L1 parent antibody. Thus, this antigen-specific nomenclature indicates whether a particular antigen-binding specificity is located in the corresponding external or internal Fab binding unit of the FIT-Ig binding protein.

In alternative nomenclature schemes, the antigen specificity of each variable domain that does not name the antigen binding site (e.g., VL_EGFR、VH_EGFR、VL_PD-L1、VH_PD-L1) Instead, the abbreviated name of the parent antibody used as the source of the domain is designated as a subscript to the corresponding VL and VH domains of the antigen binding site of the parent antibody. For example, referring again to the FIT-Ig6 binding protein of the invention described in example 1.6 and Table 6 below, the structure of the first polypeptide chain (polypeptide chain #1) can be designated VL_pani-CL-VH_3G10-CH1-Fc, wherein "VL_pani"indicates that the VL domain is derived from the anti-EGFR monoclonal antibody panitumumab," VH_3G10"indicates that the VH domain is derived from the anti-PD-L1 monoclonal antibody 3G 10. The structure of the second polypeptide chain (polypeptide chain #2) of FIT-Ig6 can be named "VH_pani-CH1 ", wherein" VH_pani"means that the VH domain is derived from panitumumab. Similarly, the third polypeptide chain of FIT-Ig6 (polypeptide chain #3) may be designated "VL_3G10-CL' in which "VL_3G10"indicates that the VL domain is derived from monoclonal antibody 3G 10. Thus, this alternative nomenclature indicates the source of antigen binding specificity for the external and internal Fab binding units of the FIT-Ig binding protein. This source naming scheme is particularly useful when attempting to compare the properties of multiple FIT-Ig binding proteins that bind to the same two antigens (or epitopes), but where one or both of the parent antibodies used as a source of antigen binding specificity are different. See the examples below.

As described above, the heavy polypeptide chain binds to each of the two different light polypeptide chains to form two complete Fab binding units, each of which comprises a typical antibody VH:: VL antigen binding site. Like a natural IgG antibody, the Fc region on one heavy chain will bind to the Fc region on the other heavy chain to form a homodimer, thereby providing a FIT-Ig binding protein comprising 6 polypeptide chains and 4 Fab binding units. Each arm of the FIT-Ig binding protein has an amino terminal or "external" Fab binding unit and a carboxy proximal or "internal" Fab binding unit. In the FIT-Ig nomenclature used herein, a particular bispecific FIT-Ig binding protein may be assigned a prefix that first indicates the antigen specificity of the outer Fab binding unit and then the antigen specificity of the inner Fab binding unit. Thus, "EGFR/PD-L1 FIT-Ig binding protein" refers to a FIT-Ig binding protein having two outer Fab binding units that bind EGFR and two inner Fab binding units that bind PD-L1.

Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, oncology, genetics and biochemistry are well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and are described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The nomenclature used, and the laboratory procedures and techniques related to, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery and patient treatment.

In order to make the present invention easier to understand, the following definitions select terms.

A "tumor" is an abnormal mass of tissue.

A "benign" tumor is a slowly growing and self-limiting mass of abnormal tissue because it has no ability to invade nearby tissue and spread beyond its original site. Benign tumors are not cancers.

The term "cancer" has a meaning known in the medical and oncology arts and includes definitions according to the national cancer institute ("NCI", a division of the national institutes of health, besseida, maryland). Thus, according to NCI, the term "cancer" is a term for a disease in which abnormal cells divide uncontrollably and may invade and damage or destroy nearby tissues. Cancer cells may also shed from the primary tumor and spread (metastasize) to other parts of the body through the blood and/or lymphatic system, and form "secondary tumors," also known as "metastatic tumors" or "metastatic cancers. Thus, the term "cancer" refers to a malignant tumor in which its cells grow uncontrollably and can penetrate and damage or destroy adjacent tissues and can migrate to distant parts of the body through the circulation and form new tumors.

An "anti-cancer" compound or drug is a compound or drug that blocks, inhibits, or halts the growth of cancer cells. Preferred anti-cancer compounds are cytotoxic to cancer cells.

Unless otherwise distinguished, the terms "intravenous" (or "intravenously") and "systemic" (or "systemically") are used interchangeably in terms of the route by which a compound or composition of the invention is introduced into the circulatory system of a cancer patient.

As used herein, the terms "treat," "treating," and "treatment" generally refer to any regimen that reduces one or more symptoms or manifestations of a cancer, inhibits the progression of a cancer, arrests the progression of a cancer or reverses the progression of a cancer, prevents the occurrence of a secondary (metastatic) cancer, provides significant killing of metastatic cancer cells, reduces the size of a primary or secondary (metastatic) cancer tumor, increases remission of one or more secondary (metastatic) tumors over a period of time, slows progression of a primary or secondary (metastatic) tumor, reduces the number of secondary (metastatic) tumors over a period of time, reduces the number of new secondary (metastatic) tumors over a period of time, increases organ or tissue function in a cancer patient, increases the vitality of a cancer patient, extends the life of a patient, or a combination thereof.

"metastasis" has the same meaning known and used by those skilled in the oncology or medical arts, and refers to the process by which cancer cells spread from a primary tumor to another location in a patient's body. A "metastatic" cancer cell is a cancer cell that has been shed or otherwise detached from a primary tumor and is in the process of moving from the primary tumor to another location within the patient's body by blood or lymph, or has moved from the primary tumor to another location within the patient's body by blood or lymph. In this case, the cancer or cells thereof are said to have "metastasized". Thus, a "metastatic" tumor is a tumor that develops from metastatic cancer cells that metastasize from a primary tumor to a different location in the patient where they establish another ("secondary", "metastatic") tumor, which is of the same type as the primary tumor. For example, metastatic intestinal tumors in the liver are initiated by and consist of intestinal cancer cells that metastasize from a primary intestinal tumor and migrate to the liver through the blood, and then the cancer cells establish a secondary (metastatic) intestinal tumor in the liver. It is also understood that metastatic tumors can also be another source of metastatic cancer cells, which can migrate to other tissues and organs and establish other metastatic tumors.

Unless otherwise specified, when the terms "about" and "approximately" are used in combination with an amount, quantity, or value, the combination describes the amount, quantity, integer, or value by itself, as well as the amount, quantity, or value plus or minus 5%. For example, the phrases "about 40" and "about 40" disclose "40" and "38 to 42, including 38 and 40".

The term "polypeptide" refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide, and also refer to a polymeric chain of amino acids. The term "polypeptide" encompasses natural or artificial proteins, protein fragments, and polypeptide analogs of the protein amino acid sequence. Unless the context contradicts, the term "polypeptide" encompasses fragments and variants thereof (including fragments of variants). For antigenic polypeptides, fragments of a polypeptide optionally comprise at least one contiguous or non-linear polypeptide epitope. The precise boundaries of at least one epitope fragment can be determined using techniques common in the art. Fragments comprise at least about 5 contiguous amino acids, e.g., at least about 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide as follows: depending on the origin or source from which it is derived, it is isolated from naturally related components that accompany it in its natural state, is substantially free of other proteins from the same species, is expressed by cells from a different species, or does not exist in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived is "isolated" from its naturally associated components. Proteins consisting of one or more polypeptide chains can also be made substantially free of naturally associated components by separation using protein purification techniques well known in the art.

The term "recovering" refers to the process of rendering a chemical substance (e.g., a polypeptide) substantially free of naturally-associated components by separation (e.g., using protein purification techniques well known in the art).

The term "biological activity" of PD-L1 or EGFR refers to any or all of the inherent biological properties of PD-L1 or EGFR, respectively.

The term "specific binding" or "specific binding" in relation to the interaction of an antibody, binding protein or peptide with a second chemical means that the interaction is dependent on the specific structure (e.g., antigenic determinant or epitope) present on the second chemical. For example, antibodies recognize and bind to specific protein structures, rather than whole proteins. If the antibody is specific for epitope "A", then in the reaction comprising labeled "A" and the antibody, the presence of a molecule comprising epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody. The bispecific FIT-Ig binding protein described herein comprises two Fab binding units that specifically bind EGFR and two Fab binding units that specifically bind PD-L1.

The term "antibody" broadly refers to any immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative thereof that retains the essential epitope-binding characteristics of an Ig molecule. Such mutants, variants or derivatives of antibody forms are known in the art. Non-limiting examples of which are discussed below.

In a full-length antibody, each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The first, second and third CDRs of the VH domain are generally listed as CDR-H1, CDR-H2 and CDR-H3; similarly, the first, second and third CDRs of the VL domain are generally enumerated as CDR-L1, CDR-L2 and CDR-L3. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

In general, the term "Fc region" or simply "Fc" refers to the C-terminal region of an antibody heavy chain, which can be produced by papain digestion of an intact antibody. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region typically comprises two constant domains, namely a CH2 domain and a CH3 domain, and optionally a CH4 domain, for example in the case of Fc regions of IgM and IgE antibodies. The Fc region of IgG, IgA, and IgD antibodies comprises a hinge region, a CH2 domain, and a CH3 domain. In contrast, the Fc region of IgM and IgE antibodies lacks a hinge region, but comprises a CH2 domain, a CH3 domain, and a CH4 domain. Variant Fc regions having amino acid residue substitutions in the Fc portion to alter antibody effector functions are known in the art (see, e.g., Winter et al, U.S. Pat. nos. 5,648,260 and 5,624,821). Unless otherwise indicated, the "Fc region" of the FIT-Ig-binding protein described herein is an Fc region derived from the human IgG1 antibody, which comprises a hinge region, a CH2 domain, and a CH3 domain, and has the amino acids in any one of tables 1-6 of the following examples disclosed herein (SEQ ID NO: 8).

The Fc region of an antibody mediates several important effector functions, such as cytokine induction, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, complement-dependent cytotoxicity (CDC), and half-life/clearance of the antibody and antigen-antibody complex. In some cases, these effector functions are desirable for therapeutic antibodies, but in other cases may be unnecessary or even detrimental, depending on the therapeutic objective. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC by binding to Fc gamma receptor (Fc γ R) and complement C1q, respectively. Unless otherwise indicated, the Fc region used in the FIT-Ig binding proteins described herein retains at least one or more or all of the same functional properties as the Fc region in its original donor antibody.

In one embodiment, at least one amino acid residue in the Fc region is substituted, thereby altering one or more effector functions of the antibody. Dimerization of two identical heavy chains of the FIT-Ig binding protein described herein is mediated by dimerization of the CH3 domain, as in IgG antibodies, and is stabilized by disulfide bonds within the hinge region linking the CH1 or CL domain of the FIT-Ig heavy chain to Fc constant domains (e.g., CH2 and CH 3). The anti-inflammatory activity of IgG is completely dependent on sialylation of the N-linked glycans of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity have been determined so that a suitable IgG1 Fc fragment can be generated, resulting in a fully recombinant sialylated IgG1 Fc with greatly enhanced potency (see Anthony et al, Science,320:373 376 (2008)). Such sialylated Fc regions may be used in the FIT-Ig binding proteins described herein.

The terms "antigen-binding portion" and "antigen-binding fragment" or "functional fragment" of an antibody are used interchangeably to refer to one or more fragments of an antibody that retain the ability to specifically bind an antigen, i.e., bind the same antigen as the full-length antibody from which the portion or fragment is derived (e.g., EGFR, PD-L1). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Embodiments of such antibodies may also be bispecific, bispecific or multispecific; which specifically binds two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) fab fragments (Fab binding units), which are monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')₂A fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) fv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al, Nature,341: 544-54)6 (1989); international publication No. WO90/05144) comprising a single variable domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker using recombinant methods to make them a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al, Science,242: 423-. Such single chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody as well as equivalent terms given above. Other forms of single chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing these domains to pair with complementary domains of the other chain, creating two antigen binding sites (see, e.g., Holliger et al, Proc. Natl. Acad. Sci. USA,90:6444-,Antibody Engineering(Springer-Verlag, New York,2001), page 790 (ISBN 3-540-. In addition, single chain antibodies also include "linear antibodies" which comprise a pair of tandem Fv fragments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen binding regions (Zapata et al, Protein Eng.,8(10): 1057-.

Unless otherwise indicated, the terms "donor" and "parent" refer to any antibody or antigen-binding fragment that is the source of antibody variable domains, antibody constant domains, Fab binding units, or Fc regions provided for the preparation of the FIT-Ig binding proteins described herein. Non-natural or engineered antibodies may also be used as donors or parent antibodies for the preparation of FIT-Ig binding proteins as described herein.

Antibody (or immunoglobulin) constant domain (C) refers to the antibody heavy chain constant domain (CH) or light chain constant domain (CL). Murine and human immunoglobulin heavy and light chain constant domain amino acid sequences are known in the art.

The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic determinant (epitope). Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences, e.g., in the CDRs, particularly in CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.

The term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant, combinatorial human antibody libraries (Hoogenboom, H.R., Trends Biotechnol.,15:62-70 (1997); Azzazy and Highsmith, Clin. biochem.,35: 425. alpha. 445 (2002); Gavilon and Larrick, BioTechniques,29: 128. alpha. 145 (2000); Hoogenboom and Chames, Immunol. Today,21: 371. phi. 378(2000)), antibodies isolated from transgenic animals of human immunoglobulin genes (e.g., mice) (see, e.g., Taylor et al, Nucl. acids Res.,20: 87. beta. 87 (5 (1992), Kennn. and Opren., 6213. gamma., 5913, Torri. 12, Torri, Torrik, 2000)); or antibodies prepared, expressed, produced or isolated by any other method, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when transgenic animals with human Ig sequences are used, in vivo somatic mutagenesis) and, thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences derived from and related to human germline VH and VL sequences, but may not naturally occur in the human antibody germline repertoire in vivo.

The term "multivalent binding protein" refers to a binding protein comprising two or more antigen binding sites. Multivalent binding proteins are preferably engineered to have three or more antigen binding sites and are not typically naturally occurring antibodies. The term "bispecific binding protein" refers to a binding protein capable of binding to two different specific targets.

The term "activity" includes properties such as the ability to specifically bind to a target antigen, the affinity of an antibody or binding protein for an antigen, the ability to neutralize the biological activity of a target antigen, the ability to inhibit the interaction of a target antigen with its natural receptor or natural ligand, and the like. The activity of the EGFR/PD-L1FIT-Ig binding protein of the present invention may include, but is not limited to, inhibiting binding of EGFR to its cognate ligand (EGF), inhibiting EGFR signaling, inhibiting binding of PD-L1 to PD-1, inhibiting PD-1/PD-L1 signaling, upregulating T cell response to cancer, killing cancer cells, inhibiting cancer cell growth, inhibiting cancer cell survival, and inhibiting cancer cell spread.

As used herein, the term "Kon" (also referred to as "Kon", "Kon") refers to the binding rate constant of a binding protein (e.g., an antibody) binding to an antigen to form a binding complex (e.g., an antibody/antigen complex as known in the art). As used interchangeably herein, "kon" has also been termed the term "association rate constant" or "ka". For example, the value may represent the rate of binding of an antibody to its target antigen or the rate of complex formation between an antibody and antigen, as shown in the following equation:

antibody ("Ab") + antigen ("Ag") → Ab-Ag.

As used herein, the term "Koff" (also referred to as "Koff", "Koff") refers to the off-rate constant, or "off-rate constant," of a binding protein (e.g., an antibody) disassociating from a binding complex (e.g., an antibody/antigen complex) as known in the art. For example, the value may represent the off-rate of an antibody from its target antigen, or the off-rate of an Ab-Ag complex separating into free antibody and antigen over time, as shown in the following equation:

Ab+Ag←Ab-Ag。

as used herein, the term "K_D"(also referred to as" Kd ") means" equilibrium dissociation constant "which means that either a titration measurement is performed at equilibrium or by comparing the dissociation rate constant (k)_off) Divided by the binding rate constant (k)_on) The value obtained. Binding Rate constant (k)_on) Dissociation rate constant (k)_off) And equilibrium dissociation constant (K)_D) Used to indicate the binding affinity of an antibody or binding protein to an antigen. Methods for determining binding and dissociation rate constants are well known in the art. The use of fluorescence-based techniques can provide high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental methods and instruments may be used, for example(biomolecule interaction analysis) assay (e.g., instruments available from BIAcore International AB, GE Healthcare, Uppsala, Sweden). Using e.g.The biolayer interferometry (BLI) of the RED96 system (Pall Forte Bio LLC) is another affinity determination technique. In addition, it is also possible to use(kinetic exclusion assay) assay (available from Sapidyne Instruments (Boise, Idaho)).

The term "isolated nucleic acid" refers to a polynucleotide (e.g., a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof) that has been isolated by human intervention from all or a portion of the polynucleotide with which it is naturally occurring, operably linked to a polynucleotide that is not naturally associated, or does not occur as part of a larger sequence in nature.

The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). After introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell and thereby replicated together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence is "operably linked" to a coding sequence in a manner that achieves expression of the coding sequence under conditions compatible with the control sequences. Sequences "operably linked" include expression control sequences that are contiguous with the gene of interest and expression control sequences that control the gene of interest in trans or remote action. As used herein, the term "expression control sequences" refers to polynucleotide sequences necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, terminator, promoter and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and sequences that enhance protein secretion when desired. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and may also include other components whose presence is advantageous, such as leader sequences and fusion partner sequences.

The term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which exogenous DNA has been introduced. In one embodiment, the host cell comprises two or more (e.g., a plurality of) nucleic acids encoding an antibody, such as the host cell described in U.S. patent No. 7,262,028. Such terms are not intended to refer only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in the progeny due to mutation or environmental impact, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In one embodiment, the host cell comprises a prokaryotic and eukaryotic cell selected from any of the kingdoms of life. In another embodiment, eukaryotic cells include protists, fungi, plant and animal cells. In another embodiment, host cells include, but are not limited to, the prokaryotic cell line E.coli; mammalian cell lines CHO, HEK293, COS, NS0, SP2 and per.c 6; insect cell line Sf 9; and the fungal cell Saccharomyces cerevisiae.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, cell culture, tissue culture, and transformation (e.g., transfection, electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly done in the art or as described herein. The foregoing techniques and steps may generally be performed according to conventional methods well known in the art, and as referenced and discussed throughout the present specification in various general and more specific referencesThe method is described in the literature. See, e.g., Sambrook et al,Molecular Cloning:A Laboratory Manual2 nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

As used herein, the term "agonist" refers to a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of an activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the agonist. As used herein, the terms "antagonist" and "inhibitor" refer to a modulator that, when contacted with a molecule of interest, causes a decrease in the magnitude of an activity or function of the molecule as compared to the magnitude of the activity or function observed in the absence of the antagonist. Particular antagonists of the invention are EGFR/PD-L1FIT-Ig described herein that blocks or inhibits binding of EGFR to EGF, blocks or inhibits binding of PD-L1 to PD-1, blocks or inhibits EGFR signaling, blocks or inhibits PD-L1 signaling, blocks or inhibits the pro-cancer activity of EGFR-dependent signaling, blocks or inhibits the pro-cancer activity of PD-L1-dependent signaling, and one or more combinations thereof.

As used herein, the term "effective amount" refers to an amount sufficient to reduce or lessen the severity and/or duration of the condition or one or more symptoms thereof; preventing progression of the condition; causing regression of the condition; preventing the recurrence, development, or progression of one or more symptoms associated with the disorder; detecting the condition; or an amount of a therapy that enhances or improves the prophylactic or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent).

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definition. Furthermore, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" are intended to encompass both elements and components comprising one unit and elements and components comprising more than one subunit, unless specifically stated otherwise.

A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are required, but that other elements or steps may be added within the scope of the composition or method. To avoid redundancy, it should also be understood that any composition or method described herein as "comprising" (or "which comprises") one or more named elements or steps also describes a corresponding, more limited "composition or method consisting essentially of" or "consisting essentially of … …" that includes the named elements or steps, and may also include other elements or steps that do not materially affect the basic and novel characteristics of the composition and method. It will also be understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes a corresponding, more limited and closed composition or method "consisting of (or consisting of) the named elements or steps to the exclusion of any other unnamed element or step. In any compositions or methods disclosed herein, known or disclosed equivalents of any named essential elements or steps may be substituted for the elements or steps.

Characterization of preferred EGFR/PD-L1FIT-Ig binding proteins of the present invention

The preferred EGFR/PD-L1FIT-Ig binding protein of the present invention, referred to as "FIT-Ig 6" (or "EGFR/PD-L1 FIT-Ig 6"), binds EGFR and PD-L1 and comprises:

a first polypeptide chain (heavy chain) comprising a sequence according to SEQ ID NO: 1;

a second polypeptide chain (first light chain) comprising a sequence according to SEQ ID NO: 2;

a third polypeptide chain (second light chain) comprising a sequence according to SEQ ID NO: 3.

The above-described binding of the first, second and third polypeptide chains provides a "half FIT" molecule comprising an amino-terminal or "external" Fab binding unit specific for EGFR, which is linked in tandem to a carboxy-proximal or "internal" Fab binding specific for PD-L1. As in the native IgG1 antibody, the Fc of the carboxy-terminal region of the first polypeptide chain (hinge-CH 2-CH3) can bind to the Fc of the other half of the FIT molecule, forming a fully assembled six polypeptide chain FIT-Ig binding protein comprising two external EGFR-specific Fab binding units and two internal PD-L1-specific Fab binding units.

The specificity of the Fab binding unit of the FIT-Ig binding protein is derived from the parent antibody which serves as the source of the antibody heavy and light chain variable domains (VH, VL) that form the specific antigen binding site.

The EGFR/PD-L1FIT-Ig binding protein of the present invention binds EGFR and PD-L1 simultaneously. In another embodiment, the EGFR/PD-L1FIT-Ig binding protein binds both EGFR protein and both PD-L1 protein simultaneously.

The EGFR/PD-L1FIT-Ig binding protein according to the invention binds EGFR and PD-L1 with similar affinity to each parent antibody from which EGFR and PD-L1 specificity is derived.

The affinity of the EGFR/PD-L1FIT-Ig binding protein according to the present invention for EGFR and PD-L1 may be measured using any of a variety of systems, including biolayer interferometry (e.g., usingRED96 system, Pall Forte Bio LLC), surface plasmon resonance (e.g. using(analysis of biomolecular interactions) assay systems, BIAcore International AB, GE Healthcare, Uppsala, Sweden) or kinetic exclusion assays (e.g., usingAnalytical System, Sapidyne Instruments, Boixie, Edaho)。

The EGFR/PD-L1FIT-Ig binding protein of the present invention binds EGFR and has an association rate constant (k) to human EGFR_on) Is at least 1 × 10⁵M^-1s^-1More preferably at least 2X 10⁵M^-1s^-1As determined by bio-layer interferometry. In a further embodiment, the k of the EGFR/PD-L1FIT-Ig binding protein according to the invention is to human EGFR_onK to human EGFR over parent antibody_onApproximately 40% lower, the anti-EGFR specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody.

The EGFR/PD-L1FIT-Ig binding protein of the present invention binds to human EGFR and has an off-rate constant (k) for human EGFR_off) Less than 1.1X 10^-4sec^-1As determined by bio-layer interferometry. In a further embodiment, the k of the EGFR/PD-L1FIT-Ig binding protein of the present invention to human EGFR_offK to human EGFR over parent antibody_offApproximately 50% lower, the anti-EGFR specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody.

The EGFR/PD-L1FIT-Ig binding protein of the present invention binds human EGFR and has a dissociation constant (KD) less than 1x 10 for human EGFR^-9M, preferably less than 7X 10^-10M, more preferably less than 6X 10^-10M, and still more preferably less than or equal to 5X 10^-10M, as determined by biolayer interferometry. In a further embodiment, the K of the EGFR/PD-L1FIT-Ig binding protein of the present invention to human EGFR_DK to human EGFR with parent antibodies_DSubstantially identical (i.e., identical or within 25%), the anti-EGFR specificity of the EGFR/PD-L1FIT-Ig binding protein being derived from the parent antibody.

The EGFR/PD-L1FIT-Ig binding protein of the present invention binds to PD-L1 and has an association rate constant (k) to human PD-L1_on) Is at least 5 x 10⁵M^-1s^-1More preferably at least 7X 10⁵M^-1s^-1Even more preferably 8X 10⁵M^-1s^-1As determined by bio-layer interferometry. In a further embodiment, according to the inventionEGFR/PD-L1FIT-Ig binding protein of (g) k to human PD-L1_onK to parent antibody to human PD-L1_onThe anti-PD-L1 specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody, which is the same as or within about 90% of it.

The EGFR/PD-L1FIT-Ig binding protein of the present invention binds to human PD-L1 and has an off-rate constant (k) for human PD-L1_off) Less than 2 x 10^-2sec^-1More preferably less than 1.5X 10^-2sec^-1As determined by bio-layer interferometry. In a further embodiment, the EGFR/PD-L1FIT-Ig binding protein of the present invention is k to human PD-L1_offK to human PD-L1 over the parent antibody_offAbout 20% higher, the anti-PD-L1 specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody.

In one embodiment, the EGFR/PD-L1FIT-Ig binding protein of the present invention binds to human PD-L1 and has a dissociation constant (k) for PD-L1_D) Less than 2 x 10^-8M, more preferably less than 1.7X 10^-8M, as determined by biolayer interferometry. In a further embodiment, the K of the EGFR/PD-L1FIT-Ig binding protein of the present invention to PD-L1_DK to PD-L1 of parent antibody_DSubstantially identical (i.e., identical or within 30%), the anti-PD-L1 specificity of the EGFR/PD-L1FIT-Ig binding protein is derived from the parent antibody.

The preferred EGFR/PD-L1FIT-Ig binding protein of the present invention did not show significant aggregate formation after one-step purification from cell culture media using protein A affinity chromatography. As shown herein, after protein a affinity chromatography purification, column eluates containing EGFR/PD-L1FIT-Ig binding protein can be analyzed for the presence of aggregates using size exclusion chromatography (e.g., size exclusion chromatography using a High Performance Liquid Chromatography (HPLC) column). Size Exclusion Chromatography (SEC) will separate molecules according to size, and therefore, molecules with the expected molecular weight of the fully assembled hexa-polypeptide chain EGFR/PD-L1FIT-Ig binding protein (also known as the hexa-chain "monomer") from other substances with higher or lower molecular weights. The six-chain monomer has a molecular weight of about 240,000 daltons. The preferred EGFR/PD-L1FIT-Ig binding protein of the present invention that has been purified from culture medium using protein A affinity chromatography has less than or equal to 0.1% aggregates. That is, at least 99.9% of the EGFR/PD-L1FIT-Ig binding protein of the present invention produced in mammalian cell culture will be present as fully assembled six-chain monomers. Levels of protein aggregates of less than or equal to 0.1% (≦ 0.1%) are considered to be insignificant amounts that do not interfere with effective preclinical and clinical evaluation of EGFR/PD-L1FIT-Ig binding protein as an anti-cancer drug. In contrast, as shown herein, the amount of aggregates found in other previously produced EGFR/PD-L1 and PD-1/EGFR FIT-Ig binding proteins ranged from 1.2% to greater than 70%. Thus, the EGFR/PD-L1FIT-Ig binding protein of the invention is more stable in terms of aggregate formation and has a significantly lower percentage of aggregates (e.g., at least 10-fold lower) than previously produced FIT-Ig binding proteins that bind EGFR and PD-L1. See table 7 in example 1.7, below.

Generation of EGFR/PD-L1FIT-Ig binding protein of the invention

The present invention provides methods of producing an EGFR/PD-L1FIT-Ig binding protein described herein, comprising culturing an isolated host cell comprising one or more vectors encoding three polypeptide chains of an EGFR/PD-L1FIT-Ig binding protein under conditions sufficient for the production of the EGFR/PD-L1FIT-Ig binding protein. The desired EGFR/PD-L1FIT-Ig binding protein is expressed as a six polypeptide chain FIT-Ig binding protein comprising two outer EGFR-specific Fab binding units and two inner PD-L1-specific Fab binding units.

A variety of expression systems comprising an expression vector and a compatible prokaryotic or eukaryotic host cell can be used to express the recombinant heterologous protein. An example of a prokaryotic host cell often used for expression of recombinant proteins is an E.coli cell. Eukaryotic host cells that can be used to express the recombinant protein include, but are not limited to, mammalian host cells, insect host cells, plant host cells, fungal host cells, algal host cells, nematode host cells, protozoan host cells, and fish host cells. Fungal host cells useful for expression of recombinant proteins include, but are not limited to: aspergillus (Aspergillus), Neurospora (Neurospora), Saccharomyces cerevisiae (Saccharomyces), Pichia pastoris (Pichia), Hansenula (Hansenula), Schizosaccharomyces (Schizosaccharomyces), Kluyveromyces (Kluyveromyces), Yarrowia (Yarrowia), and Candida (Candida). A preferred Saccharomyces cerevisiae host cell for expression of recombinant proteins is a Saccharomyces cerevisiae (Saccharomyces cerevisiae) cell. The insect cell used as a host cell according to the present invention is an insect Sf9 cell.

The FIT-Ig binding protein is preferably produced using a mammalian cell expression system. Construction and expression of FIT-Ig binding proteins has been previously described in International publication Nos. WO 2015/103072A 1 and WO 2017/136820A 2. Similar materials and methods can be used to produce the EGFR/PD-L1FIT-Ig binding protein of the present invention. Such methods are typically used to express recombinant antibodies and engineered binding proteins in selected host cells.

Typically, each polypeptide chain of the FIT-Ig binding protein is encoded on an isolated nucleic acid molecule along with an amino-terminal signal sequence (signal peptide) that directs the nascent polypeptide chain to the lumen of the Endoplasmic Reticulum (ER) and then into the golgi apparatus for secretion. Individual nucleic acid molecules encoding the polypeptide chains of the FIT-Ig binding proteins described herein can be prepared using chemical DNA synthesis methods, using recombinant DNA methods, or using a combination of both methods.

Each nucleic acid molecule encoding one of the polypeptide chains of the FIT-Ig binding protein is then inserted into a separate expression vector, operably linked to appropriate transcription and/or translation sequences, to allow expression of that polypeptide chain in a host cell compatible with the expression vector.

The vector may be an autonomously replicating vector or a vector which incorporates the isolated nucleic acid present in the vector into the genome of the host cell. Preferred vectors for expressing Nucleic Acids described herein include, but are not limited to, pcDNA, pTT (Durocher et al Nucleic Acids Res.,30(2e9):1-9(2002)), pTT3 (pTT with other multiple cloning sites), pEFBOS (Mizushima and Nagata, Nucleic Acids Res.,18(17):5322(1990)), pBV, pJV, pcDNA3.1 TOPO, pEF6 TOPO, and pBJ, as well as modifications thereof as required for expression of the EGFR/PD-L1FIT-Ig binding proteins described herein in particular host cells.

In a preferred embodiment for producing a FIT-Ig binding protein, the FIT-Ig binding protein is expressed in mammalian host cells transfected with three expression vectors, wherein each expression vector comprises nucleic acid encoding one of the three component polypeptide chains of the FIT-Ig binding protein.

Preferably, the isolated mammalian host cell comprising the vector described herein is selected from the group consisting of: chinese Hamster Ovary (CHO) cells, COS cells, Vero cells, SP2/0 cells, NS/0 myeloma cells, human embryonic kidney (HEK293) cells, mouse kidney (BHK) cells, HeLa cells, human B cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells, and MDCK cells.

Transfected HEK293 cells are typically used as transient transfection systems that can provide short term production of recombinant proteins, including engineered antibodies and binding proteins, e.g., 4 to 10 days post transfection. Transfected HEK293 cells are commonly used for initial laboratory cloning, production and analysis of recombinant proteins, thereby avoiding the time and labor required to isolate stably transfected production cell lines (e.g., stably transfected CHO cell lines). For those familiar with the production of engineered antibodies and binding proteins, expression levels below 10mg/L in cultures of transiently transfected cells are too low to expect a sufficient amount of binding protein to be useful for preliminary preclinical evaluation, such as bioactivity studies, preliminary stability studies, Pharmacokinetic (PK) studies, and efficacy in animal models. Furthermore, those skilled in the art also recognize that expression levels below 10mg/L in cultures of transiently transfected HEK293 cells indicate that even considerable time and effort is not likely to be spent successfully isolating stably expressing CHO cells with high expression (e.g., greater than 1 g/L). Conversely, it is believed that expression levels of FIT-Ig binding protein above 10mg/L in cultures of transiently transfected HEK293 cells are sufficiently high to provide an amount of binding protein that allows for evaluation of the early discovery phase prior to generation of stably transfected CHO cells, and also suggests that it is possible to successfully isolate stably transfected CHO cells to generate higher amounts for later preclinical and clinical phase evaluation.

As shown herein, the EGFR/PD-L1FIT-Ig binding protein according to the present invention can be expressed in transfected HEK293 cells at a level higher than 10mg (>10mg/L) of EGFR/PD-L1FIT-Ig binding protein per liter of cell culture. This level of expression is unexpected given that the previously produced EGFR/PD-L1 and PD-1/EGFR FIT-Ig binding proteins are only expressed at levels of about 1mg/L to about 8 mg/L. In addition, not only was the production level gradually increased compared to other FIT-Ig binding proteins, the expression level of EGFR/PD-L1FIT-Ig binding protein of the present invention in transfected HEK293 cell cultures ensured that the binding protein could be used in sufficient amounts for preclinical and clinical phase assessments as a new anti-cancer therapeutic.

Pharmaceutical composition

The pharmaceutical compositions of the invention comprise an EGFR/PD-L1FIT-Ig binding protein as described herein and one or more pharmaceutically acceptable components, such as pharmaceutically acceptable carriers (carriers, buffers), excipients, and/or other ingredients. By "pharmaceutically acceptable" is meant that the carrier, compound, component, or other ingredient of the composition is compatible with the physiology of the human subject and is not deleterious to the desired binding specificity of the EGFR/PD-L1FIT-Ig binding protein, or to any other desired property or activity of any other component present in the composition administered to the human subject. Examples of pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In some cases, it is preferred to include isotonic agents, including, but not limited to, sugars; polyols, such as mannitol or sorbitol; sodium chloride; and combinations thereof.

The pharmaceutically acceptable compositions of the present invention may further comprise one or more excipients, minor amounts of auxiliary substances, such as wetting or emulsifying agents, fillers, preservatives or buffers, to prolong the shelf-life or effectiveness of the pharmaceutical composition. An excipient is generally any compound or combination of compounds that provides a pharmaceutical composition with a beneficial property or characteristic other than the primary therapeutic compound or activity. For pharmaceutical compositions comprising the EGFR/PD-L1FIT-Ig binding protein (the primary therapeutic compound) of the present invention, the excipients provide desirable beneficial characteristics in addition to the desired binding specificity of the EGFR/PD-L1FIT-Ig binding protein or the anti-cancer activity elicited by the EGFR/PD-L1 FTI-Ig binding protein.

In another embodiment, the pharmaceutical composition of the invention comprises an EGFR/PD-L1FIT-Ig binding protein as described herein, a pharmaceutically acceptable carrier, and an adjuvant, wherein the adjuvant provides general stimulation of the human immune system.

The pH in the pharmaceutical composition may be adjusted as desired, for example, to promote or maintain solubility of the component ingredients, to maintain stability of one or more component ingredients in the formulation, and/or to prevent unwanted microbial growth potentially introduced into the composition.

Pharmaceutical compositions comprising the EGFR/PD-L1 FTI-Ig binding protein of the present invention may be prepared to provide sustained or delayed release of the binding protein. Various methods for preparing such controlled release or time delay compositions are known to those skilled in the art, including but not limited to implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and combinations thereof, may also be used to prepare controlled-or time-release compositions comprising the EGFR/PD-L1 FIT-Ig-binding protein of the present invention.

Pharmaceutical compositions comprising the EGFR/PD-L1 FTI-Ig binding protein described herein may further comprise one or more additional therapeutically active compounds (therapeutic agents). Examples of such other therapeutic agents that may be incorporated into the pharmaceutical compositions of the present invention include, but are not limited to, anti-cancer agents other than the EGFR/PD-L1 FTI-Ig binding proteins described herein (e.g., cytotoxic metal-containing anti-cancer compounds or cytotoxic radioisotope-based anti-cancer compounds and combinations thereof), antibiotics, anti-viral compounds, sedatives, stimulants, local anesthetics, anti-inflammatory steroids (e.g., natural or synthetic anti-inflammatory steroids and combinations thereof), analgesics (e.g., acetylsalicylic acid, acetaminophen, naproxen, ibuprofen, COX-2 inhibitors, morphine, oxycodone and combinations thereof), antihistamines, non-steroidal anti-inflammatory drugs ("acetylsalicylic acid", NSAIDs, naproxen, COX-2 inhibitors and combinations thereof), and combinations thereof.

The pharmaceutical compositions according to the present invention are formulated for administration by any of a variety of routes known in the art. Such routes include, but are not limited to, parenteral, intravenous (systemic), subcutaneous, intramuscular, oral (i.e., gastrointestinal), sublingual, buccal, intranasal (e.g., inhalation), transdermal (e.g., topical), intratumoral, transmucosal, intraarticular, intrabronchial, intracapsular, intracartilaginous, intracavitary, intracervical, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, vaginal, and rectal.

Preferably, the pharmaceutical composition according to the invention is formulated for intravenous administration to a human subject suffering from cancer. Intravenous administration of a pharmaceutical composition comprising an EGFR/PD-L1 FTI-Ig binding protein provides the EGFR/PD-L1 FTI-Ig binding protein throughout the circulatory system for access to tissues and organs via the circulating blood. Typically, compositions for intravenous administration are solutions in sterile, isotonic, aqueous buffers. If desired, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to reduce pain at the injection site.

Subcutaneous administration of the pharmaceutical compositions of the invention is a route by which EGFR/PD-L1 FTI-Ig binding protein can be provided to the lymphatic system. Accordingly, pharmaceutical compositions comprising the EGFR/PD-L1 FTI-Ig binding protein of the present invention may be formulated for subcutaneous administration.

The pharmaceutical compositions of the present invention may be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions or solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient (i.e., EGFR/PD-L1FIT-Ig binding protein of the present invention) may be in powder form (e.g., lyophilized form) for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use.

The pharmaceutical compositions of the present invention may be formulated for delivery as depot formulations of the depot type. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the pharmaceutical compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

In another embodiment, the EGFR/PD-L1FIT-Ig binding protein described herein may be a crystallized EGFR/PD-L1FIT-Ig binding protein that retains the binding activity of the amorphous EGFR/PD-L1FIT-Ig binding protein to EGFR and PD-L1. This crystallized EGFR/PD-L1FIT-Ig binding protein also provides carrier-free controlled release of EGFR/PD-L1FIT-Ig binding protein when administered to an individual. The crystalline EGFR/PD-L1FIT-Ig binding protein of the invention may also exhibit a greater in vivo half-life when administered to an individual compared to the non-crystalline form. The crystallized binding proteins of the present invention may be produced according to methods known in the art, as disclosed in international publication No. WO 02/072636(Shenoy et al), which is incorporated herein by reference.

A pharmaceutical composition for releasing crystallized EGFR/PD-L1FIT-Ig binding protein, wherein the composition comprises the crystallized EGFR/PD-L1FIT-Ig binding protein described herein, an excipient ingredient, and at least one polymeric carrier. Preferably, the excipient ingredient is selected from: albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-beta-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol. Preferably, the polymeric carrier is a polymer selected from one or more of the following: poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (beta-hydroxybutyrate), poly (caprolactone), poly (dioxanone); polyethylene glycol, poly (hydroxypropyl) methacrylamide, poly [ (organo) phosphazene ], poly (orthoester), polyvinyl alcohol, poly (vinyl pyrrolidone), maleic anhydride/alkyl vinyl ether copolymers, pluronic polyols, albumin, alginates, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosaminoglycans, sulfated polysaccharides, blends thereof, and copolymers thereof.

Methods and uses of EGFR/PD-L1FIT-Ig binding protein of the present invention

The ability of the EGFR/PD-1FIT-Ig binding protein to bind to EGFR and PD-L1 has received major attention in providing anti-cancer therapies. Binding of the EGFR/PD-L1FIT-Ig binding protein to EGFR and PD-L1 is presumed to inhibit or block binding of EGFR and PD-L1 to their respective ligands (e.g., EGFR and PD-1), and thereby inhibit or block the respective independent signaling pathways (i.e., EGFR/EGF signaling and PD-L1/PD-1 signaling) involved in cancer progression, cancer cell growth, and cancer cell spreading (metastasis). The EGFR/PD-L1FIT-Ig binding protein of the present invention is preferably capable of blocking human EGFR or human PD-L1 signaling activity in vitro and in vivo. Thus, the EGFR/PD-L1FIT-Ig binding protein of the invention can be used to inhibit or block human EGFR and/or human PD-L1 signaling in human subjects or other mammalian subjects presumed to have EGFR and PD-L1 to which the EGFR/PD-L1FIT-Ig binding protein of the invention cross reacts.

Methods of inhibiting or blocking EGFR signaling in a cell comprise contacting a cell expressing EGFR with an EGFR/PD-L1FIT-Ig binding protein of the present invention.

A method of inhibiting or blocking PD-L1 signaling in a cell comprises contacting a cell expressing PD-L1 with EGFR/PD-L1FIT-Ig binding.

Methods of inhibiting or blocking EGFR signaling and PD-L1 signaling comprise contacting a cell population comprising EGFR-expressing cells and PD-L1-expressing cells with an EGFR/PD-L1FIT-Ig binding protein of the invention.

In the above method, a useful FIT-Ig binding protein is an EGFR/PD-L1FIT-Ig binding protein comprising a polypeptide comprising an amino acid sequence according to SEQ ID NO: 1; comprising a sequence according to SEQ ID NO: 2; and comprising a sequence according to SEQ ID NO: 3, or a second (light) polypeptide chain.

The present invention also provides a method of treating cancer in a human subject in need of such treatment, the method comprising administering to the subject an EGFR/PD-L1FIT-Ig binding protein or a pharmaceutical composition comprising the EGFR/PD-L1FIT-Ig binding protein, wherein the EGFR/PD-L1FIT-Ig binding protein comprises a polypeptide comprising an amino acid sequence according to SEQ ID NO: 1; comprising a sequence according to SEQ ID NO: 2; and comprising a sequence according to SEQ ID NO: 3, or a second (light) polypeptide chain.

In the method of treating cancer in a human subject according to the invention, the cancer may be an epithelial cancer.

In another embodiment, the cancer treated in the methods of the invention is selected from: melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.

In one embodiment, the invention provides a method of restoring activity (reversal of inhibition) of activated T cells, the method comprising contacting cells expressing human PD-L1 with an EGFR/PD-L1FIT-Ig binding protein of the invention such that PD-L1/PD-1 initiated T cell inhibition is inhibited. In another embodiment, the invention provides a method of inhibiting carcinogenesis induced by EGFR/EGF binding comprising contacting cells expressing human EGFR with an EGFR/PD-L1FIT-Ig binding protein of the invention such that EGFR/EGF mediated signaling is inhibited or blocked.

In another embodiment, the invention provides a method for treating a human subject suffering from a disease in which EGFR and/or PD-L1 activity is detrimental, the method comprising administering to the subject an EGFR/PD-L1 binding protein of the invention such that activity mediated by PD-L1/PD1 binding and/or EGFR/EGF binding is reduced in the subject.

In the above method, a useful FIT-Ig binding protein is an EGFR/PD-L1FIT-Ig binding protein comprising a polypeptide comprising an amino acid sequence according to SEQ ID NO: 1; comprising a sequence according to SEQ ID NO: 2; and comprising a sequence according to SEQ ID NO: 3, or a second (light) polypeptide chain.

As used herein, the term "disease in which EGFR and/or PD-L1 activity is detrimental" is intended to include diseases in which EGFR interaction with its ligand (EGR) or PD-L1 interaction with ligand (PD-1) in a subject suffering from a disorder is responsible for or contributes to the pathophysiology of the disease. Thus, a disease in which EGFR and/or PD-L1 activity is detrimental is one in which inhibition of EGFR and/or PD-L1 activity is expected to reduce the symptoms and/or progression of the disease.

In view of the binding of the EGFR/PD-L1-FIT-Ig binding protein of the present invention to human EGFR and PD-L1, the EGFR/PD-L1 binding protein can also be used, for example, to detect EGFR or PD-L1, or both, in a biological sample that contains cells expressing one or both of those target proteins. For example, the EGFR/PD-L1 binding protein of the present invention can be used in conventional immunoassays, such as enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA) or immunohistochemistry of tissues. The invention provides a method for detecting EGFR or PD-L1 in a biological sample, the method comprising contacting the biological sample with an EGFR/PD-L1FIT-Ig binding protein of the invention, and detecting whether binding to a target antigen (EGFR or PD-L1) occurs, thereby detecting the presence or absence of the target in the biological sample. The binding protein may be directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody/fragment/binding protein. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin(ii) a Examples of suitable fluorescent substances include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; examples of suitable radioactive materials include³H、¹⁴C、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I、¹⁷⁷Lu、¹⁶⁶Ho or¹⁵³Sm。

Having now described the invention in detail, it will be more clearly understood by reference to the following examples, which are included merely for purposes of illustration and are not intended to limit the invention.

Examples

Example 1: production of FIT-Ig binding protein that binds EGFR and PD-L1.

Six bispecific Fabs-in-Tandem immunoglobulin (FIT-Ig) binding proteins recognizing human EGFR and human PD-L1 were constructed using the binding sites from anti-PD-L1 and anti-EGFR parent antibodies.

anti-PD-L1 monoclonal antibodies (mabs) 1B12, 10a5, and 3G10 have been previously described. See, for example, U.S. patent No. 7,943,743B 2.

The use of the specific amino acid sequence of the anti-EGFR mAb panitumumab to prepare FIT-Ig binding proteins has been previously described. See, for example, international publication No. WO 2017/136820 a 2.

Example 1.1: FIT-Ig 1.

PD-L1/EGFR FIT-Ig (also referred to as "PD-L1/EGFR FIT-Ig 1") designated "FIT-Ig 1" was constructed using the coding sequences of the immunoglobulin domains from the parent antibody mAb 1B12 and panitumumab. FIT-Ig1 is a hexamer composed of three component polypeptide chains:

polypeptide chain #1 has the domain formula: VL_1B12Direct fusion of-CL to VH_pani-CH1, and VH_pani-CH1 is fused directly to the hinge-CH 2-CH3 (human IgG1 Fc region);

polypeptide chain #2 has the domain formula: VH_1B12-CH 1; and

polypeptide chain #3 has the structureDomain formula: VL_pani-CL。

The amino acid sequences of the three expressed FIT-Ig1 polypeptide chains, including the N-terminal signal sequence, are shown in Table 1 below:

TABLE 1 amino acid sequence of FIT-Ig1 component polypeptide chain

Example 1.2: FIT-Ig2

EGFR/PD-L1FIT-Ig (also referred to as "EGFR/PD-L1 FIT-Ig 2") designated "FIT-Ig 2" was constructed using coding sequences from immunoglobulin domains of the parent antibodies panitumumab and mAb 1B 12. FIT-Ig2 is a hexamer composed of three component polypeptide chains:

polypeptide chain #1 has the domain formula: VL_paniDirect fusion of-CL to VH_1B12-CH1, and VH_1B12-CH1 is fused directly to the hinge-CH 2-CH3 (human IgG1 Fc region);

polypeptide chain #2 has the domain formula: VH_pani-CH 1; and

polypeptide chain #3 has the domain formula: VL_1B12-CL。

The amino acid sequences of the three expressed FIT-Ig2 polypeptide chains, including the N-terminal signal sequence, are shown in Table 2 below:

TABLE 2 amino acid sequence of FIT-Ig2 component polypeptide chain

Example 1.3: FIT-Ig3

PD-L1/EGFR FIT-Ig (also referred to as "PD-L1/EGFR FIT-Ig 3") designated "FIT-Ig 3" was constructed using coding sequences from the immunoglobulin domains of the parent antibody 10A5 and panitumumab. FIT-Ig3 is a hexamer composed of three component polypeptide chains:

polypeptide chain #1 has the domain formula: VL_10A5Direct fusion of-CL to VH_pani-CH1, and VH_pani-CH1 is fused directly to the hinge-CH 2-CH3 (human IgG1 Fc region);

polypeptide chain #2 has the domain formula: VH_10A5-CH 1; and

polypeptide chain #3 has the domain formula: VL_pani-CL。

The amino acid sequences of the three expressed FIT-Ig3 polypeptide chains, including the N-terminal signal sequence, are shown in Table 3 below:

TABLE 3 amino acid sequence of FIT-Ig3 component polypeptide chain

Example 1.4: FIT-Ig4

EGFR/PD-L1FIT-Ig (also referred to as "EGFR/PD-L1 FIT-Ig 4") designated "FIT-Ig 4" was constructed using coding sequences from the immunoglobulin domains of the parent antibodies panitumumab and mAb 10a 5. FIT-Ig4 is a hexamer composed of three component polypeptide chains:

polypeptide chain #1 has the domain formula: VL_paniDirect fusion of-CL to VH_10A5-CH1, and VH_10A5-CH1 is fused directly to the hinge-CH 2-CH3 (human IgG1 Fc region);

polypeptide chain #2 has the domain formula: VH_pani-CH 1; and

polypeptide chain #3 has a knotThe domain formula is as follows: VL_10A5-CL。

The amino acid sequences of the three expressed FIT-Ig4 polypeptide chains, including the N-terminal signal sequence, are shown in Table 4 below:

TABLE 4 amino acid sequence of FIT-Ig4 component polypeptide chain

Example 1.5: FIT-Ig5

PD-L1/EGFR FIT-Ig (also referred to as "PD-L1/EGFR FIT-Ig 5") designated "FIT-Ig 5" was constructed using coding sequences from the immunoglobulin domains of the parent antibody mAb3G10 and panitumumab. FIT-Ig5 is a hexamer composed of three component polypeptide chains:

polypeptide chain #1 has the domain formula: VL_3G10Direct fusion of-CL to VH_pani-CH1, and VH_pani-CH1 is fused directly to the hinge-CH 2-CH3 (human IgG1 Fc region);

polypeptide chain #2 has the domain formula: VH_3G10-CH 1; and

polypeptide chain #3 has the domain formula: VLpani-CL.

The amino acid sequences of the three expressed FIT-Ig5 polypeptide chains, including the N-terminal signal sequence, are shown in Table 5 below:

TABLE 5 amino acid sequence of FIT-Ig5 component polypeptide chain

Example 1.6: FIT-Ig6

EGFR/PD-L1FIT-Ig (also referred to as "EGFR/PD-L1 FIT-Ig 6") designated "FIT-Ig 6" was constructed using coding sequences from the immunoglobulin domains of the parent antibodies panitumumab and mAb3G 10. FIT-Ig6 is a hexamer composed of three component polypeptide chains:

polypeptide chain #1 has the domain formula: VL_paniDirect fusion of-CL to VH_3G10-CH1, and VH_3G10-CH1 is fused directly to the hinge-CH 2-CH3 (human IgG1 Fc region);

polypeptide chain #2 has the domain formula: VH_pani-CH 1; and

polypeptide chain #3 has the domain formula: VL_3G10-CL。

The amino acid sequences of the three expressed FIT-Ig6 polypeptide chains, including the N-terminal signal sequence, are shown in Table 6 below:

TABLE 6 amino acid sequence of FIT-Ig6 component polypeptide chain

Example 1.7: expression of FIT-Ig binding protein

Six FIT-Ig constructs FIT-Ig1, FIT-Ig2, FIT-Ig3, FIT-Ig4, FIT-Ig5, FIT-Ig6 are a class of bispecific multivalent binding proteins known as non-linker Fabs-in-Tandem immunoglobulins (or non-linker FIT-Ig), described generally in WO2015/103072 and WO 2017/136820. The binding protein is produced by co-expressing three component polypeptide chains in a mammalian host cell transfected with an all three-chain expression vector. The design of binding proteins requires that the first polypeptide chain (or "heavy chain") pair with the second polypeptide chain (or "first light chain") and the third polypeptide chain (or "second light chain") to form the Fab portion in functional tandem, and that the heavy chain also be designed to dimerize via the Fc region (hinge-CH 2-CH3) to form a six-chain binding protein showing four complete Fab binding sites. Synthetic amino acid linker peptides were not used to link immunoglobulin domains, hence the designation "linker-free FIT-Igs"; this binding protein was found to be well expressed in host cells, similar to recombinantly produced monoclonal antibodies, and the absence of a linker prevented the introduction of a potential immunogenic site, which could lead to faster clearance of FIT-Ig with the linker. It was also found that the linker-free FIT-Ig exhibited comparable binding properties to its target antigen to the parent antibody, wherein based on VH-CH1 and VL-CL, steric hindrance between "internal" and "external" binding sites found in previously engineered antibodies with tandem arrangements of antigen binding sites was unexpectedly avoided. However, as shown herein, despite the use of the non-linker FIT-Ig model to be effective in routine preclinical and clinical assays required for the evaluation of therapeutic anti-cancer drugs, previous FIT-Ig protein constructs that bind PD-L1 and EGFR (e.g., FIT-Ig 1-5) exhibit abnormally low levels of expression and/or undesirably high percentages of aggregates.

In the binding proteins FIT-Ig1, FIT-Ig3 and Fit-Ig5, the N-terminal or "external" Fab binding site binds to PD-1, while the adjacent "internal" Fab binding site binds to EGFR. The outer Fab fragment (anti-PD-L1 mAb 1B12, 10a5 or 3G10) is linked to the inner Fab fragment (of anti-EGFR panitumumab) only via the heavy chain by direct fusion of VL-CL at its C-terminus (anti-PD-L1 mAb 1B12, 10a5 or 3G10) to the N-terminus of VH-CH1 (of anti-EGFR panitumumab) without the use of a linker linking the immunoglobulin domains.

In the binding proteins FIT-Ig2, FIT-Ig4 and Fit-Ig6, the N-terminal or "outer" Fab binding site binds EGFR, while the adjacent "inner" Fab binding site binds PD-L1. The outer Fab fragment (anti-EGFR panitumumab) is linked to the inner Fab fragment (of anti-PD-L1 mAb 1B12, 10a5 or 3G10) only by the heavy chain via direct fusion of the VL-CL of (anti-EGFR panitumumab) at its C-terminus to the N-terminus of VH-CH1 of (anti-PD-L1 mAbs 1B12, 10a5 or 3G10) without the use of a linker to link the immunoglobulin domains.

Each FIT-Ig was transiently expressed using transfected human embryonic kidney 293E (HEK293) cells. The HEK293E cell line is a derivative of HEK293 expressing EBNA-1 and provides increased levels of expression of recombinant proteins encoded by the vector.

The expression vector allows the expression of each of the three polypeptide chains of any FIT-Ig binding protein, wherein the first (heavy) polypeptide chain has the following structural formula: VL_A-CL-VH_B-CH1-Fc, the second (first light chain) polypeptide chain having the following structural formula: VH_A-CH1, and the third (second light) polypeptide chain has the following structural formula: VL_B-CL wherein VL_AAnd VH_AA variable domain that is an antigen binding site of a first parent antibody, and VL_BAnd VH_BIs a variable domain of the antigen binding site of the second parent antibody.

As shown in FIG. 1, to express the first polypeptide chain of the FIT-Ig binding protein ("heavy chain"), VL encoding the first polypeptide chain was synthesized_A-CL-VH_BDNA molecules of the fragments ("DNA synthesis"). The DNA molecule is then inserted into the Multiple Cloning Site (MCS) of the pcDNA3.1 expression vector in E.coli cells using homologous recombination. The homologous recombination method relies on the principle that recombinase-positive E.coli cells can recombine homologous sequences with high specificity and high speed. Linear DNA fragments containing the coding sequence of interest were generated by Polymerase Chain Reaction (PCR) to include sequences on the 5 'and 3' ends that are homologous to the end sequences on the linearized vector. When the PCR product is mixed with a linear vector and transformed into competent E.coli cells, endogenous bacterial recombinase activity can join the two DNA fragments to form a circular plasmid. The inserted DNA molecule is then positioned downstream of the strong Cytomegalovirus (CMV) enhancer promoter of the vector, also downstream of and in-frame with the DNA fragment encoding the amino-terminal Signal Peptide (SP), and upstream of and in-frame with the inserted DNA molecule encoding the CH1 domain of the antibodyThe CH1 domain is linked to an antibody Fc region comprising a hinge region-CH 2-CH3 domain (designated "h-CH 2-CH 3" in FIG. 1).

As also shown in FIG. 1, to express the second polypeptide chain of the FIT-Ig binding protein ("light chain #1), the antibody VH was synthesized encoded_AThe DNA fragment of the domain, which is then inserted into the Multiple Cloning Site (MCS) of the pcdna3.1 expression vector, such that the inserted DNA molecule is located downstream of the vector's strong CMV enhancer promoter, also downstream of and in frame with the DNA fragment encoding the amino terminal Signal Peptide (SP), and upstream and in frame with the inserted DNA molecule encoding the antibody CH1 domain.

As shown in FIG. 1, to express the third polypeptide chain of the FIT-Ig binding protein (light chain #3), the antibody VH was synthesized_BThe DNA fragment of the domain is then inserted into the Multiple Cloning Site (MCS) of the pcdna3.1 expression vector such that the inserted DNA molecule is located downstream of the vector's strong CMV enhancer promoter, also downstream of and in frame with the DNA fragment encoding the amino terminal Signal Peptide (SP), and upstream and in frame with the inserted DNA molecule encoding the antibody CL domain.

The sequence of the resulting expression vector was confirmed by DNA sequencing.

Heavy chain: light chain #1 light chain #2 at a molar ratio of 1:3, and the resulting expression vectors encoding the three component polypeptide chains of each FIT-Ig were transfected into HEK293E cells. The aim of this design was to express proportionally more light chains #1 and #2 relative to the heavy chain, which in turn would reduce the occurrence of VL-CL and VH-CH1 fragments on the heavy chain that are not paired with the corresponding light chains and would therefore lead to the failure to form functional Fab fragments. See WO 2015/103072. HEK293E cells were transfected with expression vectors using Polyethyleneimine (PEI) as transfection agent. In this transfection protocol, FreeStyle is used^TM293 expression medium and PEI, wherein the final concentration ratio of DNA to PEI is 1:2, incubated at room temperature for 15-20 minutes, then added to HEK293E cells (1.0-1.2X 10) at 60. mu.g DNA/120ml culture medium⁶Perml, cell viability>95%). After 6-24 hours of incubation in a shaker, peptone was added to the transfected cellsIntracellular, final concentration 5%, at 37 ℃ in 8% CO₂Next, the mixture was shaken at 125 rpm/min. On days 6-7, the supernatant was collected by centrifugation and filtration, and the FIT-Ig protein was purified using protein A chromatography (GE healthcare, US) according to the manufacturer's instructions. Proteins were analyzed by SDS-PAGE and their concentration determined by UV absorbance at 280nm and bicinchoninic acid protein assay (BCA) (Pierce BCA protein assay kit, Thermo Fisher Scientific).

FIT-Ig protein expression product was purified by protein A chromatography. The purified FIT-Ig was then analyzed for composition and purity by Size Exclusion Chromatography (SEC). PBS containing purified FIT-Ig was applied to a TSKgel SuperSW3000, 300X 4.6mm column (TOSOH). HPLC instrument, model U3000 (DIONEX) for SEC, using UV detection at 280nm and 214 nm. Substances detected by SEC, including larger (higher molecular weight) aggregates and smaller substances (including fragments of the FIT-Ig6 monomer), are impurities, with the exception of the hexapolypeptide chain FIT-Ig6 monomer (molecular weight 240,000 daltons).

FIGS. 2-7 show SEC elution profiles for FIT-Ig1 through FIT-Ig6, respectively.

The SEC elution profile of FIT-Ig1 shown in FIG. 2 reveals multiple overlapping peaks of protein aggregates. The figure is too complex to allow detailed analysis of the material of a single peak.

The SEC elution profile for FIT-Ig2 shown in FIG. 3 indicates that there is one major peak at the expected position of the hexapolypeptide FIT-Ig2 monomer, preceded by at least two other peaks of protein aggregates.

The SEC elution profile for FIT-Ig3 shown in FIG. 4 indicates multiple overlapping peaks for protein aggregates. The figure is too complex to allow detailed analysis of the material of a single peak.

The SEC elution profile of FIT-Ig4 shown in FIG. 5 indicates that the hexapeptide FIT-Ig4 monomer has one major peak at the expected position, preceded by a minor peak of protein aggregates, followed by another minor peak of unknown species (possibly degraded products).

The SEC elution profile for FIT-Ig5 shown in FIG. 6 indicates that there is one major peak at the expected location of the hexapeptide FIT-Ig5 monomer, preceded by a minor peak for protein aggregates that is significantly smaller than the previous minor peak for any aggregates found in FIGS. 2-5.

The SEC elution profile for FIT-Ig6 shown in FIG. 7 indicates that there is a major peak at the expected position of the hexapolypeptide FIT-Ig6 monomer, preceded by a barely detectable peak in the protein aggregate region. After one-step purification using protein a affinity chromatography, it was evident that FIT-Ig 6-binding protein was obtained as a substantially completely homogeneous product without significant aggregate formation. The percentage of aggregates is estimated to be less than or equal to 0.1% (≦ 0.1%). FIT-Ig6 clearly exceeded all other FIT-Igs, with no significant percentage of aggregates.

The expression of FIT-Ig 1-6 and SEC data are shown in Table 7 below.

Table 7: expression of FIT-Ig binding protein and SEC analysis

The above data show that:

FIT-Ig1, 2 and 3 showed an unusually high percentage of aggregates and unacceptably low levels of expression in transfected HEK293 cell cultures, and therefore failed to provide the protein quality (no aggregates or insignificant, low percentage of aggregates) and quantity required for further preclinical evaluation as a therapeutic drug.

The percentage of aggregates of FIT-Ig4 was lower than FIT-Ig1, 2, and 3, but this level was not insignificant for drug development. Furthermore, FIT-Ig4 showed unacceptably low levels of expression. Thus, FIT-Ig4 also failed to provide the quantity and quality of protein required for further preclinical evaluation as a therapeutic drug.

Aggregate and expression levels of FIT-Ig5 were almost acceptable, but expression levels below 10mg/ml indicated a need for efforts to isolate stably transfected CHO cell lines to obtain the quantities required for preclinical and clinical evaluation, which would be unsuccessful or cost-ineffective.

FIT-Ig6 surprisingly showed high level expression in transfected HEK293 cell cultures and no significant amount of aggregate formation (< 0.1%). Thus, FIT-Ig6 was more stable than FIT-Ig5 in aggregate formation after one-step purification using protein A affinity chromatography, and the percentage of aggregates was at least 10-fold lower. The expression level of FIT-Ig6 in mammalian cell culture and the exceptionally low aggregate levels qualify this binding protein for use as a candidate for preclinical and clinical evaluation of anti-cancer therapeutics.

The special properties of FIT-Ig6 are due to:

1. anti-PD-L1 mAb3G10 was used as a source of VH and VL domains that form a PD-L1 specific antigen binding site in each PD-L1 specific Fab binding unit of FIT-Ig6,

2. panitumumab anti-EGFR mAb was used as a source of VH and VL domains that form EGFR-specific antigen binding sites in each EGFR-specific Fab binding unit of FIT-Ig6,

3. locating the EGFR-specific Fab binding unit as the outer Fab binding unit of FIT-Ig6, and

4. the PD-L1 specific Fab binding unit was located as an internal Fab binding unit of FIT-Ig 6.

Example 2: binding affinity of FIT-Ig5 and FIT-Ig 6.

The binding affinities of the parent anti-EGFR panitumumab, parent anti-PD-L1 mAb3G10, FIT-Ig5 and FIT-Ig6 were determined by biolayer interferometry. In short, byRED96 biolayer interferometry (Pall Forte Bio LLC) characterizes the affinity and binding kinetics of each parent mAb and FIT-Ig. Each parental mAb and FIT-Ig were captured by an anti-human IgG Fc capture (AHC) biosensor (Pall) at a concentration of 100nM for 30 seconds. The sensor was then immersed in running buffer (1X, pH 7.2, PBS, 0.05% Tween 20, 0.1% BSA) for 60 seconds to check for baseline. By immersing the sensor in a single concentration in the range of 1nM to 200nMDegree of recombinant human PD-L1(Novoprotein) or recombinant human EGFR (Sino Biological Inc) to measure binding. After that, the sensor was immersed in running buffer for 1200 seconds to dissociate. The binding and dissociation curves were fitted to 1: 1 Langmuir binding model. The results are shown in Table 8.

TABLE 8 binding affinities of parent mAb and FIT-Ig to PD-L1 and EGFR

The results show that the binding affinities of FIT-Ig5 and FIT-Ig6 to EGFR and PD-L1 target antigens are similar to the binding affinities of the parent panitumumab and parent mAb3G10, respectively. In particular, K of FIT-Ig6 against EGFR_DK over parent panitumumab_DAbout 25% lower, while FIT-Ig6 has a K for PD-L1_DK over parent mAb3G10_DAbout 30% higher. K of FIT-Ig6 and parent mAb_DThese levels of difference in values may be due to assay differences. Thus, FIT-Ig6 has substantially the same affinity for EGFR and PD-L1 (i.e., the same or within 30% of the affinity for each affinity antibody) as each parent antibody of the corresponding specific origin.

Thus, these data indicate that the FIT-Ig6 binding protein retained the binding affinity of the parent mAb. Furthermore, the binding affinity of the FIT-Ig6 binding protein is acceptable for continued preclinical and clinical evaluation of FIT-Ig6 as an anti-cancer drug.

Example 3 pharmacokinetic study of FIT-Ig6 in male Sprague-Dawley rats.

FIT-Ig6 was evaluated for pharmacokinetic properties in male Sprague-Dawley (SD) rats. Male SD rats were administered FIT-Ig protein in a single intravenous dose of 5 mg/kg. Serum samples were collected at various time points during the 28 day period, with samples taken continuously through the tail vein at 0 minutes, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days, and 28 days and analyzed by conventional ELISA. Briefly, goat anti-antibody at 125 ng/wellHuman IgG Fc antibody (Rockland, Cat: 609-101-017) ELISA plates were coated overnight at 4 ℃ with 1 XPBS/1% BSA/0.05% Tween-20/0.05% ProClin^TM300 are closed. All serum samples were first diluted 20-fold in blocking buffer. Additional dilutions were made in 5% pooled rat serum and incubated for 60 minutes on plates at 37 ℃. Detection was performed with peroxidase-conjugated anti-human IgG (Fab fragment) (Sigma; cat # A0293) and concentration was determined by standard curve using four-parameter logarithmic fit. Values of pharmacokinetic parameters were determined by a non-compartmental model (non-comparative model) using WinNonlin software (Pharsight Corporation, Mountain View, Calif.).

FIG. 8 shows a plot of serum concentration versus time for FIT-Ig6 in three SD rats.

Analysis of the results shown in figure 8 for two of the animals (rat #1 and rat #3) resulted in the PK parameters shown in table 9 below. (due to the unresolved problems associated with the first two data points (which were excluded by the software, although the remaining time points were within the usual range), the complete data set for rat #2 could not be analyzed along with rat #1 and rat # 3).

TABLE 9 PK parameters for FIT-Ig6 in Male Sprague-Dawley rats

PK parameters	Unit cell	Rat #1	Rat #3	Mean value of
					CL	mL/day/kg	7.52	6.02	6.77
Vss	mL/kg	101	105	103
					V1	mL/kg	58.0	56.0	57.0
Alpha t1/2	Sky	0.148	0.189	0.168
					Beta t1/2	Sky	9.43	12.3	10.9
AUC	Mu g/mL day	665	830	747
					MRT	Sky	13.4	17.5	15.5

CL (Total clearance gap), Vss (Steady State volume distribution), V1 (initial volume distribution), α t_1/2(half-life of distribution), beta.t_1/2(elimination half-life), AUC (area under curve), MRT (mean residence time).

The above PK data indicate that FIT-Ig6 is stable in SD rats and has PK parameters similar to those of conventional mAb.

Importantly, the relatively long elimination half-life (. beta.t) of FIT-Ig6_1/2Day 10.9) and low clearance (CL ═ 6.77 mL/day/kg) would allow for lower dosing frequency for treatment of chronic indications, similar to therapeutic mabs.

The contents of all references (including literature references, patents, patent applications, and web sites) cited throughout this application are expressly incorporated herein by reference in their entirety. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology and cell biology, which are well known in the art.

The present invention may be embodied in other specific forms without departing from its essential characteristics. Accordingly, the foregoing embodiments are to be considered illustrative rather than limiting of the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.