PD-1 binding proteins, vegf and pd-1 bispecific binding proteins, compositions, and methods of use

Abstract

Disclosed are bispecific antibodies that bind VEGF and PD-1, pharmaceutical compositions thereof, and methods of use thereof, including methods of inhibiting VEGF and PD-1 biological activity.

Description

Background Technology

[0001] Vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells and has been reported as a key regulator of both normal and abnormal angiogenesis. VEGF is a major regulator of angiogenesis during growth and development, as well as in disease states such as cancer, diabetes, and macular degeneration. It is also essential for cyclical angiogenesis in the female reproductive tract, as well as for bone growth and cartilage formation. Cancer is one of the most common indications for current VEGF therapy, with FDA and EMA-approved therapies targeting a variety of cancers. Some examples of current VEGF therapies used to address these diseases and conditions include axitinib, bevacizumab, busizumab, carboplatinib, lapatinib, lenvatinib, pazopanib, ponatinib, ramoximumab, ranibizumab, regorafenib, sorafenib, sunitinib, and vastanib.

[0002] Programmed death receptor 1 (PD-1) is an immunosuppressive receptor primarily expressed on activated lymphocytes, such as peripheral CD4+ and CD8+ T cells, B cells, and monocytes. Interactions with its ligands have been shown to attenuate T cell responses both in vitro and in vivo.

[0003] The role of PD-1 in cancer is well-established in the literature. PD-1 ligands (programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2)) are constitutively expressed or induced in various cell types, including non-hematopoietic tissues and various tumor types. For example, blocking the interaction between PD-1 and PD-L1 has been shown to enhance tumor-specific CD8+ T cell immunity and may therefore contribute to the immune system's clearance of tumor cells. Overall, the PD-1 / PD-L1 pathway is a well-validated target for developing antibody therapeutics for cancer treatment. Summary of the Invention

[0004] This disclosure relates in part to PD-1 binding proteins and bispecific binding proteins that bind (1) VEGF and (2) PD-1.

[0005] In one aspect, a programmed death receptor 1 (PD-1) binding protein comprising an anti-PD1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) is provided, the anti-PD-1 VH comprising the following complementarity-determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 445; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 456, 461, or 446; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 457 or 447; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 451; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 458, 462, or 452; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 459 or 453; or CDR-H1 comprising the amino acid sequence of SEQ ID NO: 443; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 460, 463, or 455; and CDR-H2 comprising the amino acid sequence of SEQ ID NO: CDR-H3 with amino acid sequence 457 or 447, provided that: if CDR-H2 contains amino acid sequence SEQ ID NO: 446, then CDR-H3 does not contain amino acid sequence 447; if CDR-H2 contains amino acid sequence SEQ ID NO: 452, then CDR-H3 does not contain amino acid sequence 453; and if CDR-H2 contains amino acid sequence SEQ ID NO: 455, then CDR-H3 does not contain amino acid sequence 447.

[0006] In some embodiments, CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of the following: respectively, SEQ ID NO 445, 456, and 457; respectively, SEQ ID NO 451, 458, and 459; or respectively, SEQ ID NO 443, 460, and 457. In some embodiments, the PD-1 binding protein further comprises an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL), the anti-PD-1 VL comprising the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 454; CDR-L2 comprising the amino acid sequence of LAS; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; or CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450.In some embodiments, anti-PD-1 VH and anti-PD-1 VL have an amino acid sequence that is at least 85% identical to the amino acid sequences of the following: respectively, SEQ ID NO: 302 and 335; respectively, SEQ ID NO: 302 and 336; respectively, SEQ ID NO: 302 and 340; respectively, SEQ ID NO: 302 and 342; respectively, SEQ ID NO: 302 and 343; respectively, SEQ ID NO: 302 and 346; respectively, SEQ ID NO: 306 and 337; respectively, SEQ ID NO: 306 and 339; respectively, SEQ ID NO: 306 and 341; respectively, SEQ ID NO: 306 and 344; respectively, SEQ ID NO: 306 and 345; respectively, SEQ ID NO: 306 and 347; respectively, SEQ ID NO: 308 and 340; respectively, SEQ ID NO: 309 and 341; respectively, SEQ ID NO: 310 and 336; respectively, SEQ ID NO: 311 and 337; respectively, SEQ ID NO: 313 and 336; respectively, SEQ ID NO: 313 and 342; respectively, SEQ ID NO: 313 and 343; respectively, SEQ ID NO: 316 and 337; respectively, SEQ ID NO: 316 and 344; respectively, SEQ ID NO: 316 and 345; respectively, SEQ ID NO: 318 and 336; respectively, SEQ ID NO: 318 and 340; respectively, SEQ ID NO: 318 and 346; respectively, SEQ ID NO: 319 and 336; respectively, SEQ ID NO: 319 and 340; respectively, SEQ ID NO: 319 and 346; respectively, SEQ ID NO: 320 and 337; respectively, SEQ ID NO: 320 and 341; respectively, SEQ ID NO: 320 and 347; respectively, SEQ ID NO: 320 and 743; respectively, SEQ ID NO: 321 and 337; respectively, SEQ ID NO: 321 and 341; or respectively, SEQ ID NO: 321 and 347.In some embodiments, anti-PD-1VH and anti-PD-1VL have the amino acid sequences of the following: respectively, SEQ ID NO: 302 and 335; respectively, SEQ ID NO: 302 and 336; respectively, SEQ ID NO: 302 and 340; respectively, SEQ ID NO: 302 and 342; respectively, SEQ ID NO: 302 and 343; respectively, SEQ ID NO: 302 and 346; respectively, SEQ ID NO: 306 and 337; respectively, SEQ ID NO: 306 and 339; respectively, SEQ ID NO: 306 and 341; respectively, SEQ ID NO: 306 and 344; respectively, SEQ ID NO: 306 and 345; respectively, SEQ ID NO: 306 and 347; respectively, SEQ ID NO: 308 and 340; respectively, SEQ ID NO: 309 and 341; respectively, SEQ ID NO: 310 and 336; respectively, SEQ ID NO: 311 and 337; respectively, SEQ ID NO: 313 and 336; respectively, SEQ ID NO: 313 and 342; respectively, SEQ ID NO: 313 and 343; respectively, SEQ ID NO: 316 and 337; respectively, SEQ ID NO: 316 and 344; respectively, SEQ ID NO: 316 and 345; respectively, SEQ ID NO: 318 and 336; respectively, SEQ ID NO: 318 and 340; respectively, SEQ ID NO: 318 and 346; respectively, SEQ ID NO: 319 and 336; respectively, SEQ ID NO: 319 and 340; respectively, SEQ ID NO: 319 and 346; respectively, SEQ ID NO: 320 and 337; respectively, SEQ ID NO: 320 and 341; respectively, SEQ ID NO: 320 and 347; respectively, SEQ ID NO: 320 and 743; respectively, SEQ ID NO: 321 and 337; respectively, SEQ ID NO: 321 and 341; or respectively, SEQ ID NO: 321 and 347.

[0007] In some embodiments, CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of the following: respectively, SEQ ID NO 445, 456, and 447; respectively, SEQ ID NO 451, 458, and 453; or respectively, SEQ ID NO 443, 460, and 447. In some embodiments, the PD-1 binding protein further comprises an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL), the anti-PD-1 VL comprising the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 454; CDR-L2 comprising the amino acid sequence of LAS; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; or CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450. In some embodiments, anti-PD-1 VH and anti-PD-1 VL have an amino acid sequence that is at least 85% identical to the amino acid sequences of: respectively, SEQ ID NO: 303 and 336; respectively, SEQ ID NO: 325 and 347; or respectively, SEQ ID NO: 325 and 743. In some embodiments, anti-PD-1 VH and anti-PD-1 VL have the amino acid sequences of: respectively, SEQ ID NO: 303 and 336; respectively, SEQ ID NO: 325 and 347; or respectively, SEQ ID NO: 325 and 743.

[0008] In some embodiments, CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of the following: respectively, SEQ ID NO 445, 461, and 447; respectively, SEQ ID NO 451, 462, and 453; or respectively, SEQ ID NO 443, 463, and 447. In some embodiments, the PD-1 binding protein further comprises an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL), the anti-PD-1 VL comprising the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 454; CDR-L2 comprising the amino acid sequence of LAS; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; or CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450. In some embodiments, anti-PD-1 VH and anti-PD-1 VL have an amino acid sequence that is at least 85% identical to the amino acid sequences of the following: respectively, SEQ ID NO: 326 and 347; or respectively, SEQ ID NO: 326 and 743. In some embodiments, anti-PD-1 VH and anti-PD-1 VL have the amino acid sequences of the following: respectively, SEQ ID NO: 326 and 347; or respectively, SEQ ID NO: 326 and 743.

[0009] In some embodiments, CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of the following: respectively, SEQ ID NO 445, 446, and 457; respectively, SEQ ID NO 451, 452, and 459; or respectively, SEQ ID NO 443, 455, and 457. In some embodiments, the PD-1 binding protein further comprises an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL), the anti-PD-1 VL comprising the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 454; CDR-L2 comprising the amino acid sequence of LAS; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; or CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450. In some embodiments, anti-PD-1 VH and anti-PD-1 VL have an amino acid sequence that is at least 85% identical to the amino acid sequences of the following: respectively, SEQ ID NO: 327 and 347; or respectively, SEQ ID NO: 327 and 743. In some embodiments, anti-PD-1 VH and anti-PD-1 VL have the amino acid sequences of the following: respectively, SEQ ID NO: 327 and 347; or respectively, SEQ ID NO: 327 and 743.

[0010] In some embodiments, CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of the following: respectively, SEQ ID NO 445, 461, and 457; respectively, SEQ ID NO 451, 462, and 459; or respectively, SEQ ID NO 443, 463, and 457. In some embodiments, the PD-1 binding protein further comprises an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL), the anti-PD-1 VL comprising the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 454; CDR-L2 comprising the amino acid sequence of LAS; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; or CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450. In some embodiments, anti-PD-1 VH and anti-PD-1 VL have an amino acid sequence that is at least 85% identical to the amino acid sequences of the following: respectively, SEQ ID NO: 303 and 335; respectively, SEQ ID NO: 303 and 342; respectively, SEQ ID NO: 303 and 343; respectively, SEQ ID NO: 307 and 337; respectively, SEQ ID NO: 307 and 344; respectively, SEQ ID NO: 307 and 345; respectively, SEQ ID NO: 314 and 336; respectively, SEQ ID NO: 314 and 342; respectively, SEQ ID NO: 314 and 343; respectively, SEQ ID NO: 317 and 337; respectively, SEQ ID NO: 317 and 344; respectively, SEQ ID NO: 317 and 345; or respectively, SEQ ID NO: 324 and 347.In some embodiments, anti-PD-1 VH and anti-PD-1 VL have the following amino acid sequences: respectively, SEQ ID NO: 303 and 335; respectively, SEQ ID NO: 303 and 342; respectively, SEQ ID NO: 303 and 343; respectively, SEQ ID NO: 307 and 337; respectively, SEQ ID NO: 307 and 344; respectively, SEQ ID NO: 307 and 345; respectively, SEQ ID NO: 314 and 336; respectively, SEQ ID NO: 314 and 342; respectively, SEQ ID NO: 314 and 343; respectively, SEQ ID NO: 317 and 337; respectively, SEQ ID NO: 317 and 344; respectively, SEQ ID NO: 317 and 345; or respectively, SEQ ID NO: 324 and 347.

[0011] In some embodiments, anti-PD-1 VH has a frame having an amino acid sequence that is at least 85% identical to the amino acid sequence in any one of SEQ ID NO: 464-470, and anti-PD-1 VL has a frame having an amino acid sequence that is at least 85% identical to the amino acid sequence in any one of SEQ ID NO: 471-476.

[0012] In some embodiments, anti-PD-1 VH has a framework having an amino acid sequence of any one of SEQ ID NO: 464-470, and anti-PD-1 VL has a framework having an amino acid sequence of any one of SEQ ID NO: 471-476.

[0013] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 464, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 471.

[0014] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 465, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 472.

[0015] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 465, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 471.

[0016] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 465, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 473.

[0017] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 464, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 472.

[0018] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 464, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 473.

[0019] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 464, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 474.

[0020] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 464, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 475.

[0021] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 467, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 471.

[0022] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 467, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 474.

[0023] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 467, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 475.

[0024] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 468, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 471.

[0025] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 468, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 474.

[0026] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 468, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 475.

[0027] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 466, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 471.

[0028] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 470, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 474.

[0029] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 469, and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 476.

[0030] In some embodiments, anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 465 except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 472 except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 465 except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 471 except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 465 except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 473 except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 464 except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 472 except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 473, except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; The amino acid sequence of NO: 467, except for one amino acid substitution; and the anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution; the anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution;Furthermore, the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 466, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution; or anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 470, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution, wherein one amino acid substitution in VH is from a non-cysteine ​​residue to a cysteine ​​residue, and one amino acid substitution in VL is from a non-cysteine ​​residue to a cysteine ​​residue.

[0031] In some embodiments, an amino acid in anti-PD-1 VH is substituted at residue H44, and an amino acid in anti-PD-1 VL is substituted at residue L100, wherein the numbering is based on Kabat.

[0032] In one aspect, a PD-1 binding protein is provided comprising: (a) an anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the complementarity-determining regions comprise the amino acid sequences of: (1) SEQ ID NO 445, 456, and 447, respectively; (2) SEQ ID NO 451, 458, and 453, respectively; or (3) SEQ ID NO 443, 460, and 457, respectively; and (b) an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL) comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the complementarity-determining regions comprise the amino acid sequences of: (1) SEQ ID NO 448, 449, and 450, respectively; (2) SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450. In some embodiments, (a) anti-PD-1 VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 325; and (b) anti-PD-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347. In some embodiments, (a) anti-PD-1 VH has the amino acid sequence of SEQ ID NO: 325; and (b) anti-PD-1 VL has the amino acid sequence of SEQ ID NO: 347.

[0033] In one aspect, a PD-1 binding protein is provided comprising: (a) an anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the complementarity-determining regions comprise the amino acid sequences of: (1) SEQ ID NO 445, 456, and 457, respectively; (2) SEQ ID NO 451, 458, and 459, respectively; or (3) SEQ ID NO 443, 460, and 457, respectively; and (b) an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL) comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the complementarity-determining regions comprise the amino acid sequences of: (1) SEQ ID NO 448, 449, and 450, respectively; (2) SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450. In some embodiments, (a) anti-PD-1 VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 320; and (b) anti-PD-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347. In some embodiments, (a) anti-PD-1 VH has the amino acid sequence of SEQ ID NO: 320; and (b) anti-PD-1 VL has the amino acid sequence of SEQ ID NO: 347.

[0034] In one aspect, a PD-1 binding protein is provided comprising: (a) an anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the complementarity-determining regions comprise the amino acid sequences of: (1) SEQ ID NO 445, 461, and 457, respectively; (2) SEQ ID NO 451, 462, and 459, respectively; or (3) SEQ ID NO 443, 463, and 457, respectively; and (b) an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL) comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the complementarity-determining regions comprise the amino acid sequences of: (1) SEQ ID NO 448, 449, and 450, respectively; (2) SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450. In some embodiments, (a) anti-PD-1 VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 324; and (b) anti-PD-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347. In some embodiments, (a) anti-PD-1 VH has the amino acid sequence of SEQ ID NO: 324; and (b) anti-PD-1 VL has the amino acid sequence of SEQ ID NO: 347.

[0035] In one aspect, a PD-1 binding protein is provided comprising: (a) an anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein the complementarity-determining regions comprise the amino acid sequences of: (1) SEQ ID NO 445, 461, and 447, respectively; (2) SEQ ID NO 451, 462, and 453, respectively; or (3) SEQ ID NO 443, 463, and 447, respectively; and (b) an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL) comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the complementarity-determining regions comprise the amino acid sequences of: (1) SEQ ID NO 448, 449, and 450, respectively; (2) SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450. In some embodiments, (a) anti-PD-1 VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 326; and (b) anti-PD-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347. In some embodiments, (a) anti-PD-1 VH has the amino acid sequence of SEQ ID NO: 326; and (b) anti-PD-1 VL has the amino acid sequence of SEQ ID NO: 347.

[0036] In one aspect, a PD-1 binding protein is provided comprising: an anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, wherein these complementarity-determining regions comprise the amino acid sequences of: respectively, SEQ ID NO 445, 446, and 457; respectively, SEQ ID NO 451, 452, and 459; or respectively, SEQ ID NO 443, 455, and 457; and an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL) comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein these complementarity-determining regions comprise the amino acid sequences of: respectively, SEQ ID NO 448, 449, and 450; respectively, SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or respectively, SEQ ID NO: 448, 449 and 450.

[0037] In some embodiments, anti-PD-1 VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 327; and anti-PD-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347.

[0038] In some embodiments, anti-PD-1 VH has the amino acid sequence of SEQ ID NO: 327; and anti-PD-1 VL has the amino acid sequence of SEQ ID NO: 347.

[0039] In some embodiments, anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 467, except for up to one amino acid substitution; and anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 475, except for up to one amino acid substitution.

[0040] In some embodiments, one amino acid substitution in both anti-PD-1 VH and anti-PD-1 VL is the H44-L100 cysteine ​​mutation.

[0041] In one aspect, a programmed death receptor 1 (PD-1) binding protein comprising an anti-PD1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) and an anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 VL) is provided, wherein: the anti-PD-1 VH comprises the following complementarity-determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 445; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 446; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 447; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 451; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 452; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 453; or CDR-H1 comprising the amino acid sequence of SEQ ID NO: 443; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 455; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 453; CDR-H3 containing the amino acid sequence of SEQ ID NO: 447; CDR-L3 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; CDR-L1 containing the amino acid sequence of SEQ ID NO: 454; CDR-L2 containing the amino acid sequence of LAS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; or CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450, wherein the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 465; and the anti-PD-1 VL has a frame containing the amino acid sequence of SEQ ID NO: 465. The amino acid sequence of anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 465; and the framework of anti-PD-1 VL has an amino acid sequence of SEQ ID NO: 471; the framework of anti-PD-1 VH has an amino acid sequence of SEQ ID NO: 465; the framework of anti-PD-1 VL has an amino acid sequence of SEQ ID NO: 473; and the framework of anti-PD-1 VH has an amino acid sequence of SEQ ID NO: 464.Furthermore, the anti-PD-1 VL has a frame containing the amino acid sequence of SEQ ID NO: 472; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 464; the anti-PD-1 VL has a frame containing the amino acid sequence of SEQ ID NO: 473; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 464; the anti-PD-1 VL has a frame containing the amino acid sequence of SEQ ID NO: 474; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 464; the anti-PD-1 VL has a frame containing the amino acid sequence of SEQ ID NO: 475; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 467; the anti-PD-1 VL has a frame containing the amino acid sequence of SEQ ID NO: 471; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 472; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 473; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 474; the anti-PD-1 VL has a frame containing the amino acid sequence of SEQ ID NO: 475; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 475; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 477; the anti-PD-1 VL has a frame containing the amino acid sequence of SEQ ID NO: 471; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 473; the anti-PD-1 VH has a frame containing the amino acid sequence of SEQ ID NO: 474; the anti-PD-1 VL has a frame containing the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO: 467; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 474, the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 467; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 475, the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 471, the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 474, the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 475, the anti-PD-1 VH ...75, the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 474, the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 475, the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 474, the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 475, the anti-PD The anti-PD-1 VL has an amino acid sequence of SEQ ID NO: 475; the anti-PD-1 VH has an amino acid sequence of SEQ ID NO: 466; and the anti-PD-1 VL has an amino acid sequence of SEQ ID NO: 471, or the anti-PD-1 VH has an amino acid sequence of SEQ ID NO: 470; and the anti-PD-1 VL has an amino acid sequence of SEQ ID NO: 474.

[0042] In one aspect, a programmed death receptor 1 (PD-1) binding protein comprising an anti-PD1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) and an anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 VL) is provided, wherein the anti-PD-1 VH comprises the following complementarity-determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 445; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 446; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 447; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 451; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 452; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 453; or CDR-H1 comprising the amino acid sequence of SEQ ID NO: 443; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 455; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 453; The CDR-H3 of the amino acid sequence 447, the anti-PD-1 VL, comprises the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 454; CDR-L2 comprising the amino acid sequence of LAS; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450; or CDR-L1 comprising the amino acid sequence of SEQ ID NO: 448; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 449; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 450, and wherein the anti-PD-1 VH has a frame comprising the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution; and the anti-PD-1 VL has a frame comprising the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution; and the anti-PD-1 VL has a frame comprising the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution. The amino acid sequence of SEQ ID NO: 472, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 473, except for one amino acid substitution;The anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 472, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 473, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution; anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution; The amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution; the anti-PD-1 VH has a frame having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and the anti-PD-1 VL has a frame having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution;The anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and the anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution; the anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 466, except for one amino acid substitution; and the anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution; or the anti-PD-1 VH has a framework having the amino acid sequence of SEQ ID NO: 470, except for one amino acid substitution; and the anti-PD-1 VL has a framework having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution, wherein one amino acid substitution in VH is from a non-cysteine ​​residue to a cysteine ​​residue, and one amino acid substitution in VL is from a non-cysteine ​​residue to a cysteine ​​residue.

[0043] In some embodiments, an amino acid in anti-PD-1 VH is substituted at residue H44, and an amino acid in anti-PD-1 VL is substituted at residue L100, wherein the numbering is based on Kabat.

[0044] In one aspect, a programmed death receptor 1 (PD-1) binding protein comprising an anti-PD1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) and an anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 VL) is provided, wherein: the anti-PD-1 VH comprises the following complementarity-determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 445; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 446; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 447; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 451; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 452; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 453; or CDR-H1 comprising the amino acid sequence of SEQ ID NO: 443; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 455; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 453; CDR-H3 of the amino acid sequence of SEQ ID NO: 447; anti-PD-1 VL contains the following complementarity-determining regions: CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; CDR-L1 containing the amino acid sequence of SEQ ID NO: 454; CDR-L2 containing the amino acid sequence of LAS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; or CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450, and wherein anti-PD-1 VH and anti-PD-1 VL have at least 85% identical amino acid sequences to the following: respectively, SEQ ID NO: 301 and 335; respectively, SEQ ID SEQ ID NO: 301 and 336; respectively, SEQ ID NO: 301 and 342; respectively, SEQ ID NO: 301 and 343; respectively, SEQ ID NO: 301 and 348; respectively, SEQ ID NO: 305 and 337; respectively, SEQ ID NO: 305 and 344; respectively, SEQ ID NO: 305 and 345; respectively, SEQ ID NO: 312 and 335; respectively, SEQ ID NO: 312 and 336; respectively, SEQ ID NO: 312 and 342; respectively, SEQ ID NO: 312 and 343;Specifically, SEQ ID NO: 315 and 337; SEQ ID NO: 315 and 344; SEQ ID NO: 315 and 345; SEQ ID NO: 322 and 346; SEQ ID NO: 323 and 347; or SEQ ID NO: 323 and 743.

[0045] In some embodiments, anti-PD-1 VH and anti-PD-1 VL have the amino acid sequences of the following: respectively, SEQ ID NO: 301 and 335; respectively, SEQ ID NO: 301 and 336; respectively, SEQ ID NO: 301 and 342; respectively, SEQ ID NO: 301 and 343; respectively, SEQ ID NO: 301 and 348; respectively, SEQ ID NO: 305 and 337; respectively, SEQ ID NO: 305 and 344; respectively, SEQ ID NO: 305 and 345; respectively, SEQ ID NO: 312 and 335; respectively, SEQ ID NO: 312 and 336; respectively, SEQ ID NO: 312 and 342; respectively, SEQ ID NO: 312 and 343; respectively, SEQ ID NO: 315 and 337; respectively, SEQ ID NO: 315 and 344; respectively, SEQ ID NO: 315 and 345; respectively, SEQ ID NO: 322 and 346; respectively, SEQ ID NO: 323 and 347; or respectively, SEQ ID NO: 323 and 743.

[0046] In some embodiments, the PD-1 binding protein further includes an Fc region.

[0047] In some embodiments, the Fc region includes means for extending the half-life of the PD-1 binding protein.

[0048] In some embodiments, half-life extension means include Fc modification.

[0049] In some embodiments, the Fc modification is selected from the group consisting of: M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF) and L309D / Q311H / N434S (DHS).

[0050] In some embodiments, the Fc modification is M252Y / S254T / T256E (YTE) or M428L / N434S (LS).

[0051] In some embodiments, the Fc modification is M428L / N434S(LS).

[0052] In some embodiments, the Fc region is an IgG1, IgG2, or IgG4 Fc region.

[0053] In some embodiments, the PD-1 binding protein is an antibody or its antigen-binding fragment.

[0054] In some embodiments, the PD-1 binding protein is a human or humanized antibody or its antigen-binding fragment.

[0055] In some embodiments, the antigen-binding fragment is Fab, F(ab')2, Fab', single-chain Fv (scFv), Fv fragment, Fd fragment, or a biantibody.

[0056] In one aspect, a bispecific protein comprising the PD-1 binding protein as described in any one of claims 1-48 is provided, the bispecific protein further comprising a VEGF binding region comprising an anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF VH), the anti-VEGF VH comprising the following complementarity-determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 433; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 434; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 435; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 439; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 440; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 441; or CDR-H1 comprising the amino acid sequence of SEQ ID NO: 443; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 444; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 435.

[0057] In some embodiments, the bispecific protein further comprises an anti-VEGF immunoglobulin light chain variable domain (anti-VEGF VL), the anti-VEGF VL comprising the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 436; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 437; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 438; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 442; CDR-L2 comprising the amino acid sequence of FTS; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 438; or CDR-L1 comprising the amino acid sequence of SEQ ID NO: 436; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 437; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 438.

[0058] In some embodiments, the anti-VEGF VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 431; and the anti-VEGF VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 432.

[0059] In some embodiments, anti-VEGF VH has the amino acid sequence of SEQ ID NO: 431; and anti-VEGF-1VL has the amino acid sequence of SEQ ID NO: 432.

[0060] In one aspect, a bispecific protein comprising a PD-1 binding region and a VEGF binding region is provided, wherein (a) the PD-1 binding region comprises (i) an anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 445, 456, and 447; (2) respectively, SEQ ID NO 451, 458, and 453; or (3) respectively, SEQ ID NO 443, 460, and 447; and (ii) an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL) comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 448, 449, and 450; (2) respectively, SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450; and (b) the VEGF binding region comprises (i) an anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 433, 434, and 435; (2) respectively, SEQ ID NO: 439, 440, and 441; or (3) respectively, SEQ ID NO: 443, 444, and 435, and (ii) an anti-VEGF immunoglobulin light chain variable domain (anti-VEGF VL), which comprises the anti-VEGF VL contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 436, 437, and 438, respectively; (2) SEQ ID NO: 442, FTS, and SEQ ID NO: 438, respectively; or (3) SEQ ID NO: 436, 437, and 438, respectively.In some embodiments, (a) anti-PD-1 VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 325; and anti-PD-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347; and (b) anti-VEGF VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 431; and anti-VEGF-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 432. In some embodiments, (a) anti-PD-1 VH has the amino acid sequence of SEQ ID NO: 325; and anti-PD-1 VL has the amino acid sequence of SEQ ID NO: 347; and (b) anti-VEGF VH has the amino acid sequence of SEQ ID NO: 431; and anti-VEGF-1 VL has the amino acid sequence of SEQ ID NO: 432.

[0061] In one aspect, a bispecific protein comprising a PD-1 binding region and a VEGF binding region is provided, wherein (a) the PD-1 binding region comprises (i) an anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 445, 456, and 457; (2) respectively, SEQ ID NO 451, 458, and 459; or (3) respectively, SEQ ID NO 443, 460, and 457; and (b) an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL) comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 448, 449, and 450; (2) respectively, SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450; and (b) the VEGF binding region comprises (i) an anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 433, 434, and 435; (2) respectively, SEQ ID NO: 439, 440, and 441; or (3) respectively, SEQ ID NO: 443, 444, and 435, and (ii) an anti-VEGF immunoglobulin light chain variable domain (anti-VEGF VL), which comprises the anti-VEGF VL contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 436, 437, and 438, respectively; (2) SEQ ID NO: 442, FTS, and SEQ ID NO: 438, respectively; or (3) SEQ ID NO: 436, 437, and 438, respectively.In some embodiments, (a) anti-PD-1 VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 320; and anti-PD-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347; and (b) anti-VEGF VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 431; and anti-VEGF-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 432. In some embodiments, (a) anti-PD-1 VH has the amino acid sequence of SEQ ID NO: 320; and anti-PD-1 VL has the amino acid sequence of SEQ ID NO: 347; and (b) anti-VEGF VH has the amino acid sequence of SEQ ID NO: 431; and anti-VEGF-1 VL has the amino acid sequence of SEQ ID NO: 432.

[0062] In one aspect, a bispecific protein comprising a PD-1 binding region and a VEGF binding region is provided, wherein (a) the PD-1 binding region comprises (i) an anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 445, 461, and 457; (2) respectively, SEQ ID NO 451, 462, and 459; or (3) respectively, SEQ ID NO 443, 463, and 457; and (ii) an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL) comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 448, 449, and 450; (2) respectively, SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450; and (a) the VEGF binding region comprises (i) an anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 433, 434, and 435; (2) respectively, SEQ ID NO: 439, 440, and 441; or (3) respectively, SEQ ID NO: 443, 444, and 435, and (ii) an anti-VEGF immunoglobulin light chain variable domain (anti-VEGF VL), which comprises the anti-VEGF VL contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 436, 437, and 438, respectively; (2) SEQ ID NO: 442, FTS, and SEQ ID NO: 438, respectively; or (3) SEQ ID NO: 436, 437, and 438, respectively.In some embodiments, (a) anti-PD-1 VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 324; and anti-PD-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347; and (b) anti-VEGF VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 431; and anti-VEGF-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 432. In some embodiments, (a) anti-PD-1 VH has the amino acid sequence of SEQ ID NO: 324; and anti-PD-1 VL has the amino acid sequence of SEQ ID NO: 347; and (b) anti-VEGF VH has the amino acid sequence of SEQ ID NO: 431; and anti-VEGF-1 VL has the amino acid sequence of SEQ ID NO: 432.

[0063] In one aspect, a bispecific protein comprising a PD-1 binding region and a VEGF binding region is provided, wherein (a) the PD-1 binding region comprises (i) an anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 445, 461, and 447; (2) respectively, SEQ ID NO 451, 462, and 453; or (3) respectively, SEQ ID NO 443, 463, and 447; and (ii) an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL) comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 448, 449, and 450; (2) respectively, SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450; and (a) the VEGF binding region comprises (i) an anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF VH) comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 433, 434, and 435; (2) respectively, SEQ ID NO: 439, 440, and 441; or (3) respectively, SEQ ID NO: 443, 444, and 435, and (ii) an anti-VEGF immunoglobulin light chain variable domain (anti-VEGF VL), which comprises the anti-VEGF VL contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 436, 437, and 438, respectively; (2) SEQ ID NO: 442, FTS, and SEQ ID NO: 438, respectively; or (3) SEQ ID NO: 436, 437, and 438, respectively.In some embodiments, (a) anti-PD-1 VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 326; and anti-PD-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347; and (b) anti-VEGF VH has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 431; and anti-VEGF-1 VL has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 432. In some embodiments, (a) anti-PD-1 VH has the amino acid sequence of SEQ ID NO: 326; and anti-PD-1 VL has the amino acid sequence of SEQ ID NO: 347; and (b) anti-VEGF VH has the amino acid sequence of SEQ ID NO: 431; and anti-VEGF-1 VL has the amino acid sequence of SEQ ID NO: 432.

[0064] In one aspect, a bispecific protein comprising a PD-1 binding region and a VEGF binding region is provided, wherein the PD-1 binding region comprises an anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 VH), the anti-PD-1 VH comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, the complementarity-determining regions comprising the amino acid sequences of: (1) SEQ ID NO 445, 446, and 457, respectively; (2) SEQ ID NO 451, 452, and 459, respectively; or (3) SEQ ID NO 443, 455, and 457, respectively; and an anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 VL), the anti-PD-1 VL comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, the complementarity-determining regions comprising the amino acid sequences of: (1) SEQ ID NO 448, 449, and 450, respectively; (2) SEQ ID NO 448, 449, and 450 ...1, 452, and 459, respectively. SEQ ID NO: 454, LAS and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449 and 450; and the VEGF binding region comprises an anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF VH), which comprises complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 433, 434 and 435; (2) respectively, SEQ ID NO: 439, 440 and 441; or (3) respectively, SEQ ID NO: 443, 444 and 435, and an anti-VEGF immunoglobulin light chain variable domain (anti-VEGF VL), which comprises complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, which comprise the amino acid sequences of: (1) respectively, SEQ ID NO 454, LAS and 450; and the VEGF binding region comprises an anti-VEGF immunoglobulin heavy ... NO 436, 437 and 438; (2) respectively, SEQ ID NO: 442, FTS and SEQ ID NO: 438; or (3) respectively, SEQ ID NO: 436, 437 and 438.

[0065] In some embodiments, anti-PD-1 VH has at least 85% of the same amino acid sequence as SEQ ID NO: 327; and anti-PD-1 VL has at least 85% of the same amino acid sequence as SEQ ID NO: 347; and anti-VEGF VH has at least 85% of the same amino acid sequence as SEQ ID NO: 431; and anti-VEGF-1 VL has at least 85% of the same amino acid sequence as SEQ ID NO: 432.

[0066] In some embodiments, anti-PD-1 VH has the amino acid sequence of SEQ ID NO: 327; and anti-PD-1 VL has the amino acid sequence of SEQ ID NO: 347; and anti-VEGF VH has the amino acid sequence of SEQ ID NO: 431; and anti-VEGF-1 VL has the amino acid sequence of SEQ ID NO: 432.

[0067] In some embodiments, the bispecific protein further includes an Fc region.

[0068] In some embodiments, the Fc region includes means for extending the half-life of the PD-1 binding protein.

[0069] In some embodiments, half-life extension means include Fc modification.

[0070] In some embodiments, the Fc modification is selected from the group consisting of: M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF) and L309D / Q311H / N434S (DHS).

[0071] In some embodiments, the Fc modification is M252Y / S254T / T256E (YTE) or M428L / N434S (LS).

[0072] In some embodiments, the Fc modification is M428L / N434S(LS).

[0073] In some embodiments, the Fc region is an IgG1, IgG2, or IgG4 Fc region.

[0074] In some embodiments, the bispecific protein is an antibody or its antigen-binding fragment.

[0075] In some embodiments, the bispecific protein is a human or humanized antibody or its antigen-binding fragment.

[0076] In some embodiments, the antigen-binding fragment is Fab, F(ab')2, Fab', single-chain Fv (scFv), Fv fragment, Fd fragment, or a biantibody.

[0077] In some embodiments, the bispecific protein comprises two PD-1 binding regions and two VEGF binding regions.

[0078] In some embodiments, the bispecific protein comprises two PD-1 binding regions and one VEGF binding region.

[0079] In some embodiments, the bispecific protein comprises a PD-1 binding region and two VEGF binding regions.

[0080] In some embodiments, each PD-1 binding region is an scFv.

[0081] In some embodiments, each VEGF binding region includes anti-VEGF VH on a first polypeptide chain and anti-VEGF VL on a second polypeptide chain, anti-VEGF VH and anti-VEGF VL within a Fab, or anti-VEGF VH and anti-VEGF VL within a CrossFab.

[0082] In some embodiments, each VEGF binding region is an scFv.

[0083] In some embodiments, each PD-1 binding region includes anti-PD1 VH on a first polypeptide chain and anti-PD1 VL on a second polypeptide chain, anti-PD1 VH and anti-PD1 VL within a Fab, or anti-PD1 VH and anti-PD1 VL within a CrossFab.

[0084] In some embodiments, each PD-1 binding region is a VHH.

[0085] In some embodiments, each VEGF binding region includes anti-VEGF VH on a first polypeptide chain and anti-VEGF VL on a second polypeptide chain, anti-VEGF VH and anti-VEGF VL within a Fab, or anti-VEGF VH and anti-VEGF VL within a CrossFab.

[0086] In one aspect, a bispecific protein comprising at least one PD-1 binding region and at least one VEGF binding region is provided, the bispecific binding protein comprising a first polypeptide chain having the sequence of SEQ ID NO: 92 and a second polypeptide chain having the sequence of SEQ ID NO: 239.

[0087] In one aspect, a bispecific protein comprising at least one PD-1 binding region and at least one VEGF binding region is provided, the bispecific binding protein comprising a first polypeptide chain having the sequence of SEQ ID NO: 73 and a second polypeptide chain having the sequence of SEQ ID NO: 220.

[0088] In one aspect, a bispecific protein comprising at least one PD-1 binding region and at least one VEGF binding region is provided, the bispecific binding protein comprising a first polypeptide chain having the sequence of SEQ ID NO: 91 and a second polypeptide chain having the sequence of SEQ ID NO: 238.

[0089] In one aspect, a bispecific protein comprising at least one PD-1 binding region and at least one VEGF binding region is provided, the bispecific binding protein comprising a first polypeptide chain having the sequence of SEQ ID NO: 94 and a second polypeptide chain having the sequence of SEQ ID NO: 241.

[0090] In one aspect, a bispecific protein comprising at least one PD-1 binding region and at least one VEGF binding region is provided, the bispecific binding protein comprising a first polypeptide chain having the sequence of SEQ ID NO: 96 and a second polypeptide chain having the sequence of SEQ ID NO: 243.

[0091] In some embodiments, the bispecific protein forms a dimer.

[0092] In various respects, isolated nucleic acids encoding one or more strands of a PD-1 binding protein or bispecific protein as disclosed herein are provided, expression vectors containing said isolated nucleic acids, and host cells containing said isolated nucleic acids or expression vectors.

[0093] In each respect, it provides isolated nucleic acid sets that co-encode PD-1 binding proteins or bispecific proteins as disclosed herein, sets of expression vectors that co-contain said isolated nucleic acid sets, and host cells that contain said isolated nucleic acid sets or said expression vector sets.

[0094] In one aspect, pharmaceutical compositions are provided that comprise a PD-1 binding protein or bispecific protein as disclosed herein and a pharmaceutically acceptable carrier.

[0095] In one aspect, methods are provided that include the step of administering an effective amount of a PD-1 binding protein, a bispecific protein, or a pharmaceutical composition as disclosed herein to a subject in need.

[0096] In some embodiments, the subject has a disease or condition associated with abnormal PD-1 and / or VEGF expression or signaling or is at risk of developing such a disease or condition.

[0097] In some embodiments, the disease or symptom is cancer.

[0098] In some embodiments, the cancer is lung cancer.

[0099] In some embodiments, lung cancer is non-small cell lung cancer.

[0100] In some embodiments, the cancer is gastrointestinal cancer.

[0101] In some embodiments, gastrointestinal cancer is colorectal cancer, bile duct cancer, gastric cancer, or hepatocellular carcinoma.

[0102] In some embodiments, the cancer is reproductive cancer.

[0103] In some embodiments, reproductive cancer is cervical cancer, endometrial cancer, or ovarian cancer.

[0104] In some embodiments, the administration step includes systemic administration of PD-1 binding protein, bispecific protein, or pharmaceutical composition.

[0105] In some embodiments, systemic administration includes intravenous administration of PD-1 binding protein, bispecific protein, or pharmaceutical composition.

[0106] In some embodiments, systemic administration includes subcutaneous administration of PD-1 binding protein, bispecific protein, or pharmaceutical composition. Attached Figure Description

[0107] These and other features, aspects, and advantages of the invention will become more readily understood with reference to the following description and accompanying drawings, in which: Figure 1A-1CC The binding activity of the antibody shown with cells expressing PD-1 was depicted.

[0108] Figure 2A-2D The surface plasmon resonance (SPR) curves of the antibody were depicted. Figure 2A-2C ) and PD-1 / PD-L1 blockade assay results ( Figure 2D ).

[0109] Figure 3A The binding activity of the antibody to human VEGF, as determined by ELISA, was described.

[0110] Figure 3B The binding activity of the antibody shown to human PD-1, as determined by ELISA, was depicted.

[0111] Figure 3CThe binding activity of the antibody shown to cynomolgus monkey PD-1, as determined by ELISA, was described.

[0112] Figures 4A-4D The SPR curves of the antibody binding to human VEGF were plotted.

[0113] Figures 5A-5D The SPR curves of the antibody binding to human PD-1 were plotted.

[0114] Figures 6A-6D The SPR curves of the antibody binding to cynomolgus monkey PD-1 were plotted.

[0115] Figures 7A-7F The SEC-MALS complex profile of the antibody shown was depicted.

[0116] Figure 8 The heavy and light chain sequences (SEQ ID NO: 128 and 275, respectively) and forms of evokinemab (“AK112”) are depicted. The variable domain from penpulimab (“AK105”) is underlined.

[0117] Figure 9 It has been demonstrated that bispecific antibodies can enhance PD-1 binding through VEGF daisy chaining.

[0118] Figure 10A-10KK The PD-1 reporter gene assay results of the antibody shown are depicted.

[0119] Figure 11A-11V The VEGF reporter gene assay results of the antibody shown are depicted.

[0120] Figure 12A-12O The antibody shown was used to depict the inhibition of VEGF-mediated proliferation of human umbilical vein endothelial cells (HUVECs).

[0121] Figure 13A-13S The PD-1 internalization results of the antibody shown are depicted.

[0122] Figure 14A A bispecific reverse construct of PD-1-VEGF (scFv) was described.

[0123] Figure 14B-14E The PD-1 binding of the PD-1-VEGF (scFv) bispecific reverse construct was described. Figure 14B ), PD-1 internalization ( Figure 14D ), PD-1 reporter gene ( Figure 14C ) and VEGF reporter gene ( Figure 14E The results of the measurement.

[0124] Figure 15A-15RData on T cell activation assessed using the indicated antibody in a co-culture assay of human PBMCs and hepatocellular carcinoma are depicted.

[0125] Figure 16A-16F Data depicted on T cell activation assessed using the indicated antibody in a mixed lymphocyte reaction assay of human monocyte-derived dendritic cells and CD4+ T cells.

[0126] Figure 17A A graph depicts the viscosity at different protein concentrations at a constant pH of 6.0.

[0127] Figure 17B A graph depicts the percentage of monomers obtained by size exclusion chromatography (SEC) at different protein concentrations.

[0128] Figure 17C-17D Depicting at 40°C ( Figure 17C ) and 25°C ( Figure 17D Stability data under ( ).

[0129] Figures 18A-18C The pharmacokinetic data of the antibody shown are described.

[0130] Figure 19 The antitumor activity of the antibody shown was described. Detailed Implementation

[0131] definition

[0132] Unless otherwise defined, all technical terms, symbols, and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art. In some cases, for clarity and / or ease of reference, terms are defined herein with their commonly understood meanings, and the inclusion of such definitions herein is not necessarily to be construed as indicating a difference from the meanings commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are commonly used with conventional methods, such as those widely used as described in, for example, the following literature: Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, unless otherwise indicated, procedures involving the use of commercially available kits and reagents are generally performed according to the manufacturer's defined protocols and conditions.

[0133] As used herein, unless otherwise stated, the singular forms “a / an” and “the” include plural references.

[0134] It should be understood that the aspects and embodiments of the invention described herein include “comprising aspects and embodiments,” “consisting of aspects and embodiments,” and “substantially consisting of aspects and embodiments.”

[0135] For all compositions described herein and all methods of using the compositions described herein, the compositions may comprise the listed components or steps, or may “consist substantially of the listed components or steps.” When a composition is described as “consistent substantially of the listed components,” the composition contains the listed components and may contain other components that do not substantially affect the condition being treated, but does not contain any other components besides those explicitly listed that substantially affect the condition being treated; or, if the composition does contain additional components besides those listed that substantially affect the condition being treated, the concentration or amount of the additional components contained in the composition is insufficient to substantially affect the condition being treated. When a method is described as “consistent substantially of the listed steps,” the method contains the listed steps and may contain other steps that do not substantially affect the condition being treated, but the method does not contain any other steps besides those explicitly listed that substantially affect the condition being treated. As a non-limiting specific example, when a composition is described as "consisting substantially of components", the composition may additionally contain any amount of pharmaceutically acceptable carriers, mediators or diluents, and other such components that do not substantially affect the condition being treated.

[0136] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the host cell genome into which they have been introduced. Some vectors are capable of directing the expression of the nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0137] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, as well as the progeny of such cells. Host cells include “transformants” (or “transformed cells”) and “transfectants” (or “transfected cells”), each comprising primary transformed or transfected cells and their derived progeny. The nucleic acid content of such progeny may not be entirely identical to that of the parent cells and may contain mutations. “Recombinant host cell” or “host cell” refers to a cell containing exogenous polynucleotides, regardless of the method used for insertion, such as direct uptake, transduction, f-crossing, or other methods known in the art for producing recombinant host cells.

[0138] As used herein, an "effective dose" or "therapeutic effective dose" refers to the amount of a therapeutic compound (such as an anti-VEGF antibody) administered to an individual as a single dose or as part of a series of doses, which, alone or in combination with another therapeutic modality, effectively produces or contributes to the desired therapeutic effect. Examples of desired therapeutic effects include enhanced immune responses, slowing or delaying tumor development; stabilizing disease; and alleviating one or more symptoms. An effective dose may be administered in one or more doses.

[0139] The term "treating" (and its variations, such as "treat" or "treatment") refers to a clinical intervention that attempts to alter the natural course of a disease or symptom in a subject in need. Treatment can be performed during clinicopathology. Ideal outcomes of treatment include preventing disease recurrence, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, alleviating or mitigating the disease state, and improving or alleviating prognosis.

[0140] The term "sufficient amount" refers to an amount that is sufficient to produce the desired effect, such as an amount sufficient to modulate the immune response of a subject.

[0141] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and, in some embodiments, refer to any mammalian subject requiring diagnosis, treatment, or therapy, particularly a human. For therapeutic purposes, “mammal” means any animal classified as a mammal, including humans, livestock, and farm animals, as well as laboratory animals, zoo animals, racing animals, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human. These terms do not require medical personnel supervision.

[0142] As used herein, the term "effective amount" means an amount of compound (e.g., the compound disclosed herein) sufficient to achieve a beneficial or desired result. An effective amount may be administered, applied, or dosed once or multiple times and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treatment" includes any effect that improves symptoms, disease, condition, etc., or reduces, alleviates, or prevents their symptoms, for example, by relieving, reducing, moderating, lessening, or eliminating them.

[0143] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier, which makes the composition particularly suitable for in vivo or in vitro diagnostic or therapeutic uses.

[0144] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0145] The terms "modulate" and "modulation" refer to reducing or suppressing, or alternatively activating or increasing, a referenced variable.

[0146] The terms “increase” and “activation” refer to the increase of the variable by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or to 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times or more.

[0147] The terms “reduction” and “suppression” refer to the reduction of the variable by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100 or less.

[0148] The term "about" indicates and covers the value shown, as well as the range above and below that value. In some embodiments, the term "about" indicates a specified value ±10%, ±5%, or ±1%. In some embodiments, where applicable, the term "about" indicates a specified value ± one standard deviation of that value.

[0149] The term “optionally” when used sequentially is intended to include combinations from one enumerated combination to all enumerated combinations, and envisions all sub-combinations.

[0150] The term "amino acid" refers to twenty common, naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0151] The term “affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless otherwise stated, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope).

[0152] As used in this article, the term "k" d (sec⁻¹) refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also known as k. off value.

[0153] As used in this article, the term "k" a (M⁻¹×sec⁻¹) refers to the association rate constant of a specific antibody-antigen interaction. This value is also known as k. on value.

[0154] As used in this article, the term "K" D "(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. K D = k d / k a In some embodiments, the affinity of an antibody is based on the K-value of the interaction between such an antibody and its antigen. D To describe. For clarity, as is known in the art, the smaller K D A higher K value indicates a stronger affinity interaction, while a larger K value indicates a weaker affinity interaction. D The value indicates a lower affinity interaction.

[0155] As used in this article, the term "K" A "(M-1)" refers to the association equilibrium constant of a specific antibody-antigen interaction. A =k a / k d .

[0156] As used herein, unless otherwise stated, the term "antibody" should be understood to mean a complete antibody (e.g., a complete monoclonal antibody) or a fragment thereof, such as an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody) or an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including modified, engineered, or chemically conjugated complete antibodies, antigen-binding fragments, or Fc fragments. Typically, an antibody is a multimeric protein containing four polypeptide chains. Two of these polypeptide chains are called immunoglobulin heavy chains (H chains), and two are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are linked by interchain disulfide bonds. The immunoglobulin heavy chain is linked by interchain disulfide bonds. The light chain consists of a variable region (VL) and a constant region (CL). The heavy chain consists of a variable region (VH) and at least three constant regions (CH1, CH2, and CH3). The variable region determines the binding specificity of the antibody. Each variable region contains three hypervariable regions called complementarity-determining regions (CDRs), flanked by four relatively conserved regions called framework regions (FRs). The extent of FRs and CDRs has been defined (Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). The three CDRs, designated CDR1, CDR2, and CDR3, contribute to antibody binding specificity. Naturally occurring antibodies have been used as starting materials for engineered antibodies, such as chimeric and humanized antibodies. Examples of antibody-based antigen-binding fragments include Fab, Fab', (Fab')2, Fv, single-chain antibodies (e.g., scFv), microantibodies, and biantibodies. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). Examples of chemically conjugated antibodies are antibodies conjugated to a toxin moiety.

[0157] "VEGF antibody" or "VEGF-specific antibody" is an antibody that specifically binds to VEGF, as described herein. "VEGF conjugate" or "VEGF-specific conjugate" is a conjugate that specifically binds to VEGF, as described herein.

[0158] "PD-1 antibody", "PD1 antibody" or "PD1-specific antibody" are antibodies that specifically bind to PD1 as provided herein. "PD-1 conjugate", "PD1 conjugate" or "PD1-specific conjugate" are conjugates that specifically bind to PD1 as provided herein.

[0159] The term "epitope" refers to a portion of an antigen that specifically binds to antibodies.

[0160] As used herein, the term “hypervariant region” or “HVR” refers to each region in the antibody variable domain that exhibits sequence hypervariability and / or forms a structurally defined loop (“hypervariant loop”).

[0161] The term "antigen-binding domain" refers to the part of an antibody that can specifically bind to an antigen or epitope.

[0162] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0163] The term "human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source (e.g., obtained from a human source or de novo designed) using a human antibody library or human antibody coding sequence. Human antibodies do not specifically include humanized antibodies.

[0164] The term "humanized antibody" refers to a protein having a sequence that differs from that of antibodies derived from non-human species through substitution, deletion, and / or addition of one or more amino acids, such that when administered to human subjects, the humanized antibody is less likely to induce an immune response and / or induce a less severe immune response compared to non-human species antibodies.

[0165] The term "multispecific antibody" refers to an antibody that contains two or more different antigen-binding domains that commonly and specifically bind to two or more different epitopes.

[0166] "Bispecific antibodies" are antibodies that contain two different antigen-binding domains, each binding to a different epitope.

[0167] A "monospecific antibody" is an antibody that contains one or more binding sites that specifically bind to a single epitope. An example of a monospecific antibody is the naturally occurring IgG molecule, which, although bivalent (i.e., has two antigen-binding domains), recognizes the same epitope at each of the two antigen-binding domains. Binding specificity can exist at any suitable valence.

[0168] The term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies. A substantially homogeneous group of antibodies includes those that are substantially similar and bind to the same epitope, except for variants that may typically occur during the monoclonal antibody production process. Such variants are usually present in only small quantities. Monoclonal antibodies are typically obtained by involving a process of selecting a single antibody from a plurality of antibodies. For example, the selection process may be to choose a unique clone from multiple clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody may be further modified, for example, to improve its affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell cultures, and / or to reduce its immunogenicity in a subject.

[0169] The term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In a particular such embodiment, the C-terminus of the Fab light chain is linked to the N-terminus of the Fab heavy chain in a single-chain Fab molecule. As described in more detail herein, scFv has a light chain variable domain (VL) that is linked from its C-terminus to the N-terminus of the heavy chain variable domain (VH) via a polypeptide chain. Alternatively, scFv comprises a polypeptide chain in which the C-terminus of the VH is linked to the N-terminus of the VL via the polypeptide chain.

[0170] The “Fab fragment” (also known as the antigen-binding fragment) contains a constant domain (CL) of the light chain and a first constant domain (CH1) of the heavy chain, as well as variable domains VL and VH on the light and heavy chains, respectively. The variable domains contain a complementarity-determining loop (CDR, also known as a hypervariable region) involved in antigen binding. The Fab' fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the CH1 domain of the heavy chain, which includes one or more cysteine ​​residues from the antibody hinge region.

[0171] The “F(ab’)2” fragment contains two Fab’ fragments linked by disulfide bonds near the hinge region. The F(ab’)2 fragment can be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody. The F(ab’) fragment can be dissociated, for example, by treatment with β-mercaptoethanol.

[0172] The “Fv” fragment is a non-covalently linked dimer of a heavy chain variable domain and a light chain variable domain.

[0173] A “single-chain Fv” or “sFv” or “scFv” comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994). HER2 antibody scFv fragments are described in WO 93 / 16185, U.S. Patent No. 5,571,894, and U.S. Patent No. 5,587,458.

[0174] The “scFv-Fc” fragment contains an scFv attached to an Fc domain. For example, the Fc domain may be attached to the C-terminus of the scFv. Depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL-VH), the Fc domain may follow either VH or VL. Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain includes the IgG4 Fc domain.

[0175] The term "single-domain antibody" or "sdAb" refers to a molecule in which one of the antibody's variable domains specifically binds to an antigen in the absence of another variable domain. Single-domain antibodies and their fragments were described by Arabi Ghahroudi et al. (1998). FEBS Letters [Circular of the Federation of European Biochemical Societies] 414:521-526 and Muyldermans et al. (2001) Trends in Biochem. Sci In [Trends in Biochemistry] 26:230-245, each of which is incorporated herein by reference in its entirety. Single-domain antibodies are also known as sdAbs, VHHs, or nanobodies. sdAbs are quite stable and readily expressed as fusion-paired forms with the Fc chain of the antibody (Harmsen MM, De Haard HJ (2007) "Properties, production, and applications of camelid single-domain antibody fragments"). Appl. Microbiol Biotechnol [Applied Microbiology and Biotechnology] 77(1): 13-22).

[0176] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably herein and refer to an antibody having a structure substantially similar to that of naturally occurring antibodies and having a heavy chain containing an Fc region. For example, when used to refer to an IgG molecule, a “full-length antibody” is an antibody that comprises two heavy chains and two light chains.

[0177] The term "antibody fragment" refers to an antibody that contains a portion of a complete antibody, such as the antigen-binding region or variable region of the complete antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0178] The term "Fc domain" or "Fc region" as used in this article is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. This term includes both native sequence Fc regions and variant Fc regions.

[0179] The term “substantially purified” refers to the constructs described herein or variants thereof, which may be substantially or essentially free of components typically associated with or interacting with proteins found in their natural environment (i.e., native cells), or, in the case of recombinant antibodies (in some embodiments, which are substantially free of cellular material), the host cells comprising protein formulations having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating proteins.

[0180] In the context of two or more nucleic acid or polypeptide sequences, the term "percentage of identity" refers to the percentage of identical nucleotide or amino acid residues between two or more sequences or subsequences, measured by visual inspection, when comparing and aligning against the maximum correspondence, using one of the following sequence comparison algorithms (e.g., publicly available computer software such as BLAST, BLASTP, BLASTN, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTALOMEGA, or MUSCLE software, or other algorithms available to a technician). Software used for BLAST analysis (Altschul et al. (1990)) J. Mol. Biol[Journal of Molecular Biology] 215:403-410 is publicly available from the National Center for Biotechnology Information (ncbi.nlm.nih.gov). Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. Depending on the application, the percentage of "identity" may be based on regions of the compared sequences (e.g., functional domains), or alternatively, on the full length of the two sequences to be compared.

[0181] For sequence comparison, typically, one sequence serves as a reference sequence to be compared with the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, the coordinates of the subsequences are specified (if necessary), and the sequence algorithm program parameters are specified. Then, based on the specified program parameters, the sequence comparison algorithm calculates the percentage of sequence identity between the test sequence and the reference sequence.

[0182] The best alignment of sequences for comparison can be performed, for example, by Smith and Waterman (1981). Adv. Appl. Math [Advances in Applied Mathematics] 2:482 Local homology algorithm; Needleman and Wunsch (1970) J. Mol. Biol [Journal of Molecular Biology] 48:443: Homology Algorithm; Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci Similarity search methods [Proceedings of the National Academy of Sciences]. USA 85:2444; computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA from the Wisconsin Genetics Software Package at the Genetics Computer Group, 575 Science Avenue, Madison, Wisconsin); or visual inspection (generally see Ausubel et al., below).

[0183] The ranges described herein should be understood as abbreviations of all values ​​within that range, including the endpoints. For example, the range 1 to 50 should be understood as including any number, number combination, or subrange that comes from the following groups: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0184] It should be noted that, as used in the specification and appended claims, the singular forms “a / an” and “the” include plural indicators unless the context clearly indicates otherwise.

[0185] PD-1 antibody and bispecific anti-VEGF / PD-1 antibody

[0186] Basic antibody structure

[0187] The recognized immunoglobulin (antibody) genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0188] An exemplary immunoglobulin structural unit consists of two pairs of polypeptide chains, each pair having a “light” chain (approximately 25 kD) and a “heavy” chain (approximately 50-70 kD). The N-terminal domain of each chain defines a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains, respectively. The IgG1 heavy chain contains VH, CH1, CH2, and CH3 domains from its N-terminus to its C-terminus. The light chain contains VL and CL domains from its N-terminus to its C-terminus. The IgG1 heavy chain contains a hinge between the CH1 and CH2 domains. In some embodiments, the immunoglobulin construct includes at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE linked to a therapeutic polypeptide. In some embodiments, the immunoglobulin domains found in the antibodies provided herein are derived from or derived from immunoglobulin-based constructs, such as biantibodies or nanobodies. In some embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain from a heavy chain antibody, such as a camel antibody. In some embodiments, the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain from a mammalian antibody, such as a bovine antibody, human antibody, camel antibody, mouse antibody, or any chimeric antibody.

[0189] In some embodiments, the antibodies provided herein comprise a heavy chain. In one embodiment, the heavy chain is IgA. In one embodiment, the heavy chain is IgD. In one embodiment, the heavy chain is IgE. In one embodiment, the heavy chain is IgG. In one embodiment, the heavy chain is IgM. In one embodiment, the heavy chain is IgG1. In one embodiment, the heavy chain is IgG2. In one embodiment, the heavy chain is IgG3. In one embodiment, the heavy chain is IgG4. In one embodiment, the heavy chain is IgA1. In one embodiment, the heavy chain is IgA2.

[0190] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0191] Typically, natural four-chain antibodies contain six hypervariable regions (HVRs): three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). The HVRs typically contain amino acid residues from the hypervariable loop and / or from complementarity-determining regions (CDRs), which have the highest sequence variability and / or are involved in antigen recognition. Except for CDR1 in the VH, the CDRs typically contain amino acid residues that form the hypervariable loop. The HVR is also referred to as the CDR, and these terms are used interchangeably throughout this text when referring to the portion of the variable region that forms the antigen-binding region. This specific region has been described by: Kabat et al. (1983), US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest, and Chothia et al. (1987). J Mol Biol [Journal of Molecular Biology] 196:901-917, where definitions include overlaps or subsets of amino acid residues when compared. However, the application of any definition referring to a CDR of an antibody or its variants is intended to be within the scope of the terminology defined and used herein. The exact number of residues covering a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which residues contain a particular CDR based on the amino acid sequence of the variable region of the antibody.

[0192] The amino acid sequence boundaries of a CDR can be determined by one of those skilled in the art using any of the many known numbering schemes, including those described below: Kabat et al., ibid. (“Kabat” numbering scheme); Al-Lazikani et al. (1997). J. Mol. Biol[Journal of Molecular Biology], 273:927-948 (“Chothia” numbering scheme); MacCallum et al. (1996) J. Mol. Biol [Journal of Molecular Biology] 262:732-745 (“Contact” numbering scheme); Lefranc et al. (2003) Dev. Comp. Immunol [Developmental and Comparative Immunology] 27:55-77 (“IMGT” numbering scheme); and Honegge and Plückthun (2001). J. Mol. Biol [Journal of Molecular Biology] 309:657-70 (“AHo” numbering scheme); each of which is incorporated in its full text by reference.

[0193] Table 1 provides the locations of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia schemes. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0194] CDRs can be assigned, for example, using antibody numbering software (such as Abnum, available at www.bioinf.org.uk / abs / abnum / ), as described in Abhinandan and Martin (2008). Immunology In [Immunology], 45:3832-3839, this article is included in its entirety by reference.

[0195] Table 1. Residues in CDR according to the Kabat and Chothia numbering schemes.

[0196] When using the Kabat numbering convention, the C end of CDR-H1 varies between H32 and H34 depending on the length of the CDR.

[0197] When referring to residues in the constant region of the antibody heavy chain, the “EU numbering scheme” is generally used (e.g., as reported above by Kabat et al.). Unless otherwise stated, the EU numbering scheme is used to refer to residues in the constant region of the antibody heavy chain as described herein.

[0198] One example of an antigen-binding domain is an antigen-binding domain formed by the VH-VL dimer of an antibody. Another example of an antigen-binding domain is an antigen-binding domain formed by the diversification of certain loops from the tenth fibronectin type III domain of Adnectin. An antigen-binding domain may sequentially include CDRs 1, 2, and 3 from the heavy chain; and CDRs 1, 2, and 3 from the light chain.

[0199] Epitopes typically consist of surface-accessible amino acid residues and / or sugar side chains and can possess specific three-dimensional structural characteristics and specific charge properties. The difference between conformational and non-conformational epitopes is that binding to the former, but not to the latter, may be lost in the presence of denaturing solvents. Epitopes can contain amino acid residues that directly participate in binding and other amino acid residues that do not directly participate in binding. Epitopes that bind to antibodies can be determined using known epitope assay techniques, for example, testing the binding of antibodies to VEGF variants with different point mutations or to chimeric VEGF variants.

[0200] To screen for antibodies that bind to epitopes on target antigens bound by antibodies of interest (e.g., VEGF or PD-1), conventional cross-blocking assays can be performed, such as those described in *Antibodies, A Laboratory Manual*, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively or additionally, epitope mapping can be performed using methods known in the art.

[0201] Chimeric antibodies are antibodies in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from different sources or species.

[0202] Human antibodies are antibodies having an amino acid sequence corresponding to that of antibodies produced by humans or human cells or derived from non-human sources (e.g., obtained from human sources or de novo designed) that utilize human antibody libraries or human antibody coding sequences. Human antibodies do not specifically include humanized antibodies.

[0203] Humanized antibodies have sequences that differ from those of antibodies derived from non-human species through substitution, deletion, and / or addition of one or more amino acids, such that when administered to human subjects, the humanized antibody is less likely to induce an immune response and / or induce a less severe immune response compared to the non-human species antibody. In one embodiment, certain amino acids in the framework and constant domains of the heavy and / or light chains of the non-human species antibody are mutated to produce a humanized antibody. In another embodiment, a constant domain from a human antibody is fused with a variable domain from a non-human species. In yet another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the immunogenicity that may occur when the non-human antibody is administered to a human subject, wherein the altered amino acid residues are not essential for the immune-specific binding of the antibody to its antigen, or the alteration to the amino acid sequence is a conserved change such that the binding of the humanized antibody to the antigen is not significantly worse than that of the non-human antibody to the antigen. Examples of how to prepare humanized antibodies can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293. For further details, see Jones et al. (1986). Nature [Nature] 321:522-525; Riechmann et al. (1988) Nature [Nature] 332:323-329; and Presta (1992) Curr. Op. Struct. Biol [Latest Views in Structural Biology] 2:593-596, each of which is incorporated herein by reference in its entirety.

[0204] Two or more distinct epitopes can be epitopes on the same antigen (e.g., a single VEGF) or on different antigens (e.g., different VEGF molecules, or VEGF molecules and non-VEGF molecules). In some embodiments, a multispecific antibody binds to two distinct epitopes (i.e., a "bispecific antibody"). In some embodiments, a multispecific antibody binds to three distinct epitopes (i.e., a "trispecific antibody").

[0205] Anti-VEGF or PD-1 antibodies may include those described herein, such as the clones shown in the figures and / or tables. In some embodiments, the binding protein comprises an alternative scaffold. In some embodiments, the binding protein is composed of an alternative scaffold. In some embodiments, the binding protein is substantially composed of an alternative scaffold. In some embodiments, the binding protein comprises an antibody fragment. In some embodiments, the binding protein is composed of an antibody fragment. In some embodiments, the binding protein is substantially composed of an antibody fragment.

[0206] In some embodiments, the bispecific antibody is a monoclonal antibody.

[0207] In some embodiments, the bispecific antibody is a polyclonal antibody.

[0208] In some embodiments, the bispecific antibody is generated by a hybridoma. In other embodiments, the bispecific antibody is generated by recombinant cells engineered to express desired variable and constant domains.

[0209] In some embodiments, the bispecific antibody may be a single-chain antibody or other antibody derivative or variant thereof that retains antigen specificity and the lower hinge region.

[0210] In some embodiments, the bispecific antibody may be a multifunctional antibody, a recombinant antibody, a human antibody, a humanized antibody, a fragment thereof, or a variant thereof. In a particular embodiment, the antibody fragment or a derivative thereof is selected from Fab fragments, Fab'2 fragments, CDRs, and scFvs.

[0211] In some embodiments, bispecific antibodies can form immune complexes. For example, immune complexes can be tumor cells covered by bispecific antibodies.

[0212] For sequence comparison, typically, one sequence serves as a reference sequence to be compared with the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, the coordinates of the subsequences are specified (if necessary), and the sequence algorithm program parameters are specified. Then, based on the specified program parameters, the sequence comparison algorithm calculates the percentage of sequence identity between the test sequence and the reference sequence.

[0213] Bispecific antibody structure

[0214] This application provides proteins that bind VEGF and PD-1, as well as compositions (e.g., pharmaceutical compositions thereof). The bispecific antibodies disclosed herein may have any structure known in the art.

[0215] The disclosed bispecific binding proteins may take any form, including but not limited to those described herein.

[0216] In an exemplary bispecific form, the bispecific binding protein comprises (a) two immunoglobulin heavy chains, each comprising from its N-terminus to its C-terminus: (1) a heavy chain variable domain and (2) an Fc (hinge-CH2-CH3) domain, each of the immunoglobulin heavy chains being linked to an scFv at its C-terminus; and (b) two immunoglobulin light chains, each comprising from its N-terminus to its C-terminus: (1) a light chain variable domain and (2) a light chain constant domain. A linker (e.g., an amino acid linker) may be used between any of the aforementioned domains within a given immunoglobulin chain, within an scFv, and / or between an immunoglobulin heavy chain and an scFv. In this form, the heavy chain variable domain and the light chain variable domain together form a first binding region, and the scFv contains a second binding region. In some embodiments, the first binding region is a VEGF binding region, and the second binding region is a PD-1 binding region. Therefore, for example, in this form, the bispecific binding protein comprises two VEGF binding regions and two PD-1 binding regions in each construct.

[0217] However, other forms have been envisioned, such as those containing different numbers of each binding region, for example, two VEGF binding regions and one PD-1 binding region, one VEGF binding region and two PD-1 binding regions, or one VEGF binding region and one PD-1 binding region.

[0218] Another form is a bispecific binding protein comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain. In this form, (1) the first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain, a first variable heavy chain domain, and an optional first CH1 heavy chain domain; (2) the immunoglobulin light chain comprises a variable light chain domain and a constant light chain domain; and (3) the immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms an antigen-binding site that binds to VEGF. In this form, the second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second variable heavy chain domain, and a second CH1 heavy chain domain, which can pair with the same immunoglobulin light chain that pairs with the first immunoglobulin heavy chain, except that when the immunoglobulin light chain pairs with the second immunoglobulin heavy chain, the resulting antigen-binding site binds to PD-1.

[0219] In some embodiments, the bispecific binding protein is in the form of Triomab, a trifunctional bispecific binding protein that maintains an IgG-like shape. The chimera consists of two hemiantibodies, each having a light chain and a heavy chain, derived from two parent antibodies.

[0220] In some embodiments, the bispecific binding protein is in the form of a KiH common light chain (LC), which relates to the mortar and pestle (KIH) technique. KIH involves the binding of C... H The three domains are engineered to create "pestle" or "mortar" in each heavy chain to promote heterodimerization. The concept behind the "pestle-mortar (KiH)" Fc technology is to introduce a "pestle" in a CH3 domain (CH3A) by replacing small residues with large residues (e.g., T366W in EU designation). CH3A To accommodate the "pestle," a complementary "mortar" surface is created on another CH3 domain (CH3B) by replacing the nearest adjacent residue with a smaller residue (e.g., T366S / L368A / Y407V). CH3B ). Optimization of the "mortar" mutation through structured-guided phage library screening (Atwell et al. (1997), "Stableheterodimers from remodeling the domain interface of a homodimer using aphage display library to obtain stable heterodimers"). J. Mol. Biol. [Journal of Molecular Biology] 270(1):26-35). X-ray crystal structure of KiH Fc variant (Elliott et al. (2014) "Antiparallel conformation of knob and holeaglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction"). J. Mol. Biol. [Journal of Molecular Biology] 426(9):1947-57; Mimoto et al. (2014) "Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcgammaRs," Mol. Immunol.[Molecular Immunology] 58(1):132-8) proved that the hydrophobic interaction driven by the spatial complementarity at the interface between the cores of the CH3 domain is thermodynamically favorable for heterodimerization, while the mortar-mortar and mortar-mortar interfaces are unfavorable for homodimerization due to steric hindrance and the disruption of favorable interactions, respectively.

[0221] In some embodiments, the bispecific binding protein is in the form of an orthogonal Fab interface. In the Ortho-Fab method (Lewis et al. (2014) "Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface"), Nat. Biotechnol. [Nature Biotechnology] 32(2):191-8), structure-based regional design of LC and HC in only one Fab VH-CH1 A complementary mutation is introduced at the interface without making any changes to the other Fab.

[0222] In some embodiments, the bispecific binding protein is in a 2-in-1 Ig form. In some embodiments, the bispecific binding protein is in an ES form, which is a heterodimeric construct containing two different Fabs, binding target 1 and target 2, fused to the Fc. Heterodimerization is ensured by an electrostatic orientation mutation in the Fc. In some embodiments, the bispecific binding protein is... The λ-body form is a heterodimeric construct having two different Fabs fused to an Fc stabilized by a heterodimerization mutation: Fab1 targeting antigen 1 contains κLC, while the second Fab targeting antigen 2 contains λLC.

[0223] In some embodiments, bispecific binding proteins are in the form of Fab arm exchange (the antibody Fab arm is exchanged by exchanging the heavy chain and the attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which generates a bispecific antibody). In some embodiments, bispecific binding proteins are in the form of SEED bodies. Designing a chain exchange engineered domain (SEED) platform to generate asymmetric and bispecific binding protein-like molecules is a capability to expand the therapeutic applications of natural antibodies. This protein engineering platform is based on exchanging structurally relevant sequences of immunoglobulins within a conserved CH3 domain. SEED design allows for efficient generation of AG / GA heterodimers while inhibiting homodimerization of the AG and GA SEED CH3 domains. (Muda M. et al. (2011)) Protein Engineering, Design, and Selection.24(5):447-54). In some embodiments, the bispecific binding protein is in the form of LuZ-Y, where the leucine zipper is used to induce heterodimerization of two different HCs. (Wranik et al. (2012)) J. Biol. Chem. [Journal of Biochemistry] 287:43331-9).

[0224] In some embodiments, the bispecific binding protein is in the form of a Cov-X body. In the bispecific Cov-X body, two distinct peptides are linked together using a branched-chain azacyclobutanone linker and fused to the scaffold-binding protein in a site-specific manner under mild conditions. Although the pharmacophore is responsible for functional activity, the binding protein scaffold confers a long half-life and Ig-like distribution. The pharmacophore can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies. (Doppalapudi et al. (2010)) PNAS [Proceedings of the National Academy of Sciences of the United States of America] 107(52): 22611-22616.

[0225] In some embodiments, the bispecific binding protein is in the form of an Oasc-Fab heterodimer, comprising a Fab for binding target 1 fused to the Fc and an scFab for binding target 2. Heterodimerization is ensured by mutations in the Fc.

[0226] In some embodiments, the bispecific binding protein is in the form of DuetMab, a heterodimeric construct containing two distinct Fabs that bind antigens 1 and 2, and an Fc stabilized by a heterodimerization mutation. Fabs 1 and 2 contain distinct SS bridges that ensure proper LC and HC pairing.

[0227] In some embodiments, the bispecific binding protein is in the form of a CrossmAb, which is a heterodimeric construct having two different Fabs fused to binding targets 1 and 2 via heterodimerization. The CL and CH1 domains, as well as the VH and VL domains, are exchanged; for example, CH1 is fused inline with VL, while CL is fused inline with VH.

[0228] In some embodiments, the bispecific binding protein is in the form of Fit-Ig, which is a homodimeric construct in which the Fab binding antigen 2 is fused to the N-terminus of the HC of the Fab binding antigen 1. The construct contains wild-type Fc.

[0229] Tables 2A-2B list the heavy and light chains that can bind to VEGF and / or PD-1 during combination.

[0230] Sequences of PD-1 antibody and bispecific anti-VEGF / PD-1 binding protein

[0231] This article provides information on binding proteins that bind to programmed death receptor 1 (PD-1). PD-1 binding proteins typically contain one or more PD-1 binding regions, as discussed further in this article.

[0232] This article also provides bispecific binding proteins that bind to vascular endothelial growth factor (VEGF) and programmed death receptor 1 (PD-1). The provided bispecific binding proteins typically contain one or more VEGF-binding domains and one or more PD-1-binding domains.

[0233] Exemplary feature sequences of VEGF binding regions

[0234] The VEGF-binding region can bind VEGF. In some embodiments, the VEGF-binding region can bind to epitopes of human VEGF (such as human VEGF-A isotype). In some embodiments, the VEGF-binding region can bind to all human VEGF-A isotypes. In some embodiments, the VEGF-binding region antagonizes VEGF, for example by blocking the binding of VEGF to its receptor.

[0235] In some embodiments, the VEGF binding region includes a heavy chain variable structural domain, which includes complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 having sequences as shown in Table 2. In some embodiments, the VEGF binding region further includes a light chain variable structural domain, which includes complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 having sequences as shown in Table 2.

[0236] In some embodiments, the VEGF binding region comprises a heavy chain variable domain having a heavy chain variable domain sequence as shown in Table 2. In some embodiments, the VEGF binding region comprises a heavy chain variable domain that is a variant of the heavy chain variable sequence shown in Table 2 because the heavy chain variable domain has (1) CDR-H1, CDR-H2, and CDR-H3 having sequences as shown in Table 2, and (2) an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the heavy chain variable domain shown in Table 2.

[0237] In some embodiments, the VEGF binding region comprises a heavy chain variable domain (or a variant thereof) as described in Table 2 herein, and further comprises a light chain variable region having (1) CDR-L1, CDR-L2, and CDR-L3 having sequences as shown in Table 2, and (2) an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the light chain variable domain shown in Table 2.

[0238] Table 2: Feature sequences of exemplary VEGF binding regions

[0239] In some embodiments, the VEGF binding region includes a heavy chain variable structure domain, which includes complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of sequences within the heavy chain variable structure domain sequences shown in Table 5A. In some embodiments, the VEGF binding region further includes a light chain variable structure domain, which includes complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of sequences within the light chain variable structure domain sequences shown in Table 6A.

[0240] In some embodiments, the VEGF binding region comprises a heavy chain variable domain having a heavy chain variable sequence as shown in Table 5A. In some embodiments, the VEGF binding region comprises a heavy chain variable domain that is a variant of the heavy chain variable sequence shown in Table 5A because the heavy chain variable domain has (1) CDR-H1, CDR-H2, and CDR-H3 having sequences within a given heavy chain variable domain sequence shown in Table 5A, and (2) an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the same heavy chain variable domain sequence shown in Table 5A.

[0241] In some embodiments, the VEGF binding region comprises a heavy chain variable domain (or a variant thereof) as described in Table 5A herein, and further comprises a light chain variable region having (1) CDR-L1, CDR-L2, and CDR-L3 within the light chain variable domain sequence as shown in Table 6A, and (2) an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the same light chain variable domain sequence shown in Table 6A.

[0242] Exemplary feature sequences of the PD-1 binding region

[0243] PD-1 The binding region can bind to PD-1. In some embodiments, the PD-1 binding region can bind to the epitope of human PD-1. In some embodiments, the PD-1 binding region antagonizes PD-1, for example by blocking the binding of PD-1 to PD-L1.

[0244] In some embodiments, the PD-1 binding region includes a heavy chain variable structural domain comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 having sequences as shown in Table 3A for a given group. In some embodiments, the PD-1 binding region further comprises a light chain variable structural domain comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 having sequences as shown in the same group in Table 3A.

[0245] Table 3A. Exemplary sequences of complementarity-determining regions within the PD-1 binding region

[0246] In some embodiments, the PD-1 binding region includes a heavy chain variable structure domain, which includes complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of sequences within the heavy chain variable structure domain sequence shown in Table 5B. In some embodiments, the PD-1 binding region further includes a light chain variable structure domain, which includes complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of sequences within the light chain variable structure domain sequence shown in Table 6B.

[0247] In some embodiments, the PD-1 binding region comprises a heavy chain variable domain having a heavy chain variable sequence as shown in Table 5B. In some embodiments, the PD-1 binding region comprises a heavy chain variable domain that is a variant of the heavy chain variable sequence shown in Table 5B because the heavy chain variable domain has (1) CDR-H1, CDR-H2, and CDR-H3 having sequences within a given heavy chain variable domain sequence shown in Table 5B, and (2) an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the same heavy chain variable domain sequence shown in Table 5B.

[0248] In some embodiments, the PD-1 binding region comprises a heavy chain variable domain (or a variant thereof) as described in Table 5B herein, and further comprises a light chain variable region having (1) CDR-L1, CDR-L2, and CDR-L3 within the light chain variable domain sequence as shown in Table 6B, and (2) an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the same light chain variable domain sequence shown in Table 6B.

[0249] Tables 3B and 3C depict exemplary VH and VL sequences of the PD-1 binding region, respectively, illustrating representative heavy chain variable domain and light chain variable domain frame sequences within the PD-1 binding region disclosed herein. Exemplary CDR sequences within these VH and VL sequences (as defined by Kabat) are shown within the frames. However, those skilled in the art will understand that the exact boundary between the CDR and the frame region can vary depending on the antibody annotation system used. For example, a CDR can be defined as described in Kabat et al., USDept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J Mol Biol 196:901-917 (1987), where the definition includes overlap or subsets of amino acid residues when compared to each other. Other CDR definitions may also be used, such as those based on IMGT, Chothia, or Contact residues. The exact number of residues covering a particular CDR varies depending on the sequence and size of the CDR. Those skilled in the art can typically determine which residues contain a specific CDR based on the amino acid sequence of the antibody's variable region.

[0250] In some embodiments, the PD-1 binding region comprises: (1) a CDR group as described herein with respect to the PD-1 binding region; (2) a heavy chain variable domain having a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 97.5%, at least 98%, or at least 99% amino acid sequence identity with the sequence of the VH sequence shown in Table 3B; and (3) a light chain variable domain having a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 97.5%, at least 98%, or at least 99% amino acid sequence identity with the sequence of the VL sequence shown in Table 3C.

[0251] In some embodiments, the PD-1 binding region includes a heavy chain variable domain framework sequence as shown in the VH sequence in Table 3B and a light chain variable domain framework sequence as shown in the VL sequence in Table 3C.

[0252] In some embodiments, the PD-1 binding region comprises variants of the heavy chain variable domain framework sequence shown in the VH sequence as shown in Table 3B and the light chain variable domain framework sequence shown in the VL sequence as shown in Table 3C. In some embodiments, the PD-1 binding region comprises variants of the heavy chain variable domain framework sequence shown in the VH sequence as shown in Table 3B and the light chain variable domain framework sequence shown in the VL sequence as shown in Table 3C. In some embodiments, the PD-1 binding region comprises variants of the heavy chain variable domain framework sequence shown in the VH sequence as shown in Table 3B and the light chain variable domain framework sequence shown in the VL sequence as shown in Table 3C.

[0253] Examples of variants of the disclosed frame sequence include, but are not limited to: (1) sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 97.5%, at least 98%, or at least 99% amino acid sequence identity with the disclosed frame sequence across frame region residues (excluding CDR residues, as defined by any CDR used by those skilled in the art); and (2) sequences substantially identical to the disclosed frame sequence except for specific frame mutations or frameset mutation groups as discussed herein (e.g., up to four, three, two, or one amino acid substitution, deletion, or insertion within a given immunoglobulin frame region). Non-limiting examples of specific frame mutations include stabilization mutations and / or cysteine ​​mutations (e.g., at H44 and / or L100).

[0254] Any combination of the heavy chain framework sequences and light chain framework sequences disclosed herein can be used in the PD-1 binding region disclosed herein. Non-limiting examples of heavy chain and light chain framework sequence pairs that can be used within the PD-1 binding region include: (1) The VH0 and VL0 frame regions (within SEQ ID NO: 464 and 471, respectively) or variations thereof; (2) The VH1 and VL1 frame regions (within SEQ ID NO: 465 and 472, respectively) or variants thereof; (3) The VH1 and VL0 frame regions (within SEQ ID NO: 465 and 471, respectively) or variants thereof; (4) VH1 and VL2 frame regions (within SEQ ID NO: 465 and 473, respectively) or variants thereof; (5) The VH0 and VL1 frame regions (within SEQ ID NO: 464 and 472, respectively) or variants thereof; (6) The VH0 and VL2 frame regions (within SEQ ID NO: 464 and 473, respectively) or variants thereof; (7) The VH0 and VL3 frame regions (within SEQ ID NO: 464 and 474, respectively) or variants thereof; (8) The VH0 and VL4 frame regions (within SEQ ID NO: 464 and 475, respectively) or variants thereof; (9) VH3 and VL0 frame regions (within SEQ ID NO: 467 and 471, respectively) or variants thereof; (10) VH3 and VL3 frame regions (within SEQ ID NO: 467 and 474, respectively) or variants thereof; (11) VH3 and VL4 frame regions (within SEQ ID NO: 467 and 475, respectively) or variants thereof; (12) VH4 and VL0 frame regions (within SEQ ID NO: 468 and 471, respectively) or variants thereof; (13) VH4 and VL3 frame regions (within SEQ ID NO: 468 and 474, respectively) or variants thereof; (14) VH4 and VL4 frame regions (within SEQ ID NO: 468 and 475, respectively) or variants thereof; (15) VH2 and VL0 frame regions (within SEQ ID NO: 466 and 471, respectively) or variants thereof; (16) VH6 and VL3 frame regions (within SEQ ID NO: 470 and 474, respectively) or variants thereof; and (17) VH5 and VL5 frame regions (within SEQ ID NO: 469 and 476, respectively) or variants thereof; Any frame sequence disclosed herein may be combined with any group of CDRs in the PD-1 binding region as described herein or any CDRs within the VH and VL sequences shown in Tables 5B and 6B.

[0255] Table 3B. Exemplary VH sequences of the PD-1 binding region

[0256] Table 3C. Exemplary VL sequences of the PD-1 binding region

[0257] Heavy chain and light chain structural domains

[0258] In some embodiments, the binding protein comprises: a heavy chain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 1-147, 656-678, and 702-713; and a light chain sequence comprising a sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 148-194, 679-701, and 714-737. In some embodiments, the binding protein comprises: a heavy chain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 1-147, 656-678, and 702-713; and a light chain sequence comprising a sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 148-194, 679-701, and 714-737. In some embodiments, the binding protein comprises: a heavy chain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 1-147, 656-678, and 702-713; and a light chain sequence comprising a sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 148-194, 679-701, and 714-737. In some embodiments, the binding protein comprises: a heavy chain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 1-147, 656-678, and 702-713; and a light chain sequence comprising a sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 148-194, 679-701, and 714-737. In some embodiments, the binding protein comprises: a heavy chain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 1-147, 656-678, and 702-713; and a light chain sequence comprising a sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 148-194, 679-701, and 714-737. In some embodiments, the binding protein comprises: a heavy chain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 1-147, 656-678, and 702-713; and a light chain sequence comprising a sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 148-194, 679-701, and 714-737.In some embodiments, the binding protein comprises: a heavy chain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 1-147, 656-678, and 702-713; and a light chain sequence comprising a sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 148-194, 679-701, and 714-737. In some embodiments, the binding protein comprises: a heavy chain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 1-147, 656-678, and 702-713; and a light chain sequence comprising a sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 148-194, 679-701, and 714-737. In some embodiments, the binding protein comprises: a heavy chain comprising an amino acid sequence having 100% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 1-147, 656-678, and 702-713; and a light chain sequence comprising a sequence having 100% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 148-194, 679-701, and 714-737.

[0259] In some embodiments, this document provides bispecific binding proteins that bind vascular endothelial growth factor (VEGF) and programmed death receptor 1 (PD-1), comprising: a) a heavy chain sequence comprising an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 22-23, 60, 62, 65-67, 70-72, 75-77, 142-143, 667-678, and 702-713; and b) a light chain sequence comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NO: 148-194, 679-701, and 714-737.

[0260] In some embodiments, this document further provides bispecific binding proteins that bind vascular endothelial growth factor (VEGF) and programmed death receptor 1 (PD-1), comprising: a) a heavy chain sequence comprising an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO: 1-21, 24-57, 58, 59, 61, 63, 64, 68, 69, 73, 74, 78-97, 130-141, 144-147, and 656-666; and b) a light chain sequence comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NO: 148-194, 679-701, and 714-737.

[0261] In some embodiments, this document further provides bispecific binding proteins that bind vascular endothelial growth factor (VEGF) and programmed death receptor 1 (PD-1), comprising: a) a heavy chain sequence comprising the amino acid sequence of any one of SEQ ID NO: 98-127 and 129; and b) a light chain sequence comprising a sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 148-194, 679-701, and 714-737.

[0262] Table 4A. Sequences of the heavy and light chains of multispecific binding proteins

[0263] Table 4B. Sequences of the heavy and light chains of multispecific binding proteins

[0264] VH domain

[0265] In some embodiments, the binding protein provided herein comprises a first VH sequence selected from SEQ ID NO: 295-296, 301-334, and 738-742 and a second VH sequence selected from SEQ ID NO: 295-296, 301-334, and 738-742. In some embodiments, the binding protein provided herein comprises a first VH sequence selected from SEQ ID NO: 295-296 and a second VH sequence selected from SEQ ID NO: 301-334. In some embodiments, the binding protein provided herein comprises a first VH sequence containing SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 301-334.

[0266] In some embodiments, the binding protein provided herein comprises a first VH sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a VH sequence selected from SEQ ID NO: 295-296 and a second VH sequence selected from SEQ ID NO: 301-334. In some embodiments, the binding protein provided herein comprises a first VH sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a VH sequence containing SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 301-334. In some embodiments, the binding protein provided herein comprises a first VH sequence selected from SEQ ID NO: 295-296 and a second VH sequence selected from SEQ ID NO: 301-334, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some embodiments, the binding protein provided herein comprises a first VH sequence containing SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 301-334, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some embodiments, the amino acid substitutions are conserved amino acid substitutions. In some embodiments, the bispecific binding protein described in this paragraph is referred to herein as a “variant.” In some embodiments, such variants are derived from sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from sequences provided herein and can be de novo isolated, for example, according to methods provided herein for obtaining bispecific binding proteins.

[0267] Table 5A. Sequence of the VEGF heavy chain variable region (VH)

[0268] Table 5B. Sequence of the PD-1 heavy chain variable region (VH)

[0269] VL domain

[0270] In some embodiments, the binding protein provided herein comprises a first VL sequence selected from SEQ ID NO: 298-300, 335-349, and 743-744 and a second VL sequence selected from SEQ ID NO: 298-300, 335-349, and 743-744. In some embodiments, the binding protein provided herein comprises a first VL sequence containing SEQ ID NO: 298 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744. In some embodiments, the binding protein provided herein comprises a first VL sequence containing SEQ ID NO: 298 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744. In some embodiments, the binding protein provided herein comprises a first VL sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VL sequence containing SEQ ID NO: 298 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744. In some embodiments, the binding protein provided herein comprises a first VL sequence containing SEQ ID NO: 299 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744. In some embodiments, the binding protein provided herein comprises a first VL sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VL sequence containing SEQ ID NO: 299 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744. In some embodiments, the binding protein provided herein comprises a first VL sequence containing SEQ ID NO: 300 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744. In some embodiments, the binding protein provided herein comprises a first VL sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VL sequence containing SEQ ID NO: 300 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744.

[0271] In some embodiments, the binding protein provided herein comprises a first VL sequence selected from SEQ ID NO: 298-300 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some embodiments, the binding protein provided herein comprises a first VL sequence containing SEQ ID NO: 298 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[0272] Table 6A. Sequence of the variable region (VL) of the VEGF light chain

[0273] Table 6B. Sequence of the variable region (VL) of the PD-1 light chain

[0274] VH-VL combination

[0275] In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 of VH (anti-PD1 VH) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO 445, 456, and 457, respectively; (b) SEQ ID NO: 451, 458, and 459, respectively; or (c) SEQ ID NO: 443, 460, and 457, respectively; and the CDR-L1, CDR-L2, and CDR-HL of VL (anti-PD1 VL) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO: 448, 449, and 450, respectively; (b) SEQ ID NO: 454, SAS, and SEQ ID NO: 450, respectively; or (c) SEQ ID NO: 448, 449, or 450, respectively; and the anti-PD1 VH and anti-PD1 VL... VL has an amino acid sequence that is at least 85% identical to the amino acid sequences of the following: (a) SEQ ID NO: 302 and 335, respectively; (b) SEQ ID NO: 302 and 336, respectively; (c) SEQ ID NO: 302 and 340, respectively; (d) SEQ ID NO: 302 and 342, respectively; (e) SEQ ID NO: 302 and 343, respectively; (f) SEQ ID NO: 302 and 346, respectively; (g) SEQ ID NO: 306 and 337, respectively; (h) SEQ ID NO: 306 and 339, respectively; (i) SEQ ID NO: 306 and 341, respectively; (j) SEQ ID NO: 306 and 344, respectively; (k) SEQ ID NO: 306 and 345, respectively; (l) SEQ ID NO: 302 and 346, respectively; (v) SEQ ID NO: 302 and 346, respectively; (v) SEQ ID NO: 302 and 346, respectively; (v) SEQ ID NO: 306 and 345, respectively; (v) SEQ ID NO: 302 and 346 ... SEQ ID NO: 306 and 347; (m) respectively, SEQ ID NO: 308 and 340; (n) respectively, SEQ ID NO: 309 and 341; (o) respectively, SEQ ID NO: 310 and 336; (p) respectively, SEQ ID NO: 311 and 337; (q) respectively, SEQ ID NO: 313 and 336; (r) respectively, SEQ ID NO: 313 and 342; (s) respectively, SEQ ID NO: 313 and 343; (t) respectively, SEQ ID NO: 316 and 337; (u) respectively, SEQ ID NO: 316 and 344; (v) respectively, SEQ ID NO: 316 and 345; (w) respectively, SEQ ID NO: 318 and 336;(x) respectively, SEQ ID NO: 318 and 340; (y) respectively, SEQ ID NO: 318 and 346; (z) respectively, SEQ ID NO: 319 and 336; (aa) respectively, SEQ ID NO: 319 and 340; (bb) respectively, SEQ ID NO: 319 and 346; (cc) respectively, SEQ ID NO: 320 and 337; (dd) respectively, SEQ ID NO: 320 and 341; (ee) respectively, SEQ ID NO: 320 and 347; (ff) respectively, SEQ ID NO: 320 and 343; (gg) respectively, SEQ ID NO: 321 and 337; (hh) respectively, SEQ ID NO: 321 and 341; or (ii) respectively, SEQ ID NO: 321 and 347. In some embodiments, anti-PD1 VH and anti-PD1 VL have the amino acid sequences of the following: (a) respectively, SEQ ID NO: 302 and 335; (b) respectively, SEQ ID NO: 302 and 336; (c) respectively, SEQ ID NO: 302 and 340; (d) respectively, SEQ ID NO: 302 and 342; (e) respectively, SEQ ID NO: 302 and 343; (f) respectively, SEQ ID NO: 302 and 346; (g) respectively, SEQ ID NO: 306 and 337; (h) respectively, SEQ ID NO: 306 and 339; (i) respectively, SEQ ID NO: 306 and 341; (j) respectively, SEQ ID NO: 306 and 344; (k) respectively, SEQ ID NO: 306 and 345; (l) respectively, SEQ ID NO: 302 and 346. SEQ ID NO: 306 and 347; (m) respectively, SEQ ID NO: 308 and 340; (n) respectively, SEQ ID NO: 309 and 341; (o) respectively, SEQ ID NO: 310 and 336; (p) respectively, SEQ ID NO: 311 and 337; (q) respectively, SEQ ID NO: 313 and 336; (r) respectively, SEQ ID NO: 313 and 342; (s) respectively, SEQ ID NO: 313 and 343; (t) respectively, SEQ ID NO: 316 and 337; (u) respectively, SEQ ID NO: 316 and 344; (v) respectively, SEQ ID NO: 316 and 345; (w) respectively, SEQ ID NO: 318 and 336;(x) SEQ ID NO: 318 and 340 respectively; (y) SEQ ID NO: 318 and 346 respectively; (z) SEQ ID NO: 319 and 336 respectively; (aa) SEQ ID NO: 319 and 340 respectively; (bb) SEQ ID NO: 319 and 346 respectively; (cc) SEQ ID NO: 320 and 337 respectively; (dd) SEQ ID NO: 320 and 341 respectively; (ee) SEQ ID NO: 320 and 347 respectively; (ff) SEQ ID NO: 320 and 743 respectively; (gg) SEQ ID NO: 321 and 337 respectively; (hh) SEQ ID NO: 321 and 341 respectively; or (ii) SEQ ID NO: 321 and 347 respectively.

[0276] In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 of VH (anti-PD1 VH) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO 445, 456, and 447, respectively; (b) SEQ ID NO: 451, 458, and 453, respectively; or (c) SEQ ID NO: 443, 460, and 447, respectively, and the CDR-L1, CDR-L2, and CDR-HL of VL (anti-PD1 VL) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO: 448, 449, and 450, respectively; (b) SEQ ID NO: 454, SAS, and SEQ ID NO: 450, respectively; or (c) SEQ ID NO: 448, 449, or 450, respectively, and anti-PD1 VH and anti-PD1 VL... VL has an amino acid sequence that is at least 85% identical to the following: (a) SEQ ID NO: 303 and 336, respectively; (b) SEQ ID NO: 325 and 347, respectively; or (c) SEQ ID NO: 325 and 743, respectively. In some embodiments, anti-PD1 VH and anti-PD1 VL have the following amino acid sequences: (a) SEQ ID NO: 303 and 336, respectively; (b) SEQ ID NO: 325 and 347, respectively; or (c) SEQ ID NO: 325 and 743, respectively.

[0277] In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 of VH (anti-PD1 VH) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO 445, 461, and 447, respectively; (b) SEQ ID NO 451, 462, and 453, respectively; or (c) SEQ ID NO 443, 463, and 447, respectively; and the CDR-L1, CDR-L2, and CDR-HL of VL (anti-PD1 VL) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO 448, 449, and 450, respectively; (b) SEQ ID NO 454, SAS, and SEQ ID NO 450, respectively; or (c) SEQ ID NO 448, 449, or 450, respectively; and the anti-PD1 VH and anti-PD1 VL... VL has an amino acid sequence that is at least 85% identical to the following: respectively, SEQ ID NO: 326 and 347; or respectively, SEQ ID NO: 326 and 743. In some embodiments, anti-PD1 VH and anti-PD1 VL have the following amino acid sequences: respectively, SEQ ID NO: 326 and 347; or respectively, SEQ ID NO: 326 and 743.

[0278] In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 of VH (anti-PD1 VH) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO 445, 446, and 457, respectively; (b) SEQ ID NO: 451, 452, and 459, respectively; or (c) SEQ ID NO: 443, 455, and 457, respectively, and the CDR-L1, CDR-L2, and CDR-HL of VL (anti-PD1 VL) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO: 448, 449, and 450, respectively; (b) SEQ ID NO: 454, SAS, and SEQ ID NO: 450, respectively; or (c) SEQ ID NO: 448, 449, or 450, respectively, and anti-PD1 VH and anti-PD1 VL... VL has an amino acid sequence that is at least 85% identical to the following: respectively, SEQ ID NO: 327 and 347; or respectively, SEQ ID NO: 327 and 743. In some embodiments, anti-PD1 VH and anti-PD1 VL have the following amino acid sequences: respectively, SEQ ID NO: 327 and 347; or respectively, SEQ ID NO: 327 and 743.

[0279] In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 of VH (anti-PD1 VH) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO 445, 461, and 457, respectively; (b) SEQ ID NO: 451, 462, and 459, respectively; or (c) SEQ ID NO: 443, 463, and 457, respectively, and the CDR-L1, CDR-L2, and CDR-HL of VL (anti-PD1 VL) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO: 448, 449, and 450, respectively; (b) SEQ ID NO: 454, SAS, and SEQ ID NO: 450, respectively; or (c) SEQ ID NO: 448, 449, or 450, respectively, and anti-PD1 VH and anti-PD1 VL... VL has an amino acid sequence that is at least 85% identical to the amino acid sequences of the following: (a) SEQ ID NO: 303 and 335, respectively; (b) SEQ ID NO: 303 and 342, respectively; (c) SEQ ID NO: 303 and 343, respectively; (d) SEQ ID NO: 307 and 337, respectively; (e) SEQ ID NO: 307 and 344, respectively; (f) SEQ ID NO: 307 and 345, respectively; (g) SEQ ID NO: 314 and 336, respectively; (h) SEQ ID NO: 314 and 342, respectively; (i) SEQ ID NO: 314 and 343, respectively; (j) SEQ ID NO: 317 and 337, respectively; (k) SEQ ID NO: 317 and 344, respectively; (l) SEQ ID NO: 303 and 335, respectively. NO: 317 and 345; or (m) respectively, SEQ ID NO: 324 and 347.In some embodiments, anti-PD1 VH and anti-PD1 VL have the following amino acid sequences: (a) SEQ ID NO: 303 and 335, respectively; (b) SEQ ID NO: 303 and 342, respectively; (c) SEQ ID NO: 303 and 343, respectively; (d) SEQ ID NO: 307 and 337, respectively; (e) SEQ ID NO: 307 and 344, respectively; (f) SEQ ID NO: 307 and 345, respectively; (g) SEQ ID NO: 314 and 336, respectively; (h) SEQ ID NO: 314 and 342, respectively; (i) SEQ ID NO: 314 and 343, respectively; (j) SEQ ID NO: 317 and 337, respectively; (k) SEQ ID NO: 317 and 344, respectively; (l) Specifically, SEQ ID NO: 317 and 345; or (m) specifically, SEQ ID NO: 324 and 347.

[0280] In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 of VH (anti-PD1 VH) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO 445, 446, and 447, respectively; (b) SEQ ID NO: 451, 452, and 453, respectively; or (c) SEQ ID NO: 443, 455, and 447, respectively, and the CDR-L1, CDR-L2, and CDR-HL of VL (anti-PD1 VL) within the PD-1 binding region comprise the amino acid sequences of: (a) SEQ ID NO: 448, 449, and 450, respectively; (b) SEQ ID NO: 454, SAS, and SEQ ID NO: 450, respectively; or (c) SEQ ID NO: 448, 449, or 450, respectively, and anti-PD1 VH and anti-PD1 VL... VL has an amino acid sequence that is at least 85% identical to the amino acid sequences of the following: (a) SEQ ID NO: 301 and 335, respectively; (b) SEQ ID NO: 301 and 336, respectively; (c) SEQ ID NO: 301 and 342, respectively; (d) SEQ ID NO: 301 and 343, respectively; (e) SEQ ID NO: 301 and 348, respectively; (f) SEQ ID NO: 305 and 337, respectively; (g) SEQ ID NO: 305 and 344, respectively; (h) SEQ ID NO: 305 and 345, respectively; (i) SEQ ID NO: 312 and 335, respectively; (j) SEQ ID NO: 312 and 336, respectively; (k) SEQ ID NO: 312 and 342, respectively; (l) SEQ ID NO: 301 and 336, respectively; (v) SEQ ID NO: 301 and 336, respectively; (v) SEQ ID NO: 301 and 336, respectively; (v) SEQ ID NO: 301 and 342, respectively; (v) SEQ ID NO: 301 and 336 ... NO: 312 and 343; (m) respectively, SEQ ID NO: 315 and 337; (n) respectively, SEQ ID NO: 315 and 344; (o) respectively, SEQ ID NO: 315 and 345; (p) respectively, SEQ ID NO: 322 and 346; (q) respectively, SEQ ID NO: 323 and 347; or (r) respectively, SEQ ID NO: 323 and 743.In some embodiments, anti-PD1 VH and anti-PD1 VL have the amino acid sequences of the following: (a) respectively, SEQ ID NO: 301 and 335; (b) respectively, SEQ ID NO: 301 and 336; (c) respectively, SEQ ID NO: 301 and 342; (d) respectively, SEQ ID NO: 301 and 343; (e) respectively, SEQ ID NO: 301 and 348; (f) respectively, SEQ ID NO: 305 and 337; (g) respectively, SEQ ID NO: 305 and 344; (h) respectively, SEQ ID NO: 305 and 345; (i) respectively, SEQ ID NO: 312 and 335; (j) respectively, SEQ ID NO: 312 and 336; (k) respectively, SEQ ID NO: 312 and 342; (l) respectively, SEQ ID NO: 301 and 335. SEQ ID NO: 312 and 343; (m) respectively, SEQ ID NO: 315 and 337; (n) respectively, SEQ ID NO: 315 and 344; (o) respectively, SEQ ID NO: 315 and 345; (p) respectively, SEQ ID NO: 322 and 346; (q) respectively, SEQ ID NO: 323 and 347; or (r) respectively, SEQ ID NO: 323 and 743.

[0281] In some embodiments, the binding protein provided herein comprises a first VH sequence selected from SEQ ID NO: 295 or SEQ ID NO: 296 and a second VH sequence selected from SEQ ID NO: 301-334 and 738-742; and a first VL sequence selected from SEQ ID NO: 298-300 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744. In some embodiments, the binding protein provided herein comprises a first VH sequence containing SEQ ID NO: 295 and a second VH sequence selected from SEQ ID NO: 301-334 and 738-742; and a first VL sequence containing SEQ ID NO: 298 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744.

[0282] In some respects, any one of SEQ ID NO: 295 or SEQ ID NO: 296 may be combined with any one of SEQ ID NO: 298-300, and any one of SEQ ID NO: 301-334 and 738-742 may be combined with any one of SEQ ID NO: 335-349 and 743-744.

[0283] In some embodiments, the binding protein provided herein comprises a first VH sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a VH sequence selected from SEQ ID NO: 295 or SEQ ID NO: 296, and a second VH sequence selected from SEQ ID NO: 301-334 and 738-742; and a first VL sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a VL sequence provided in SEQ ID NO: 298-300, and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744. In some embodiments, the binding protein provided herein comprises a first VH sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VH sequence containing SEQ ID NO: 295 and a second VH sequence selected from SEQ ID NO: 301-334 and 738-742; and a first VL sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VL sequence containing SEQ ID NO: 298 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744.

[0284] In some embodiments, the binding protein provided herein comprises a first VH sequence provided in SEQ ID NO: 295 or SEQ ID NO: 296 and a second VH sequence selected from SEQ ID NO: 301-334 and 738-742, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions; and a first VL sequence provided in SEQ ID NO: 298-300 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions. In some embodiments, the binding protein provided herein comprises a first VH sequence of SEQ ID NO: 295 and a second VH sequence selected from SEQ ID NO: 301-334 and 738-742, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions; and a first VL sequence of SEQ ID NO: 298 and a second VL sequence selected from SEQ ID NO: 335-349 and 743-744, having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some embodiments, the amino acid substitutions are conserved amino acid substitutions. In some embodiments, the binding protein described in this paragraph is referred to herein as a “variant.” In some embodiments, such variants are derived from sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from sequences provided herein and can be de novo isolated, for example, according to methods provided herein for obtaining binding proteins.

[0285] Bispecific form

[0286] In some embodiments, the bispecific binding protein disclosed herein takes the form of any of the bispecific antibody forms described herein. In some embodiments, the bispecific binding proteins disclosed herein are selected from the group consisting of: (a) single-chain Fv (scFv), (b) tandem scFv form of a bispecific T-cell adaptor (BiTE), (c) disulfide-linked biantibody form of amphiphilic and highly targeted (DART) bsAb, (d) tandem biantibody (TandAb), (e) single-domain antibody (VHH), (f) conventional immunoglobulin G (IgG), (g) IgG having additional binding units (such as scFv), (h) IgG having additional binding units (such as VHH), (i) dual variable domain immunoglobulin (DVD-Ig), (j) quadromab bsAb, (k) mortis-and-mortis (KiH) bsAb having a common light chain, (l) KiH-CrossMabCH1-CL, and (m) bsAb obtained via controlled Fab arm exchange (cFAE). (See Shim et al. (2020)) Biomolecules [Biomolecules] 26; 10(3):360.) In some embodiments, the bispecific binding protein disclosed herein is selected from the group consisting of IgG-scFv, IgG-VHH, and DVD-Ig. In some embodiments, the bispecific binding protein disclosed herein is in the form of IgG-scFv. In some embodiments, the bispecific binding protein disclosed herein is in the form of IgG-VHH. In some embodiments, the bispecific binding protein disclosed herein is in the form of DVD-Ig. In some embodiments, the bispecific binding protein disclosed herein is in the form of kiH-CrossMabCH1-CL.

[0287] In some embodiments, the bispecific binding protein comprises an scFv characterized by one or more stabilizing features (e.g., specific amino acid residues or sequences). In some embodiments, one or more stabilizing features in the scFv are engineered disulfide bonds between the contact surfaces of the VH and VL domains. In some embodiments, the disulfide bonds are located at positions H44-L100 (Kabat numbering).

[0288] In some embodiments, the binding protein comprises a heavy chain as disclosed in Table 4A or Table 4B and a light chain as disclosed in Table 4A or Table 4B. In some embodiments, the binding protein provided herein comprises a first VH sequence as listed in Table 5A and a second VH sequence as listed in Table 5B. In some embodiments, the binding protein provided herein comprises a first VL sequence as listed in Table 6A and a second VL sequence as listed in Table 6B. In some embodiments, the binding protein provided herein comprises a heavy chain constant region as listed in Table 7. In some embodiments, the binding protein provided herein comprises a light chain constant region as listed in Table 8. In some embodiments, the binding protein provided herein comprises an scFv or VHH sequence as listed in Table 9. In some embodiments, the binding protein provided herein comprises a first VH sequence as listed in Table 5A and a second VH sequence as listed in Table 5B; a first VL sequence as listed in Table 6A and a second VL sequence as listed in Table 6B; a heavy chain constant region as listed in Table 7; a light chain constant region as listed in Table 8; and an scFv or VHH sequence as listed in Table 9.

[0289] In some embodiments, the bispecific binding protein disclosed herein is in the form of IgG-scFv. In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing any one of SEQ ID NO: 295 or SEQ ID NO: 296, a heavy chain constant region (CH(1-3)) containing any one of SEQ ID NO: 350-352 and a first adapter having at least 90% sequence identity with GGGGSGGGGSGGGGSGGGS, a first VL sequence (VL1) containing any one of SEQ ID NO: 298-300, a second adapter having at least 90% sequence identity with GGGGSGGGGSGGGSGGGS, a second VH (VH2) sequence containing any one of SEQ ID NO: 301-334 and 738-742, a second VL sequence (VL2) containing any one of SEQ ID NO: 335-349 and 743-744, and a light chain constant domain (CL) containing SEQ ID NO: 353.

[0290] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing any one of SEQ ID NO: 301-334 and 738-742, a heavy chain constant region (CH(1-3)) containing any one of SEQ ID NO: 350-352 and a first adapter having at least 90% sequence identity with GGGGSGGGGSGGGGSGGGS, a first VL sequence (VL1) containing any one of SEQ ID NO: 298-300, a second adapter having at least 90% sequence identity with GGGGSGGGGSGGGSGGGS, a second VH (VH2) sequence containing any one of SEQ ID NO: 295 or SEQ ID NO: 296, a second VL sequence (VL2) containing any one of SEQ ID NO: 335-349 and 743-744, and a light chain constant domain (CL) containing SEQ ID NO: 353.

[0291] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing any one of SEQ ID NO: 295-296, 301-334 and 738-742, a heavy chain constant region (CH(1-3)) containing any one of SEQ ID NO: 350-352 and a first adapter having at least 90% sequence identity with GGGGSGGGGSGGGGSGGGGS, an scFv or VHH containing any one of SEQ ID NO: 354-430, a first VL sequence containing any one of SEQ ID NO: 298-300 and a light chain constant domain (CL) containing SEQ ID NO: 353.

[0292] Table 7. Sequences of exemplary heavy chain constant regions 1-3

[0293] Table 8. Sequences of exemplary light constant regions

[0294] Table 9. Exemplary scFv and VHH sequences

[0295] Fc modification

[0296] The binding proteins described herein (e.g., PD-1 binding proteins or bispecific PD-1 / VEGF binding proteins) typically comprise an immunoglobulin Fc region. An Fc region typically contains one or more Fc polypeptides, such as a first Fc polypeptide and a second Fc polypeptide. IgG Fc polypeptides typically contain two constant recombinant domains (CH2 and CH3) and a hinge region connected to the CH2 domain. A typical Fc region contains two Fc polypeptides dimerized together; however, an Fc region may have a single Fc polypeptide or more than two Fc polypeptides, as can be found, for example, in some antibody forms.

[0297] In some embodiments, the binding protein described herein comprises an IgG1 Fc region (e.g., a human IgG1 Fc region), i.e., an Fc region having an amino acid sequence substantially similar to the amino acid sequence of an Fc region within wild-type IgG1 Fc, except for having specific residues at certain positions as described herein. In some embodiments, wild-type IgG1 Fc is human IgG1 Fc, wherein each Fc polypeptide comprises an amino acid sequence of SEQ ID NO: 792, 911, or 1022. In some embodiments, the binding protein described herein comprises an Fc region, wherein each Fc polypeptide has an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of an Fc polypeptide within wild-type IgG1 Fc.

[0298] In some embodiments, the human IgG1 Fc region includes the SRDEL allotype or the SREEM allotype.

[0299] In some embodiments, the binding protein described herein comprises an IgG2 Fc region (e.g., a human IgG2 Fc region), i.e., an Fc region having an amino acid sequence substantially similar to the amino acid sequence of an Fc region within wild-type IgG2 Fc, except for having specific residues at certain positions as described herein. In some embodiments, wild-type IgG2 Fc is human IgG2 Fc, wherein each Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 793 or 912. In some embodiments, the binding protein described herein comprises an Fc region, wherein each Fc polypeptide has an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of an Fc polypeptide within wild-type IgG2 Fc.

[0300] In some embodiments, the binding protein described herein comprises an IgG4 Fc region (e.g., a human IgG4 Fc region), i.e., an Fc region having an amino acid sequence substantially similar to the amino acid sequence of an Fc region within wild-type IgG4 Fc, except for having specific residues at certain positions as described herein. In some embodiments, wild-type IgG4 Fc is human IgG4 Fc, wherein each Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 794 or 913. In some embodiments, the binding protein described herein comprises an Fc region, wherein each Fc polypeptide has an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of an Fc polypeptide within wild-type IgG4 Fc.

[0301] In some embodiments, the binding protein comprises a modified Fc containing one or more modifications. In some embodiments, the one or more modifications are located in the Fc from IgG1 (e.g., human IgG1 (hIgG1)). In some embodiments, the one or more modifications are located in the Fc from IgG4 (e.g., human IgG4 (hIgG4)). In some embodiments, the one or more modifications are located in the Fc from IgG2. In some embodiments, the one or more modifications promote selective binding to the Fc-γ receptor. In any embodiment, the constant heavy chain region may contain a C-terminal lysine residue.

[0302] Exemplary amino acid sequences of the Fc region are provided in Tables 10A and 10B. Unless otherwise stated herein, amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0303] Table 10A. Exemplary Fc peptide sequences

[0304] Table 10B. Exemplary Fc polypeptide sequences

[0305] In some embodiments, any binding protein described herein may include an Fc containing one or more modifications having any of the Fc modifications described herein. In some embodiments, any binding protein described herein may include any of the Fc sequences (SEQ ID NO: 792-1029) in Tables 10A-10B. In some embodiments, the CH(1-3) domains of the binding proteins in Table 7 are replaced by any of the Fc sequences (SEQ ID NO: 792-1029) in Tables 10A-10B.

[0306] In some embodiments, one or more modifications in the modified Fc are selected from the group consisting of: S298A, E333A, K334A, K326A, F243L, R292P, Y300L, V305I, P396L, F243L, R292P, Y300L, L235V, P396L, F243L, S239D, I332E, A330L, S267E, L328F, D265S, S239E, K326A, A327H, G237F, K326E, G236A, D270L, H268D, S324T, L234F, N325L, V266L, and S267D. In some embodiments, one or more modifications in the modified Fc are selected from the group consisting of: S228P, M252Y, S254T, T256E, T256D, T250Q, H285D, T307A, T307Q, T307R, T307W, L309D, Q411H, Q311V, A378V, E380A, M428L, N434A, N434S, N297A, D265A, L234A, L235A, and N434W.

[0307] In some embodiments, the modified Fc comprises a specific combination of amino acid substitutions selected from the group consisting of: L234A / L235A; V234A / G237A; L235A / G237A / E318A; S228P / L236E; H268Q / V309L / A330S / A331S; C220S / C226S / C229S / P238S; C226S / C229S / E3233P / L235V / L235A; L234F / L235E / P331S; C226S / P230S; L234A / G237A; L234A / L235A / G237A; and L234A / L235A / P329G.

[0308] In some embodiments, the modified Fc comprises a specific combination of amino acid substitutions selected from the group consisting of: M428L / N434S (LS); M252Y / S254T / T256E (YTE); T250Q / M428L; T307A / E380A / N434A; T256D / T307Q (DQ); T256D / T307W (DW); M252Y / T256D (YD); T307Q / Q311V / A378V (QVV); T256D / H285D / T307R / Q311V / A378V (DDRVV); L309D / Q311H / N434S (DHS); S228P / L235E ( SPLE); L234A / L235A (LALA); M428L / N434A (LA); L234A / G237A (LAGA); L234A / L235A / G237A (LALAGA); L234A / L235A / P329G (LALAPG); N297A / YTE; D265A / YTE;LALA / YTE;LAGA / YTE;LALAGA / YTE;LALAPG / YTE;N297A / LS;D265A / LS;LALA / LS;LAGA / LS;LALAGA / LS;LALAPG / LS;N297A / DHS;D265A / DHS;LALA / D HS; LAGA / DHS; LALAGA / DHS; LALAPG / DHS; SP / YTE; SPLE / YTE; SP / LS; SPLE / LS; SP / DHS; SPLE / DHS; N297A / LA; ALAPG / LA; N297A / N434A; D265A / N434A; LALA / N434A; LAGA / N434A; LALAGA / N434A; LALAPG / N434A; N297A / N434W; D265A / N434W; LALA / N434W; LAGA / N434 W;LALAGA / N434W;LALAPG / N434W;N297A / DQ;D265A / DQ;LALA / DQ;LAGA / DQ;LALAGA / DQ;LALAPG / DQ;N297A / DW;D265A / DW;LALA / DW;LAGA / DW;LALAGA / DW ;LALAPG / DW; N297A / YD; D265A / YD; LALA / YD; LAGA / YD; LALAGA / YD; LALAPG / YD; N297A / QVV; D265A / QVV; LALA / QVV; LAGA / QVV, LALAGA / QVV; LALAPG / QVV;N297A / DDRVV; D265A / DDRVV; LALA / DDRVV; LAGA / DDRVV; LALAGA / DDRVV; and LALAPG / DDRVV. In some embodiments, the modified Fc comprises a specific combination of amino acid substitutions selected from the group consisting of: M428L / N434S (LS) and M252Y / S254T / T256E (YTE). In some embodiments, the modified Fc comprises M428L / N434S (LS) (e.g., SEQ ID NO: 810, 827, 834, 904, 929, 946, 953, or 1023). In some embodiments, the modified Fc comprises M252Y / S254T / T256E (YTE) (e.g., SEQ ID NO: 803, 824, 833, 905, 922, 952, or 1024).

[0309] In some embodiments, the bispecific binding proteins described herein include modifications to enhance their ability to mediate effector function. Such modifications are known in the art and include fucosylation-free binding or affinity engineering of Fc for activating receptors (primarily FCGR3a, for antibody-dependent cytotoxicity (ADCC)) and for C1q (for complement-dependent cytotoxicity (CDC)).

[0310] In some embodiments, modifications to the Fc region reduce or eliminate effector function, such as Fcγ receptor-mediated effector function. Non-limiting examples of effector-reducing mutations or mutant groups include, for example, glycosylation-free mutations (e.g., N297A, N297Q, or N297G), L234A / L235A (for the IgG1 Fc region), H268Q / V309L / A330S / P331S (for the IgG2 Fc region), and V234A / G237A / P238S / H268A / V309L / A330S / P331S (for the IgG2 Fc region). In some embodiments, effector function is reduced by modifying the attached sugar structure. Non-limiting examples of effector-reducing sugar modifications include increasing sialylation or galactosylation of the sugar chain.

[0311] In some respects, the bispecific binding proteins described herein comprise an Fc domain (e.g., IgG1) with a reduced fucose content at position Asn 297 (EU number) compared to the naturally occurring Fc domain. Such Fc domains are known to have improved ADCC. In some respects, such binding proteins do not contain any fucose at position Asn 297.

[0312] In some embodiments, the bispecific binding protein described herein comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at one or more of positions 298, 333, and 334 of the Fc region. In some embodiments, the bispecific binding protein provided herein comprises an Fc region having one or more amino acid substitutions at positions 239, 332, and 330.

[0313] In some embodiments, Fc comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NOs in Table 10A or 10B. In some embodiments, Fc comprises an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NOs in Table 10A or 10B. In some embodiments, Fc comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NOs in Table 10A or 10B. In some embodiments, Fc comprises an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NOs in Table 10A or 10B. In some embodiments, Fc comprises an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NOs in Table 10A or 10B. In some embodiments, Fc comprises an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NOs in Table 10A or 10B. In some embodiments, Fc comprises an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NOs in Table 10A or 10B. In some embodiments, Fc comprises an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NOs in Table 10A or 10B. In some embodiments, Fc comprises an amino acid sequence according to any one of SEQ ID NOs in Table 10A or 10B.

[0314] In some embodiments, the bispecific binding protein described herein comprises an Fc region to which at least one galactose residue of the oligosaccharide is attached. Such variants may have improved CDC function.

[0315] In some embodiments, the bispecific binding proteins described herein comprise one or more alterations that improve or reduce C1q binding and / or CDC.

[0316] In some embodiments, the Fc region contains one or more amino acid substitutions, wherein the one or more substitutions result in an increase in one or more of the antibody half-life, ADCC activity, ADCP activity, or CDC activity compared to an Fc region without the one or more substitutions. In some embodiments, the one or more amino acid substitutions result in an increase in the antibody half-life at pH 6.0 compared to an antibody containing a wild-type Fc region. In some embodiments, the binding protein has an increased half-life compared to antibodies containing the wild-type Fc region, which is approximately 10,000, 1,000, 500, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.50, 1.45, 1.4, 1.35, 1.3, 1.25, 1.2, 1.15, 1.1, or 1.05 times that of antibodies containing the wild-type Fc region.

[0317] In some embodiments, the Fc region contains one or more amino acid substitutions, wherein the one or more substitutions result in a reduction of one or more of ADCC activity, ADCP activity, or CDC activity compared to an Fc without the one or more substitutions.

[0318] In some embodiments, the Fc region binds to Fcγ receptors selected from the group consisting of: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region binds to the Fcγ receptor with higher affinity at pH 6.0 compared to antibodies containing the wild-type Fc region.

[0319] In some embodiments, the bispecific binding protein described herein has an extended half-life (i.e., serum half-life). In some embodiments, the bispecific binding protein described herein has a half-life of at least about 14, 28, 42, 56, 70, 84, 96, or more than 96 weeks. In some embodiments, the binding protein described herein has a half-life in the range of: about 14 days to about 96 days, about 14 days to about 84 days, about 14 days to about 70 days, about 14 days to about 56 days, about 14 days to about 42 days, about 14 days to about 28 days, about 28 days to about 96 days, about 28 days to about 84 days, about 28 days to about 70 days, about 28 days to about 56 days, about 28 days to about 42 days, about 42 days to about 96 days, about 42 days to about 84 days, about 42 days to about 70 days, or about 42 days to about 56 days. In some embodiments, the bispecific binding protein described herein has a half-life in the range of about 42 days to about 56 days. In some embodiments, the bispecific binding protein described herein has a half-life of at least about 50 days. Methods for measuring the half-life are known in the art. In some embodiments, the half-life is measured in non-human primates. In some embodiments, the half-life is measured in humans. In some embodiments, the half-life is measured after intravenous administration. In some embodiments, the half-life is measured after subcutaneous administration.

[0320] In some embodiments, the bispecific binding protein described herein has a half-life at least 20% longer than that of the reference antibody. In some embodiments, the reference antibody comprises the same complementarity-determining region and variable region but different Fc regions. In some embodiments, the half-life of the bispecific binding protein described herein is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% longer than that of the reference antibody. In some embodiments, the half-life of the bispecific binding protein described herein is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times longer than that of the reference antibody.

[0321] Combination

[0322] The affinity between molecule X and its partner Y can be determined by the dissociation equilibrium constant (K). D The affinity is indicated by (). The kinetic components contributing to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by commonly used methods known in the art, including those described herein, such as surface plasmon resonance (SPR) techniques (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0323] Regarding antibody binding to target molecules, the terms "binding" to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen, "specifically binding" to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen, "specifically binding" to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen, "specifically" binding to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen, "selectively binding" to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen, and "selectively" binding to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen, mean measurably different from nonspecific or nonselective interactions (e.g., binding to non-target molecules). Specific binding can be measured, for example, by measuring binding to a target molecule (i.e., VEGF or PD-1) and comparing it to binding to non-target molecules. Specific binding can also be determined by competition with a control molecule that mimics an epitope recognized on a target molecule. In that case, if the binding of the antibody to the target molecule is competitively inhibited by the control molecule, it indicates specific binding. In some embodiments, the binding proteins disclosed herein have an affinity for non-target molecules less than about 50% of their affinity for VEGF or PD-1. In some embodiments, the binding proteins disclosed herein have an affinity for non-target molecules less than about 40% of their affinity for VEGF or PD-1. In some embodiments, the binding proteins disclosed herein have an affinity for non-target molecules less than about 40% of their affinity for VEGF or PD-1. In some embodiments, the binding proteins disclosed herein have an affinity for non-target molecules less than about 20% of their affinity for VEGF or PD-1. In some embodiments, the binding proteins disclosed herein have an affinity for non-target molecules less than about 10% of their affinity for VEGF or PD-1. In some embodiments, the binding proteins disclosed herein have an affinity for non-target molecules less than about 1% of their affinity for VEGF or PD-1. In some embodiments, the binding proteins disclosed herein have an affinity for non-target molecules less than about 0.1% of their affinity for VEGF or PD-1.

[0324] When used herein in the context of two or more binding proteins, the term “competitive” or “cross-competitive” means that two or more binding proteins compete to bind an antigen (e.g., VEGF). In one exemplary assay, VEGF is coated onto a surface and contacted with a first anti-VEGF antibody, and then a second anti-VEGF antibody is added. In another exemplary assay, a first anti-VEGF antibody is coated onto a surface and contacted with VEGF, and then a second anti-VEGF antibody is added. In either assay, the binding proteins compete with each other if the presence of the first anti-VEGF antibody reduces the binding of the second anti-VEGF antibody. The term “competitive” also includes combinations of binding proteins in which one antibody reduces the binding of another antibody, but no competition is observed when the binding proteins are added in reverse order. However, in some embodiments, the first and second binding proteins inhibit each other’s binding regardless of the order in which they are added. In some embodiments, one antibody reduces the binding of another antibody to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, as measured in a competitive binding assay. Skilled technicians can select the concentration of binding proteins used in competitive assays based on the affinity of the binding proteins for VEGF and the valence of the binding proteins. The assays described in this definition are illustrative, and technicians can use any suitable assay to determine whether binding proteins compete with each other. Suitable assays are described in the following literature: e.g., Cox et al., “Immunoassay Methods,” in AssayGuidance Manual [Internet], updated December 24, 2014 (ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al., Cytometry [Cytochemistry], 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358; each of which is incorporated herein by reference in its entirety.

[0325] If an excess of the test antibody (e.g., at least 2x, 5x, 10x, 20x, or 100x) inhibits or blocks the binding of the reference antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as measured in a competitive binding assay, then the test antibody competes with the reference antibody. Binding proteins (competitive antibodies) identified by the competitive assay include binding proteins that bind to the same epitope as the reference antibody and binding proteins that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antibody to cause steric hindrance. For example, a second competitive antibody can be identified that competes with the first antibody described herein for binding to VEGF. In some cases, the second antibody can block or inhibit the binding of the first antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as measured in a competitive binding assay. In some cases, the second antibody can replace the first antibody by more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0326] In some embodiments, the antibody binds to human VEGF and PD-1.

[0327] In some embodiments, the antibodies provided herein are in quantities less than or equal to about 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.98, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.50, 1.45, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001 × 10⁻⁶. -8 M or smaller K D The antibody binds to VEGF or PD-1, as measured by ELISA or any other suitable method known in the art. In some embodiments, the antibodies provided herein are in doses of 5-3, 4-2, 3-1, 1.9-1.8, 1.8-1.7, 1.7-1.6, 1.6-1.5, 1.9-1.5, 1.5-1, 1-0.8, 1-0.5, 0.9-0.6, 0.7-0.4, 0.6-0.2, 0.5-0.3, 0.3-0.2, 0.2-0.1, 0.1-0.01, 0.01-0.001, or 0.001-0.0001 × 10⁻¹⁰. -8 K between M DCombined with VEGF or PD-1, as measured by ELISA or any other suitable method known in the art.

[0328] Pharmaceutical Composition

[0329] This application provides compositions comprising the disclosed binding protein (e.g., PD-1 binding protein or VEGF / PD-1 bispecific binding protein), including pharmaceutical compositions having one or more pharmaceutically acceptable excipients. In some embodiments, the composition is sterile. The pharmaceutical composition typically contains an effective amount of the binding protein.

[0330] In addition to one or more binding proteins disclosed herein, these compositions may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material may depend on the route of administration, such as oral, intravenous, skin or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0331] Pharmaceutical compositions intended for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain a solid carrier, such as gelatin or an adjuvant. Liquid pharmaceutical compositions typically contain a liquid carrier, such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may contain physiological saline solutions, dextran or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0332] For intravenous, subcutaneous, or subcutaneous injection, or injection at the site of pain, the active ingredient will be in a parenteral acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are fully capable of preparing suitable solutions using, for example, isotonic agents (such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection). Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0333] The binding protein to be administered to the individual is preferably given at a “therapeutic effective amount” or a “prophylactic effective amount” (as the case may be, although prophylaxis can be considered treatment), which is sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and timing of administration, will depend on the nature and severity of the protein aggregation disorder being treated. Treatment prescription (e.g., decisions regarding dosage, etc.) is the responsibility of general practitioners and other physicians and typically takes into account the condition to be treated, the individual patient’s symptoms, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16. th Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. (ed.), 1980.

[0334] The composition can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated.

[0335] method

[0336] Preparation method

[0337] Methods for preparing binding proteins are known in the art. See Milstein and Cuello (1983) Nature 305:537, International (PCT) Publication WO 93 / 08829, and Traunecker et al. (1991) EMBOJ, 10:3655. For further details on the production of binding proteins, see, for example, Suresh et al. (1986) Methods Enzymol, 121:210. Binding proteins include cross-linked or “heteroconjugate” or “heterodimeric” binding proteins. For example, one binding protein in a heterodimer may be coupled to avidin, and another to biotin. Heterodimeric binding proteins can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed, along with many cross-linking techniques, in U.S. Patent No. 4,676,980.

[0338] For example, the binding protein described herein can be generated using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid is provided encoding a chain of the binding protein described herein. Such a nucleic acid may encode an amino acid sequence comprising a VL sequence of an antibody and / or an amino acid sequence comprising a VH sequence of an antibody (e.g., the light chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In one embodiment, the nucleic acid is provided in a polycistronic vector. In another embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL sequence of an antibody and an amino acid sequence comprising a VH sequence of an antigen-binding polypeptide construct; or (2) a first vector and a second vector, the first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL sequence of an antigen-binding polypeptide construct, and the second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH sequence of an antigen-binding polypeptide construct. In one embodiment, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphoid cells (e.g., Y0, NSO, Sp20 cells). In one embodiment, a method for preparing the binding protein is provided, wherein the method includes culturing a host cell containing at least one strand of nucleic acid, each encoding the binding protein, as provided above, under conditions suitable for expressing the binding protein, and optionally recovering the binding protein from the host cell (or host cell culture medium).

[0339] To recombinantly generate binding proteins, a nucleic acid encoding at least one strand of an antibody, such as that described above, is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes capable of specifically binding to genes encoding both the heavy and light chains of the antibody.

[0340] When the binding protein or its variants are recombined from host cells, in some embodiments the protein is present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the cell dry weight. When the antibody or its variants are recombined from host cells, in some embodiments the protein is present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of the cell dry weight. In some embodiments, the “substantially purified” antibodies produced by the methods described herein have a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, particularly, at least about 75%, 80%, 85%, and more particularly, at least about 90%, at least about 95%, at least about 99%, or higher, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0341] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein.

[0342] Recombinant host cells, or host cells in general, are cells containing exogenous polynucleotides, regardless of the method used for insertion, such as direct uptake, transduction, f-crossing, or other methods known in the art for generating recombinant host cells. The exogenous polynucleotides may remain as non-integrating vectors, such as plasmids, or alternatively, may be integrated into the host genome. Host cells may include CHO, CHO derivatives, NSO, Sp2O, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0343] For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*.) After expression, soluble fractions of the antibody can be isolated from the bacterial cell paste and further purified.

[0344] Besides prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeasts) are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," thereby producing antibodies with partial or complete human glycosylation patterns. See Gerngross. Nat. Biotech [Nature Biotechnology] 22:1409-1414 (2004) and Li et al., Nat. Biotech [Nature Biotechnology] 24:210-215 (2006).

[0345] Suitable host cells for expressing glycosylation-binding proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfecting fall armyworm (Spodoptera frugiperda) cells.

[0346] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES™ technology for producing antibodies in transgenic plants).

[0347] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension growth can be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed from SV40 (COS-7); human embryonic kidney lines (293 or 293 cells, as shown, for example, in Graham et al.) J. Gen Virol As described in [Journal of General Virology] 36:59 (1977); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells, e.g., in Mather, Biol. Reprod [Reproductive Biology] 23:243-251 (1980) describes); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells, such as those described, for example, in Mather et al., Annals NY Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 77:4216 (1980); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0348] In one embodiment, the binding protein described herein is produced in stable mammalian cells by a method comprising the steps of: transfecting at least one stable mammalian cell with: a nucleic acid encoding an antibody at a predetermined ratio; and expressing the nucleic acid in the at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acid is determined in a transient transfection experiment to determine the relative ratio of the input nucleic acid that results in the highest percentage of antibody in the expressed product.

[0349] If necessary, the binding protein can be purified or isolated after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques performed at atmospheric or high pressure using systems such as FPLC and HPLC, which include ion exchange, hydrophobic interactions, affinity, sizing or gel filtration, and reversed-phase chromatography. Purification methods also include electrophoresis, immunology, precipitation, dialysis, and chromatographic focusing. Ultrafiltration and percolation techniques combined with protein concentration are also useful. As is well known in the art, many natural proteins bind to Fc and binding proteins, and these proteins can be used in this invention to purify binding proteins. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some binding proteins. Purification can often be achieved using specific fusion couplers. For example, if a GST fusion is used, the binding protein can be purified using glutathione resin; if a His tag is used, Ni... +2 Affinity chromatography can be used to purify the bound protein, or, if a flag tag is used, immobilized anti-flag antibodies can be used. For general guidance on suitable purification techniques, see, for example, Protein Purification: Principles and Practice, 3, which is incorporated herein by reference in its entirety. rd Ed. [Protein Purification: Principles and Practice, 3rd Edition], Scopes, Springer-Verlag, NY, 1994, which is incorporated herein by reference in its entirety. The necessary degree of purification will vary depending on the intended use of the bound protein. In some cases, purification is not required.

[0350] In some embodiments, anion exchange chromatography (including but not limited to chromatography on columns of Q-sepharose, DEAEsepharose, poros HQ, poros DEAF, Toyopearl Q, oyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, and Fractogel Q and DEAE) is used to purify the bound protein.

[0351] In certain embodiments, the proteins described herein are purified using cation exchange chromatography (including, but not limited to, SP-sepharose, CMsepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns, and their equivalents and comparables).

[0352] Furthermore, the binding proteins described herein can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., NY, and Hunkapiller et al.). Nature [Nature], 310:105-111 (1984). For example, peptides corresponding to polypeptide fragments can be synthesized using a peptide synthesizer. Furthermore, if desired, non-classical amino acids or chemical amino acid analogs can be introduced into the polypeptide sequence as substitutions or additions. Non-classical amino acids include, but are not limited to, D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, leucine, valine, hydroxyproline, sarcosine, citrulline, homocitrulline, sulfonylalanine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoroamino acids, designer amino acids such as (methyl amino acids, C-methyl amino acids, N-methyl amino acids), and general amino acid analogs. Furthermore, the amino acid can be D (dextrorotatory) or L (levorotatory).

[0353] In some embodiments, the antibodies described herein have aggregation temperatures above about 69°C, above about 70°C, above about 71°C, above about 72°C, above about 73°C, above about 74°C, above about 75°C, or above about 76°C, for example, between about 69°C and about 77°C, between about 70°C and about 76°C, or between about 71°C and about 75°C. In some embodiments, the aggregation temperature is measured using DSF.

[0354] In some embodiments, as measured by hydrophobic interaction chromatography (HIC), the antibody described herein exhibits reduced hydrophobicity compared to lerechizumab. In some embodiments, the antibody demonstrates a HIC retention time of less than about 15.2 minutes. In some embodiments, the antibody demonstrates a HIC retention time between about 13 minutes and about 15 minutes.

[0355] Usage / Treatment Method

[0356] In one aspect, this application provides a method for contacting VEGF and / or PD-1 with the binding proteins described herein.

[0357] In one aspect, this application provides a method for treating a subject with a condition or disease using the binding protein or pharmaceutical composition thereof described herein. In other aspects, this application describes a method for treating a subject in need, comprising administering a therapeutically effective amount of the binding protein or pharmaceutical composition thereof described herein to a mammalian subject. In some embodiments, this application provides a method for treating a subject with a condition or disease associated with elevated VEGF and / or PD-1 levels.

[0358] In some respects, this article describes a method for treating pathologies associated with VEGF and / or PD-1 activity, comprising administering to a subject a therapeutically effective amount of the binding protein or a pharmaceutical composition thereof described herein.

[0359] Subjects

[0360] In some embodiments, the subject is a mammal, such as a primate. In some embodiments, the subject is a human.

[0361] Subjects may have a disease or condition associated with abnormal VEGF and / or PD-1 expression and / or signaling, exhibit at least one symptom of such a disease or condition, be diagnosed with such a disease or condition, and / or be identified as being at risk of such a disease or condition. Non-limiting examples of such diseases or conditions include cancers (including solid tumors and liquid-filled tumors), such as brain cancer (e.g., Glioblastoma ), chest and thoracic cancers (e.g., esophageal cancer, pleural mesothelioma, lung cancer (e.g., non-small cell lung cancer (NSCLC), including EGRm NSCLC, non-squamous NSCLC and squamous NSCLC; and small cell lung cancer (SCLC)), gastrointestinal cancers (e.g., biliary tract cancer, colorectal cancer (all types, including MSI-H / dMMR and non-MSI-H / dMMR), gastric cancer, gastroesophageal junction (GEJ) cancer), hepatocellular carcinoma or primary peritoneal cancer), head and neck cancers (e.g., head and neck squamous cell carcinoma, nasopharyngeal carcinoma or thyroid cancer), hematologic malignancies (e.g., classical Hodgkin lymphoma or primary mediastinal large B-cell lymphoma (PMBCL)), kidney cancers (e.g., Renal cell carcinoma Hepatocellular carcinoma and biliary tract cancer (e.g., biliary tract cancer or hepatocellular carcinoma), reproductive cancer (e.g., breast cancer (including triple-negative breast cancer), cervical cancer, endometrial cancer, fallopian tube cancer, ovarian cancer (including epithelial ovarian cancer) and urothelial carcinoma), and soft tissue cancer (e.g., alveolar soft tissue carcinoma or soft tissue sarcoma).

[0362] Application method

[0363] In some embodiments, the methods provided herein can be used to treat an individual's disease or condition. In these embodiments, the individual is a person, and the binding protein described herein is applied to the person.

[0364] In some embodiments, the binding protein is administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracardiaclysm, or intranasally. An effective amount of the binding protein can be administered to treat a disease or condition. The appropriate dose of the binding protein can be determined based on the type of disease or condition to be treated, the type of binding protein, the severity and duration of the disease or condition, the individual's clinical condition, the individual's clinical history and response to treatment, and the judgment of the attending physician.

[0365] In some embodiments, the antibodies provided herein are administered in combination with at least one additional therapeutic agent. Any suitable additional therapeutic agent or immunotherapeutic agent may be administered in combination with the antibodies provided herein. Additional therapeutic agents include agents for treating or preventing diseases or conditions such as, but not limited to, cancers, such as cancers associated with elevated VEGF and / or PD-1 levels, PD-1-expressing cancers, non-small cell lung cancer, etc.

[0366] Additional therapeutic agents may be administered in any suitable manner. In some embodiments, the antibodies and additional therapeutic agents provided herein are contained in the same pharmaceutical composition. In some embodiments, the antibodies and additional therapeutic agents provided herein are contained in different pharmaceutical compositions.

[0367] In the embodiments where the binding protein and additional therapeutic agent provided herein are included in different pharmaceutical compositions, the administration of the binding protein may be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent. In some embodiments, the administration of the antibody and additional therapeutic agent provided herein occurs within approximately one month of each other. In some embodiments, the administration of the binding protein and additional therapeutic agent provided herein occurs within approximately one week of each other. In some embodiments, the administration of the binding protein and additional therapeutic agent provided herein occurs within approximately one day of each other. In some embodiments, the administration of the binding protein and additional therapeutic agent provided herein occurs within approximately twelve hours of each other. In some embodiments, the administration of the binding protein and additional therapeutic agent provided herein occurs within approximately one hour of each other.

[0368] reagent kits and products

[0369] This application provides kits comprising any one or more binding protein compositions described herein and instructions for use. In some embodiments, these kits further comprise components selected from any of the following: secondary binding proteins, reagents for immunohistochemical analysis, pharmaceutically acceptable excipients, and instructions for use, and any combination thereof. In one particular embodiment, the kit comprises a pharmaceutical composition comprising any one or more binding protein compositions described herein, and one or more pharmaceutically acceptable excipients.

[0370] This application also provides articles comprising any of the binding protein compositions or kits described herein. Examples of articles include vials (including sealed vials).

[0371] Example

[0372] This document provides examples of specific embodiments for carrying out the invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental error and deviation should certainly be allowed.

[0373] Unless otherwise stated, the practice of this invention will be carried out using conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, which are well explained in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods in Enzymology (edited by S. Colowick and N. Kaplan, Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18 th Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. [Advanced Organic Chemistry, 3rd Edition] (Plenum Press) Volumes A and B (1992).

[0374] method

[0375] Gene synthesis and plasmid construction

[0376] The coding sequences for bispecific antibodies HC and LC were generated through DNA synthesis and PCR, and these coding sequences were subsequently subcloned into plasmids for protein expression in mammalian cell systems. The gene sequences in the expression vectors were confirmed by DNA sequencing.

[0377] Expression of antibody constructs

[0378] The bispecific constructs are engineered using methods known to those skilled in the art (e.g., transient expression of bispecific antibodies by co-transfecting paired HC and LC constructs into CHO cells using the PEI method).

[0379] SEC-HPLC analysis of antibody constructs

[0380] Standard analytical SEC-HPLC was performed using a Shimadzu LC-10 HPLC instrument (Shimadzu Corp.).

[0381] Antibody binding kinetics were measured using surface plasmon resonance (SPR).

[0382] Binding kinetics and affinity constants were determined using methods known to those skilled in the art, employing a Biacore 8K SPR system (GE HealthCare) equipped with an S-series sensor chip for protein G. Samples were injected in a multi-cycle manner onto freshly captured bispecific antibodies by injecting glycine to regenerate the capture surface. Data were processed and analyzed using Biacore Insight evaluation software version 2.0.15.12933 (GE HealthCare). Responses from two buffer blank injections were then subtracted from the reference subtraction data to generate dual reference data, which was fitted to a 1:1 binding model to determine the apparent binding (k... a ) and dissociation rate constant (k d Their ratio provides the apparent equilibrium dissociation constant, or affinity constant (K). D = k d / k a ).

[0383] As a primary screening system, for example, SPR can be used to measure the K-response of antibodies against PD-1-hFc and VEGFA. D .

[0384] Assessing blockade using cell line-based assays

[0385] The blocking of the intact VEGF and / or PD-1 signaling complex was assessed using cell line-based assays and / or ELISA methods. The methods used to perform those assays are known to those skilled in the art.

[0386] PD-1 cell-based binding assay

[0387] PD-1 binding was assessed in Jurkat-hPD-1 cells by flow cytometry in the presence or absence of 2xVEGF. Briefly, Jurkat-hPD-1 cells were resuspended in FACS buffer (1X PBS containing 2% FBS). Bispecific antibody and 2xVEGF were pre-incubated at room temperature for 30 min and then added to the cells. Cells were incubated at 4°C for 1 h and washed with FACS buffer. Cells were resuspended with 100 μL of secondary antibody (1:500 dilution in FACS buffer) and incubated at 4°C for 1 h. After incubation, cells were washed with FACS buffer and analyzed by FACS. Data were plotted and analyzed in GraphPadPrism 10.

[0388] PD-1 reporter gene assay

[0389] PD-1 signaling blockade was assessed by reporter gene assays of co-cultured Jurkat-NFAT-hPD-1 and Hep3B-OS8-hPD-L1 cells. Briefly, Hep3B-OS8-PD-L1 and Jurkat (6C8)-NFAT-PD-1 cells were co-cultured in the presence of a bispecific antibody (+ / - VEGF) and incubated for 6 hours in a humidified 37°C, 5% CO2 incubator. After incubation, the ONE-Glo™ luciferase assay system was added to each well to allow for complete cell lysis, and luminescence was measured using a Thermo Fisher Varioskan LUX multimode microplate reader. Data were plotted and analyzed in a GraphPad Prism 10.

[0390] PD-1 internalization assay

[0391] PD-1 internalization was assessed by measuring PD-1 levels on Jurkat cells expressing PD-1 using flow cytometry. Briefly, Jurkat-hPD-1 cells were seeded at 2E5 cells per well in 96-well plates. Cells were pre-incubated with a bispecific antibody and a 2-fold concentration of VEGF at room temperature for 30 min. Cells were then treated with antibody (+ / -VEGF) in warm medium at 37°C for 0, 0.5, 2, 4, and 6 h. After incubation at 37°C, cells were held on ice and fixed with 2% PFA for 15 min. Cells were then labeled with anti-human IgG Fc-Alexa 647 (1:500) at 4°C for 1 h and analyzed by flow cytometry. Data were plotted and analyzed in GraphPad Prism 10.

[0392] VEGF reporter gene assay

[0393] VEGFA-induced signal blockade was assessed using human VEGF R2 (Luc) HEK293 reporter cells via reporter gene assay. In short, human VEGF R2 (Luc) HEK293 reporter cells were resuspended in assay medium and plated (3E4 / well) in 96 wells the day before assay. Bispecific antibody and 2-fold concentration of VEGF were pre-incubated at room temperature for 30 min and then added to the cells. Cells were incubated for 4 h in a humidified 37°C, 5% CO2 incubator. After incubation, the ONE-Glo™ luciferase assay system was added to each well to allow for complete cell lysis, and luminescence was measured using a Thermo Fisher Varioskan LUX multimode microplate reader. Data were plotted and analyzed in a GraphPad Prism 10.

[0394] Human umbilical vein endothelial cell (HUVEC) proliferation assay

[0395] The inhibition of VEGF-mediated cell proliferation was assessed in HUVEC cultures. Briefly, HUVECs were digested with 3 ml TrypLE at 37°C for 3 min and washed. Cells were then resuspended in assay medium and seeded in assay plates, and incubated overnight at 37°C. Bispecific antibody and 2-fold concentration of VEGF were pre-incubated at room temperature for 30 min.

[0396] Antibody (+ / -VEGFA) was added to the cells and incubated at 37°C for 3 days. After incubation, CellTiter-Glo luminescence buffer was added to the plate and incubated at room temperature for 10 min. Luminescence was then measured using a Thermo Varioskan LUX. Data were plotted and analyzed in GraphPad Prism 10.

[0397] T cell activation in co-culture of human PBMCs and hepatocellular carcinoma cell lines

[0398] In a co-culture assay of human PBMCs and Hep3B-OS8-hPD-L1 cells, T cell activation was assessed by measuring IL-2 secretion in the presence or absence of 2xVEGFA. Briefly, Hep3B-OS8-PD-L1 cells were resuspended in mitomycin C 10 μg / ml (in 5–10 ml 10% FBS + RPMI 1640 medium) and incubated at 37°C for 1.5 h. Bispecific antibody and 2x concentration of VEGFA were pre-incubated at room temperature for 30 min. During incubation, PBMCs from healthy volunteers were resuspended in 10% FBS + RPMI 1640 medium. PBMC cells (5E4 / well), Hep3B-OS8-PD-L1 cells (5E3 / well), and antibody (+ / -VEGF) were added to 96-well plates and incubated in a humidified 37°C, 5% CO2 incubator for 72 h. After 72 h, cell culture supernatant was collected, and the concentrations of IL-2 and IFN were measured using a human IL-2 ELISA kit. Data were plotted and analyzed in GraphPad Prism 10.

[0399] T cell activation in human monocyte-derived dendritic cells and CD4+ T cells obtained by mixed lymphocyte reaction (MLR) assay

[0400] T cell activation was assessed in a mixed lymphocyte reaction (MLR) of human monocyte-derived dendritic cells (moDCs) and CD4+ T cells by measuring IL-2 and IFNγ secretion. Briefly, CD14+ monocytes were isolated from human PBMCs using a CD14 microbeads isolation kit (MILTENYI-130-050-201) according to the manufacturer's protocol. The isolated CD14+ monocytes were then added to 6-well plates, followed by the addition of 2 mL / well of hGM-CSF and hIL-4 with assay medium to induce dendritic cell differentiation. The plates were then incubated at 37°C in 5% CO2 for 6 days. After incubation, the cells were treated with rhGM-CSF (50 ng / mL), IL-4 (25 ng / mL), and LPS (1 μg / mL) to mature the dendritic cells (DCs). The DCs were then harvested and diluted to 0.2 × 10⁻⁶. 6 cells / mL. CD4+ T cells were isolated from human PBMCs using the manufacturer's protocol with a human CD4+ T cell isolation kit. The isolated CD4+ T cells were cultured in assay medium at 2 × 10⁻⁶ cells / mL. 6 Resuspend cells / mL, then feed at 50 μL / well (0.1 × 10⁻⁶ cells / mL). 6The bispecific antibody and 2-fold concentration of VEGF were dispensed into 96-well plates. The mixture was pre-incubated at room temperature for 30 min. The antibody mixture (+ / -VEGF) was transferred to T cell cultures, thoroughly mixed, and incubated at 37°C for 30 min. After incubation, monocyte-derived dendritic cells were dispensed at 50 μL / well (0.2 × 10⁻⁶ cells / well). 6 Cells / mL, 0.01 × 10 6 A volume of cells / well was added to the plate and incubated at 37°C with 5% CO2. After a 48-hour incubation period, 50 μL of supernatant was transferred to a new 96-well plate for IL-2 quantification using microsphere immunoassay (CBA), and after a 96-hour incubation period, 50 μL of supernatant was transferred to a new 96-well plate for IFNγ quantification using CBA. Data were plotted and analyzed in GraphPad Prism 10.

[0401] Reference point

[0402] In some of the examples provided herein, references were used to evaluate the biological activity of the bispecific antibodies disclosed herein. In some cases, evosimib (also referred to herein as PAL054-0001.1L or AK112) antibodies were used. AK112 is a PD-1 / VEGF bispecific antibody comprising a heavy chain (SEQ ID NO: 128) and a light chain (SEQ ID NO: 275).

[0403] AK112 contains the VH and VL domains derived from bevacizumab and penaplimab (AK105). Figure 8 ).

[0404] Other reference sequences (controls and references are provided in Table 11).

[0405] Table 11.

[0406] Example 1: Determining PD-1 binding on PD-1 expressing cells

[0407] As described above, PD-1 binding was assessed in Jurkat-hPD-1 cells by flow cytometry in the presence and absence of 2xVEGFA. Greater binding with PD-1-expressing cells was observed in the presence of VEGFA (interchangeably referred to as VEGF or VEGFA). Results showed... Figure 1A-1CC And in Table 12.

[0408] Table 12.

[0409] Example 2: Determining the affinity of an antibody for PD-1

[0410] The binding affinity (Kb) of pembrolizumab and clonal variants with specified Fc modifications to PD-1 was determined by surface plasmon resonance. D () Figure 2A-2C And Table 13), and analyzed the antibody's blocking effect on PD-1 / PD-L1 by PD-1 reporter gene assay (and Table 13). Figure 2D ).

[0411] Table 13.

[0412] Example 3: Binding of bispecific antibodies to human VEGF, human PD-1, and cynomolgus monkey PD-1 as confirmed by ELISA.

[0413] PD-1 binding assays were performed to measure the affinity of AK112 and the VEGF / PD-1 bispecific antibody disclosed herein for PD-1-expressing cells. The half-maximal effective concentration (MCP) of the test sample for binding to human VEGF, human PD-1, and cynomolgus monkey PD-1 was determined by ELISA. 50 Data can be found in Figures 3A-3C And in Table 14.

[0414] Table 14.

[0415] Example 4: Determining the affinity of bispecific antibodies for human VEGF

[0416] The binding affinity (Kb) of the bispecific antibody to human VEGF was determined by capturing the test sample using a protein G chip via surface plasmon resonance and employing mobile phases of 100, 33.33, 11.11, 3.7, and 1.23 nM. D () Figures 4A-4D (and Table 15).

[0417] Table 15.

[0418] Example 5: Determining the affinity of bispecific antibodies for human PD-1

[0419] The binding affinity (Kb) of the bispecific antibody to human PD-1 was determined by capturing the test sample using a protein G chip via surface plasmon resonance and employing mobile phases of 200, 66.67, 22.22, 7.41, and 2.47 nM. D () Figures 5A-5D (and Table 16).

[0420] Table 16.

[0421] Example 6: Determining the affinity of bispecific antibodies for cynomolgus monkey PD-1

[0422] The binding affinity (Kb) of the bispecific antibody to cynomolgus monkey PD-1 was determined by capturing the test sample using a protein G chip via surface plasmon resonance and using 200, 66.67, 22.22, 7.41, and 2.47 nM or 200, 66.67, 22.22, 7.41, 2.47, and 0.82 nM as the mobile phase. D () Figures 6A-6D (and Table 17).

[0423] Table 17.

[0424] Example 7: Size exclusion chromatography of bispecific variants, controls, and references

[0425] Size exclusion chromatography with multi-angle static light scattering (SEC-MALS) was used to characterize and compare the reference bispecific antibody AK112 with controls and the VEGF / PD-1 bispecific antibody as disclosed herein. The exemplary clones disclosed herein exhibited SEC-MAL complex profiles similar to AK112 in the presence of VEGFA. Figures 7A-7F (Table 18). Unbound by the mechanism of action, the results clearly show that bispecific antibodies can enhance PD-1 binding through VEGFA daisy chain linkage (…). Figure 9 ).

[0426] Table 18.

[0427] Example 8: Functional activity of bispecific antibodies in reporter gene assays

[0428] PD-1 reporter gene assay

[0429] The PD-1 reporter gene assay was performed on the VEGF / PD-1 bispecific antibody disclosed in this paper, and the assay demonstrated that the VEGF / PD-1 bispecific antibody enhanced the functional efficacy of blocking the PD-1 / PD-L1 signaling pathway in the presence of VEGFA. Figure 10A-10KK (and Tables 19A-19C).

[0430] Table 19A. Operation 1

[0431] Table 19B. Operation 2

[0432] Table 19C. Running 3

[0433] VEGF reporter gene assay

[0434] The VEGF / PD-1 bispecific antibody disclosed in this article was subjected to VEGF reporter gene assay, and the resulting IC50 was... 50 Value provided Figure 11A-11V In Tables 20A and 20B.

[0435] Table 20A.

[0436] Table 20B.

[0437] Example 9: Effects of VEGF on HUVEC proliferation

[0438] HUVEC proliferation assays were performed on the VEGF / PD-1 bispecific antibody disclosed in this paper, and the resulting IC50 values ​​were analyzed. 50 Value provided Figure 12A-12O And in Table 21.

[0439] Table 21. HUVEC proliferation IC 50 value

[0440] Example 10: The effect of VEGF on PD-1 internalization

[0441] A PD-1 internalization assay was performed on the VEGF / PD-1 bispecific antibody disclosed in this paper, and this assay revealed enhanced PD-1 internalization in the presence of VEGF. The results showed... Figure 13A-13S middle.

[0442] Example 11: Binding and activity of bispecific reverse constructs

[0443] On the PD-1-VEGF (scFv) reverse construct ( Figure 14A Cell binding, internalization, PD-1 reporter gene assays, and VEGF reporter gene assays were performed as known to those skilled in the art and described herein, and the results showed... Figure 14B-14E The results did not show a significant enhancement of PD-1 bioactivity in the presence of VEGFA.

[0444] Example 12: T cell activation in co-culture of human PBMCs and hepatocellular carcinoma cell lines

[0445] T cell activation was assessed by measuring IL-2 secretion in the presence or absence of 2xVEGFA (2VEGF) in a co-culture assay with human PBMCs and Hep3B-OS8-hPD-L1. For each bispecific antibody clone, various controls were used, including untreated (-), allotype control (hIgG1), PAL054-0004.4 (pembrolizumab), and / or PAL054-0001.1L (also known as edoximib or AK112). Data are available in [link to relevant documentation]. Figure 15A-15R In the data, the bispecific antibody-VEGF complex was found to enhance T cell activation in the co-culture assay.

[0446] Example 13: T cell activation in human monocyte-derived dendritic cells and CD4+ T cells obtained by mixed lymphocyte reaction (MLR) assay

[0447] In addition, T cell activation was assessed in the mixed lymphocyte response (MLR) of human monocyte-derived dendritic cells (moDCs) and CD4+ T cells by measuring IL-2 and IFNγ secretion. Data were collected from four independent donor pairs, and the data are available in […]. Figure 16A-16F As seen in the data, the bispecific antibody-VEGF complex enhanced T cell activation in the DC-CD4+ T cell MLR assay.

[0448] Example 14: Titer, purity, and thermostability data of reference and exemplary bispecific antibodies

[0449] Determine the titer, purity, and thermal stability data for the reference and bispecific antibodies as disclosed herein. Use standard analytical techniques well-known in the art to determine the titer and purity of the antibody samples.

[0450] Two different methods were used to determine thermal stability.

[0451] Method 1: Thermal stability determined using UNCLE

[0452] To determine thermal stability, 9 μL of each antibody sample was loaded into Uni microvolume cuvettes specifically designed for use with UNCLE instruments from Unchained Labs. The samples were subjected to controlled thermal homogenization from 20°C to 95°C at a rate of 0.5°C / min. Dynamic light scattering (DLS) measurements were performed before and after heating, each consisting of four acquisitions lasting five seconds, to assess particle size and polydispersity. The melting temperature (Tm) of each sample was determined using UNCLE software by analyzing the change in the first derivative of the centroidal mean (BCM) of fluorescence intensity with temperature. The aggregation initiation temperature (Tagg) was calculated based on the intensity of scattered light measured at wavelengths of 266 nm or 473 nm. Size and polydispersity data were extracted from the DLS correlation function. This method provides a comprehensive assessment of antibody thermal stability and aggregation behavior. The data are shown in Table 22.

[0453] Table 22.

[0454] Method 2: Thermal stability determined using MicroCal™ VP DSC

[0455] To assess thermal stability and unfolding behavior, antibody samples were analyzed using a MicroCal™ VP-capillary differential scanning calorimeter (DSC) from Malvern. Prior to analysis, samples were diluted to a final concentration of 1 mg / mL using the appropriate reference buffer. A total of 400 μL of reference buffer was aliquoted into the odd-numbered wells of a 96-well plate, while 400 μL of sample solution was added to the even-numbered wells.

[0456] The DSC instrument was programmed to perform thermal scans from 10°C to 110°C at a constant scan rate of 200°C per hour. Thermal transitions were continuously monitored throughout the scan. Data acquisition and analysis were performed using MicroCal™ VP-Capillary DSC automated data analysis software. The midpoint of the thermal transition (Tm) was determined by identifying the peak of the heat capacity curve, while the onset of defolding (T_onset) was calculated based on the deviation from the baseline preceding the transition peak.

[0457] This method enables precise quantification of protein thermostability and unfolding characteristics under controlled conditions. Data corresponding to these measurements are presented in Table 23.

[0458] Table 23.

[0459] The viscosity, aggregation, and stability of PAL054-0170.1L were further determined. Data can be found in... Figures 17A-17DIn this study, it was demonstrated that PAL054-0170.1L had improved viscosity compared to evosimib. Figure 17A ) and aggregation ( Figure 17B ) and at 40°C ( Figure 17C ) and 25°C ( Figure 17D Stability under ( ).

[0460] Example 15: Pharmacokinetics in Non-human Primates (NHP)

[0461] In vivo pharmacokinetic (PK) studies were conducted on the bispecific antibody described in this paper compared to evokinemab (AK112). Cynomolgus monkeys were used. Macaca fascicularis The study was conducted using intravenous (IV) administration of antibodies. All animals were male and weighed between 2.5 and 3.9 kg (n = 5 / group). On day 0, animals were administered PAL054-0001.1L (AK112), PAL054-0104.1L (104), PAL054-0157.1L (157), PAL054-0167.1L (167), PAL054-0168.1L (168), PAL054-0169.1L (169), PAL054-0170.1L (170), or PAL054-0171.1L (171) via intravenous (IV) bolus, as shown in Table 24, and serum samples were collected periodically throughout the study. PK parameters were determined from cynomolgus monkey serum samples up to day 28.

[0462] Table 24.

[0463] By resuspending cynomolgus monkey PBMCs in FACS buffer and using 2 × 10⁻⁶ ppm... 5 Cells / well were seeded into 96-well V plates to determine receptor occupancy (RO). Cells were then incubated at 4°C for 1 h with a saturated concentration (200 nM) of hIgG (isotype control) or the test sample. After incubation, cells were washed and stained with fixative viability stain 510, APC-Cy™ 7 mouse anti-human CD3, Brilliant Violet 605™ anti-human CD4 antibody, Alexa Fluor® 700 anti-human CD8a antibody, Brilliant Violet 421™ anti-human CD95 (Fas), and goat F(ab')2 anti-human IgG-Fc (PE), and incubated at 4°C for 45 min. Cells were then washed and resuspended in FACS buffer for FACS analysis. PD-1 RO was calculated according to the following equation. RO (%) = [%CD3+CD95+ (control hIgG) / %CD3+CD95+ (bispecific)] × 100 The results are summarized in Figures 18A-18C PK analysis showed that the half-life of PAL054-0170.1L was 3.57 days, while that of edoximab was 1.50 days. Furthermore, edoximab and PAL054-0170.1L showed similar total PK and intact PK, as well as receptor occupancy (RO), in NHP. PK analysis demonstrated that PAL054-0170.1L had an improved half-life compared to edoximab (Table 24). Figures 18A-18B ).

[0464] Example 16: Efficacy study in a mouse tumor model co-inoculated with HCC827 and hPBMCs subcutaneously

[0465] Seven- to nine-week-old female SCID / Beige mice (Vital River Laboratory Animal Technology) were divided into five groups of eight mice each. On day 0, HCC827 tumor cells (4 × 10⁻⁶) were subcutaneously injected into the right posterior flank. 6 ), PBMCs from healthy donors (8 × 10) 5 The mixture of the drug and the specified dose of antibody was administered. The specified antibody was then injected intravenously on days 7, 14, 21, 28, and 35 (Table 25). Tumor volume was measured twice weekly until day 68.

[0466] The bispecific antitumor activity of VEGF / PD-1 was evaluated in PBMC humanized SCID / Beige mice with subcutaneous HCC827 xenograft tumors and compared with that of bevacizumab. Treatment with PAL054-0170.1L resulted in strong tumor growth inhibition. At an equivalent molar concentration of 10 mg / kg, the antitumor activity was greater than that of bevacizumab. Figure 19 ).

[0467] Table 25. Mouse cohorts used in in vivo studies

[0468] Numbered Examples

[0469] Example 1. A bispecific antibody that binds vascular endothelial growth factor (VEGF) and programmed death receptor 1 (PD-1), the bispecific antibody comprising: a) A heavy chain sequence comprising an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 22-23, 60, 62, 65-67, 70-72, 75-77, 142-143, 667-678, and 702-713; and b) A light chain sequence comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NO: 148-194, 679-701 and 714-737.

[0470] Example 2. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:22; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:169.

[0471] Example 3. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:23; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:170.

[0472] Example 4. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 65; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 212.

[0473] Example 5. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 66; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 213.

[0474] Example 6. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 67; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 214.

[0475] Example 7. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 60; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 207.

[0476] Example 8. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 62; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 209.

[0477] Example 9. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 217.

[0478] Example 10. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:71; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:218.

[0479] Example 11. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:72; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:219.

[0480] Example 12. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:75; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:222.

[0481] Example 13. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:76; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:223.

[0482] Example 14. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:77; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:224.

[0483] Example 15. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 142; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 289.

[0484] Example 16. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 143; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 290.

[0485] Example 17. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 656; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 679.

[0486] Example 18. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 657; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 680.

[0487] Example 19. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 658; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 681.

[0488] Example 20. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 659; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 682.

[0489] Example 21. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 660; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 683.

[0490] Example 22. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 661; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 684.

[0491] Example 23. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 662; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 685.

[0492] Example 24. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 663; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 686.

[0493] Example 25. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 664; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 687.

[0494] Example 26. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 665; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 688.

[0495] Example 27. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 666; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 689.

[0496] Example 28. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 667; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 690.

[0497] Example 29. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 668; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 691.

[0498] Example 30. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 669; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 692.

[0499] Example 31. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 670; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 693.

[0500] Example 32. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 671; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 694.

[0501] Example 33. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 672; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 695.

[0502] Example 34. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 673; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 696.

[0503] Example 35. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 674; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 697.

[0504] Example 36. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 675; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 698.

[0505] Example 37. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 676; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 699.

[0506] Example 38. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 677; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 700.

[0507] Example 39. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:702; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:714; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:726.

[0508] Example 40. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:703; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:715; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:727.

[0509] Example 41. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:704; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:716; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:728.

[0510] Example 42. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:705; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:717; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:729.

[0511] Example 43. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:706; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:718; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:730.

[0512] Example 44. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:707; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:719; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:731.

[0513] Example 45. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:708; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:720; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:732.

[0514] Example 46. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:709; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:721; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:733.

[0515] Example 47. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:710; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:722; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:734.

[0516] Example 48. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:711; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:723; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:735.

[0517] Example 49. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:712; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:724; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:736.

[0518] Example 50. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:713; and the first light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:725; and the second light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:737.

[0519] Example 51. The bispecific antibody as described in Example 1, wherein the heavy chain sequence comprises a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 678; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 701.

[0520] Example 52. A bispecific antibody that binds vascular endothelial growth factor (VEGF) and programmed death receptor 1 (PD-1), the bispecific antibody comprising: a) A heavy chain sequence comprising an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO: 1-21, 24-57, 58, 59, 61, 63, 64, 68, 69, 73, 74, 78-97, 130-141, 144-147, and 656-666; and b) A light chain sequence comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NO: 148-194, 679-701 and 714-737.

[0521] Example 53. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:148.

[0522] Example 54. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:149.

[0523] Example 55. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:3; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:150.

[0524] Example 56. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:4; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:151.

[0525] Example 57. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 152.

[0526] Example 58. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 153.

[0527] Example 59. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:7; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:154.

[0528] Example 60. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 8; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 155.

[0529] Example 61. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 9; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 156.

[0530] Example 62. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 10; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 157.

[0531] Example 63. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 11; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 158.

[0532] Example 64. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 159.

[0533] Example 65. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 13; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 160.

[0534] Example 66. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 14; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 161.

[0535] Example 67. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 15; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 162.

[0536] Example 68. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 16; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 163.

[0537] Example 69. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 17; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 164.

[0538] Example 70. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:18; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:165.

[0539] Example 71. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:19; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:166.

[0540] Example 72. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:20; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:167.

[0541] Example 73. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:21; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:168.

[0542] Example 74. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:24; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:171.

[0543] Example 75. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:25; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:172.

[0544] Example 76. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:26; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:173.

[0545] Example 77. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:27; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:174.

[0546] Example 78. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:28; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:175.

[0547] Example 79. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:29; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:176.

[0548] Example 80. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:30; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:177.

[0549] Example 81. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:31; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:178.

[0550] Example 82. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:32; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:179.

[0551] Example 83. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:33; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:180.

[0552] Example 84. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:34; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:181.

[0553] Example 85. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:35; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:182.

[0554] Example 86. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:36; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:183.

[0555] Example 87. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:37; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:184.

[0556] Example 88. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:38; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:185.

[0557] Example 89. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:39; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:186.

[0558] Example 90. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:40; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:187.

[0559] Example 91. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:41; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:188.

[0560] Example 92. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:42; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:189.

[0561] Example 93. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:43; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:190.

[0562] Example 94. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:44; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:191.

[0563] Example 95. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:45; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:192.

[0564] Example 96. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:46; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:193.

[0565] Example 97. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:47; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:194.

[0566] Example 98. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:48; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:195.

[0567] Example 99. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:49; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:196.

[0568] Example 100. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 50; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 197.

[0569] Example 101. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 51; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 198.

[0570] Example 102. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 52; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 199.

[0571] Example 103. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 53; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 200.

[0572] Example 104. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 54; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 201.

[0573] Example 105. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 55; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 202.

[0574] Example 106. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 56; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 203.

[0575] Example 107. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 57; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 204.

[0576] Example 108. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 58; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 205.

[0577] Example 109. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 59; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 206.

[0578] Example 110. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 61; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 208.

[0579] Example 111. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 63; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 210.

[0580] Example 112. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 64; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 211.

[0581] Example 113. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 68; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 215.

[0582] Example 114. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 69; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 216.

[0583] Example 115. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 73; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 220.

[0584] Example 116. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 74; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 221.

[0585] Example 117. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 78; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 225.

[0586] Example 118. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 79; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 226.

[0587] Example 119. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 80; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 227.

[0588] Example 120. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 81; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 228.

[0589] Example 121. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 82; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 229.

[0590] Example 122. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 83; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 230.

[0591] Example 123. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 84; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 231.

[0592] Example 124. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 85; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 232.

[0593] Example 125. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 86; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 233.

[0594] Example 126. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 87; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 234.

[0595] Example 127. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 88; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 235.

[0596] Example 128. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 89; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 236.

[0597] Example 129. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 90; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 237.

[0598] Example 130. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 91; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 238.

[0599] Example 131. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 92; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 239.

[0600] Example 132. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 93; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 240.

[0601] Example 133. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 94; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 241.

[0602] Example 134. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 95; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 242.

[0603] Example 135. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 96; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 243.

[0604] Example 136. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 97; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 244.

[0605] Example 137. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 130; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 277.

[0606] Example 138. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 131; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 278.

[0607] Example 139. A bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 132; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 279.

[0608] Example 140. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 133; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 280.

[0609] Example 141. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 134; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 281.

[0610] Example 142. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 135; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 282.

[0611] Example 143. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 136; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 283.

[0612] Example 144. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 137; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 284.

[0613] Example 145. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 138; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 285.

[0614] Example 146. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 139; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 286.

[0615] Example 147. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 140; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 287.

[0616] Example 148. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 141; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 288.

[0617] Example 149. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 144; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 291.

[0618] Example 150. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 145; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 292.

[0619] Example 151. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 146; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 293.

[0620] Example 152. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 147; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 294.

[0621] Example 153. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 656; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 679.

[0622] Example 154. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 657; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 680.

[0623] Example 155. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 658; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 681.

[0624] Example 156. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 659; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 682.

[0625] Example 157. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 660; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 683.

[0626] Example 158. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 661; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 684.

[0627] Example 159. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 662; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 685.

[0628] Example 160. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 663; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 686.

[0629] Example 161. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 664; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 687.

[0630] Example 162. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 665; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 688.

[0631] Example 163. The bispecific antibody as described in Example 52, wherein the heavy chain sequence comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 666; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 689.

[0632] Example 164. A bispecific antibody that binds vascular endothelial growth factor (VEGF) and programmed death receptor 1 (PD-1), the bispecific antibody comprising: a) A heavy chain sequence comprising the amino acid sequence of any one of SEQ ID NO: 98-127 and 129; and b) A light chain sequence comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NO: 148-194, 679-701 and 714-737.

[0633] Example 165. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 98; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 245.

[0634] Example 166. The bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 99; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 246.

[0635] Example 167. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 100; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 247.

[0636] Example 168. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 101; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 248.

[0637] Example 169. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 102; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 249.

[0638] Example 170. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 103; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 250.

[0639] Example 171. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 104; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 251.

[0640] Example 172. The bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 105; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 252.

[0641] Example 173. The bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 106; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 253.

[0642] Example 174. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 107; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 254.

[0643] Example 175. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 108; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 255.

[0644] Example 176. The bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 109; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 256.

[0645] Example 177. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 110; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 257.

[0646] Example 178. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 111; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 258.

[0647] Example 179. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 112; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 259.

[0648] Example 180. The bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 113; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 260.

[0649] Example 181. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 114; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 261.

[0650] Example 182. The bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 115; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 262.

[0651] Example 183. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 116; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 263.

[0652] Example 184. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 117; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 264.

[0653] Example 185. The bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 118; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 265.

[0654] Example 186. The bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 119; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 266.

[0655] Example 187. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 120; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 267.

[0656] Example 188. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 121; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 268.

[0657] Example 189. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 122; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 269.

[0658] Example 190. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 123; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 270.

[0659] Example 191. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 124; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 271.

[0660] Example 192. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 125; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 272.

[0661] Example 193. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 126; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 273.

[0662] Example 194. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 127; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 274.

[0663] Example 195. A bispecific antibody as described in Example 164, wherein the heavy chain sequence comprises the amino acid sequence of SEQ ID NO: 129; and the light chain sequence comprises a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 276.

[0664] Example 196. A bispecific antibody as described in any one of Examples 1-195, wherein the bispecific antibody is a humanized antibody, a human antibody, or a chimeric antibody.

[0665] Example 197. A bispecific antibody as described in any one of Examples 1-196, wherein the bispecific antibody is a humanized antibody.

[0666] Example 198. A bispecific antibody as described in any one of Examples 1-197, wherein the bispecific antibody comprises a heavy chain human constant region selected from the classes of IgG, IgA, IgD, IgE and IgM.

[0667] Example 199. A bispecific antibody as described in Example 198, wherein the human Fc region comprises a human heavy chain constant region of IgG class and subclasses selected from IgG1, IgG2, IgG3 and IgG4.

[0668] Example 200. A bispecific antibody as described in Example 199, wherein the human Fc region comprises human IgG1 Fc. [0...

Claims

1. A variable domain comprising anti-PD1 immunoglobulin heavy chain (anti-PD-1 V) H The programmed death receptor 1 (PD-1) binding protein, this anti-PD-1 V H Includes the following complementary determinant regions: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 445; CDR-H2 containing the amino acid sequence of SEQ ID NO: 456, 461, or 446; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 457 or 447; (b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 451; CDR-H2 containing the amino acid sequence of SEQ ID NO: 458, 462, or 452; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 459 or 453; or (c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 443; CDR-H2 containing the amino acid sequence of SEQ ID NO: 460, 463, or 455; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 457 or 447, The premise is: (i) If CDR-H2 contains the amino acid sequence of SEQ ID NO: 446, then CDR-H3 does not contain the amino acid sequence of 447; (ii) If CDR-H2 contains the amino acid sequence of SEQ ID NO: 452, then CDR-H3 does not contain the amino acid sequence of 453; and (iii) If CDR-H2 contains the amino acid sequence of SEQ ID NO: 455, then CDR-H3 does not contain the amino acid sequence of 447.

2. The PD-1 binding protein of claim 1, wherein the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of: (a) SEQ ID NO 445, 456 and 457, respectively; (b) SEQ ID NO 451, 458 and 459, respectively; or (c) SEQ ID NO 443, 460 and 457, respectively.

3. The PD-1 binding protein of claim 2, wherein the PD-1 binding protein further comprises an anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V). L ), the anti-PD-1 V L Includes the following complementary determinant regions: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; (b) CDR-L1 containing the amino acid sequence of SEQ ID NO: 454; CDR-L2, which contains the amino acid sequence of LAS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; or (c) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO:

450.

4. The PD-1 binding protein of claim 3, wherein the anti-PD-1 V H and anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the following: (a) respectively, SEQ ID NO: 302 and 335; (b) respectively, SEQ ID NO: 302 and 336; (c) respectively, SEQ ID NO: 302 and 340; (d) Specifically, SEQ ID NO: 302 and 342; (e) respectively, SEQ ID NO: 302 and 343; (f) respectively, SEQ ID NO: 302 and 346; (g) respectively, SEQ ID NO: 306 and 337; (h) respectively, SEQ ID NO: 306 and 339; (i) respectively, SEQ ID NO: 306 and 341; (j) respectively, SEQ ID NO: 306 and 344; (k) respectively, SEQ ID NO: 306 and 345; (l) respectively, SEQ ID NO: 306 and 347; (m) respectively, SEQ ID NO: 308 and 340; (n) respectively, SEQ ID NO: 309 and 341; (o) respectively, SEQ ID NO: 310 and 336; (p) respectively, SEQ ID NO: 311 and 337; (q) respectively, SEQ ID NO: 313 and 336; (r) respectively, SEQ ID NO: 313 and 342; (s) respectively, SEQ ID NO: 313 and 343; (t) respectively, SEQ ID NO: 316 and 337; (u) respectively, SEQ ID NO: 316 and 344; (v) respectively, SEQ ID NO: 316 and 345; (w) respectively, SEQ ID NO: 318 and 336; (x) respectively, SEQ ID NO: 318 and 340; (y) respectively, SEQ ID NO: 318 and 346; (z) respectively, SEQ ID NO: 319 and 336; (aa) Specifically, SEQ ID NO: 319 and 340; (bb) Specifically, SEQ ID NO: 319 and 346; (cc) Specifically, SEQ ID NO: 320 and 337; (dd) Specifically, SEQ ID NO: 320 and 341; (ee) respectively, SEQ ID NO: 320 and 347; (ff) Specifically, SEQ ID NO: 320 and 743; (gg) Specifically, SEQ ID NO: 321 and 337; (hh) Specifically, SEQ ID NO: 321 and 341; or (ii) SEQ ID NO: 321 and 347, respectively.

5. The PD-1 binding protein of claim 4, wherein the anti-PD-1 V H and anti-PD-1 V L It has the following amino acid sequences: (a) respectively, SEQ ID NO: 302 and 335; (b) respectively, SEQ ID NO: 302 and 336; (c) respectively, SEQ ID NO: 302 and 340; (d) Specifically, SEQ ID NO: 302 and 342; (e) respectively, SEQ ID NO: 302 and 343; (f) respectively, SEQ ID NO: 302 and 346; (g) respectively, SEQ ID NO: 306 and 337; (h) respectively, SEQ ID NO: 306 and 339; (i) respectively, SEQ ID NO: 306 and 341; (j) respectively, SEQ ID NO: 306 and 344; (k) respectively, SEQ ID NO: 306 and 345; (l) respectively, SEQ ID NO: 306 and 347; (m) respectively, SEQ ID NO: 308 and 340; (n) respectively, SEQ ID NO: 309 and 341; (o) respectively, SEQ ID NO: 310 and 336; (p) respectively, SEQ ID NO: 311 and 337; (q) respectively, SEQ ID NO: 313 and 336; (r) respectively, SEQ ID NO: 313 and 342; (s) respectively, SEQ ID NO: 313 and 343; (t) respectively, SEQ ID NO: 316 and 337; (u) respectively, SEQ ID NO: 316 and 344; (v) respectively, SEQ ID NO: 316 and 345; (w) respectively, SEQ ID NO: 318 and 336; (x) respectively, SEQ ID NO: 318 and 340; (y) respectively, SEQ ID NO: 318 and 346; (z) respectively, SEQ ID NO: 319 and 336; (aa) Specifically, SEQ ID NO: 319 and 340; (bb) Specifically, SEQ ID NO: 319 and 346; (cc) Specifically, SEQ ID NO: 320 and 337; (dd) Specifically, SEQ ID NO: 320 and 341; (ee) respectively, SEQ ID NO: 320 and 347; (ff) Specifically, SEQ ID NO: 320 and 743; (gg) Specifically, SEQ ID NO: 321 and 337; (hh) Specifically, SEQ ID NO: 321 and 341; or (ii) SEQ ID NO: 321 and 347, respectively.

6. The PD-1 binding protein of claim 1, wherein the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of: (a) SEQ ID NO 445, 456 and 447, respectively; (b) SEQ ID NO 451, 458 and 453, respectively; or (c) SEQ ID NO 443, 460 and 447, respectively.

7. The PD-1 binding protein of claim 6, wherein the PD-1 binding protein further comprises an anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V). L ), the anti-PD-1 V L Includes the following complementary determinant regions: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; (b) CDR-L1 containing the amino acid sequence of SEQ ID NO: 454; CDR-L2, which contains the amino acid sequence of LAS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; or (c) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO:

450.

8. The PD-1 binding protein of claim 7, wherein the anti-PD-1 V H and anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the following: (a) respectively, SEQ ID NO: 303 and 336; (b) respectively, SEQ ID NO: 325 and 347; or (c) respectively, SEQ ID NO: 325 and 743.

9. The PD-1 binding protein of claim 8, wherein the anti-PD-1 V H and anti-PD-1 V L It has the following amino acid sequences: (a) respectively, SEQ ID NO: 303 and 336; (b) respectively, SEQ ID NO: 325 and 347; or (c) respectively, SEQ ID NO: 325 and 743.

10. The PD-1 binding protein of claim 1, wherein the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of: (a) SEQ ID NO 445, 461 and 447, respectively; (b) SEQ ID NO 451, 462 and 453, respectively; or (c) SEQ ID NO 443, 463 and 447, respectively.

11. The PD-1 binding protein of claim 10, wherein the PD-1 binding protein further comprises an anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V). L ), the anti-PD-1 V L Includes the following complementary determinant regions: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; (b) CDR-L1 containing the amino acid sequence of SEQ ID NO: 454; CDR-L2, which contains the amino acid sequence of LAS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; or (c) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO:

450.

12. The PD-1 binding protein of claim 11, wherein the anti-PD-1 V H and anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the following: (a) respectively, SEQ ID NO: 326 and 347; or (b) respectively, SEQ ID NO: 326 and 743.

13. The PD-1 binding protein of claim 12, wherein the anti-PD-1 V H and anti-PD-1 V L It has the following amino acid sequences: (a) respectively, SEQ ID NO: 326 and 347; or (b) SEQ ID NO: 326 and 743, respectively.

14. The PD-1 binding protein of claim 1, wherein the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of: (a) SEQ ID NO 445, 446 and 457, respectively; (b) SEQ ID NO 451, 452 and 459, respectively; or (c) SEQ ID NO 443, 455 and 457, respectively.

15. The PD-1 binding protein of claim 14, wherein the PD-1 binding protein further comprises an anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V). L ), the anti-PD-1 V L Includes the following complementary determinant regions: (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; (e) CDR-L1 containing the amino acid sequence of SEQ ID NO: 454; CDR-L2, which contains the amino acid sequence of LAS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; or (f) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO:

450.

16. The PD-1 binding protein of claim 14, wherein the anti-PD-1 V H and anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the following: (a) respectively, SEQ ID NO: 327 and 347; or (b) respectively, SEQ ID NO: 327 and 743.

17. The PD-1 binding protein of claim 15, wherein the anti-PD-1 V H and anti-PD-1 V L It has the following amino acid sequences: (a) respectively, SEQ ID NO: 327 and 347; or (b) respectively, SEQ ID NO: 327 and 743.

18. The PD-1 binding protein of claim 1, wherein the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of: (a) SEQ ID NO 445, 461 and 457, respectively; (b) SEQ ID NO 451, 462 and 459, respectively; or (c) SEQ ID NO 443, 463 and 457, respectively.

19. The PD-1 binding protein of claim 18, wherein the PD-1 binding protein further comprises an anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V). L ), the anti-PD-1 V L Includes the following complementary determinant regions: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; (b) CDR-L1 containing the amino acid sequence of SEQ ID NO: 454; CDR-L2, which contains the amino acid sequence of LAS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; or (c) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO:

450.

20. The PD-1 binding protein of claim 19, wherein the anti-PD-1 V H and anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the following: (a) respectively, SEQ ID NO: 303 and 335; (b) respectively, SEQ ID NO: 303 and 342; (c) respectively, SEQ ID NO: 303 and 343; (d) respectively, SEQ ID NO: 307 and 337; (e) respectively, SEQ ID NO: 307 and 344; (f) respectively, SEQ ID NO: 307 and 345; (g) respectively, SEQ ID NO: 314 and 336; (h) respectively, SEQ ID NO: 314 and 342; (i) respectively, SEQ ID NO: 314 and 343; (j) respectively, SEQ ID NO: 317 and 337; (k) respectively, SEQ ID NO: 317 and 344; (l) respectively, SEQ ID NO: 317 and 345; or (m) respectively, SEQ ID NO: 324 and 347.

21. The PD-1 binding protein of claim 20, wherein the anti-PD-1 V H and anti-PD-1 V L It has the following amino acid sequences: (a) Specifically, SEQ ID NO: 303 and 335; (b) Specifically, SEQ ID NO: 303 and 342; (c) Specifically, SEQ ID NO: 303 and 343; (d) Specifically, SEQ ID NO: 307 and 337; (e) Specifically, SEQ ID NO: 307 and 344; (f) Specifically, SEQ ID NO: 307 and 345; (g) Specifically, SEQ ID NO: 314 and 336; (h) Specifically, SEQ ID NO: 314 and 342; (i) respectively, SEQ ID NO: 314 and 343; (j) Specifically, SEQ ID NO: 317 and 337; (k) Specifically, SEQ ID NO: 317 and 344; (l) respectively, SEQ ID NO: 317 and 345; or (m) respectively, SEQ ID NO: 324 and 347.

22. The PD-1 binding protein as described in any one of claims 3, 7, 11, 15, or 19, wherein... (a) This anti-PD-1 V H It has a frame having an amino acid sequence that is at least 85% identical to the amino acid sequence in any one of SEQ ID NO: 464-470; and (b) The anti-PD-1 V L It has a frame having an amino acid sequence that is at least 85% identical to the amino acid sequence in any one of SEQ ID NO: 471-476.

23. The PD-1 binding protein of claim 22, wherein... (a) This anti-PD-1 V H It has a framework having an amino acid sequence of any one of SEQ ID NO: 464-470; and (b) The anti-PD-1 V L It has a framework having an amino acid sequence of any one of SEQ ID NO: 471-476.

24. The PD-1 binding protein as described in any one of claims 3, 7, 11, 15, or 19, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

471.

25. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

472.

26. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

471.

27. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

473.

28. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

472.

29. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

473.

30. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

474.

31. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

475.

32. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

471.

33. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

474.

34. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

475.

35. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

471.

36. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

474.

37. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

475.

38. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 466; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

471.

39. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 470; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

474.

40. The PD-1 binding protein of claim 23, wherein... (a) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 469; and (b) The anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

476.

41. The PD-1 binding protein of claim 22, wherein: (i) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 472, except for one amino acid substitution. (ii) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution. (iii) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 473, except for one amino acid substitution. (iv) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 472, except for one amino acid substitution. (v) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 473, except for one amino acid substitution. (vi) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution. (vii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution. (viii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution. (ix) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution. (x) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution. (xi) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution. (xii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution. (xiii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution. (xiv) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution. (xv) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 466, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution, or (xvi) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 470, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution. Among them, V H The substitution of one amino acid in V is from a non-cysteine ​​residue to a cysteine ​​residue, and V L The substitution of one amino acid in this is from a non-cysteine ​​residue to a cysteine ​​residue.

42. The PD-1 binding protein of claim 41, wherein the anti-PD-1 V H The amino acid substitution occurs at residue H44, and this anti-PD-1 V L The amino acid substitution is at residue L100, where the numbering is based on Kabat.

43. A PD-1 binding protein, the PD-1 binding protein comprising (a) Anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 V) H ), the anti-PD-1 V H The device contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 445, 456, and 447, respectively; (2) SEQ ID NO 451, 458, and 453, respectively; or (3) SEQ ID NO 443, 460, and 457, respectively. (b) Anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 V) L ), the anti-PD-1 V L It contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) respectively, SEQ ID NO 448, 449, and 450; (2) respectively, SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450.

44. The PD-1 binding protein of claim 43, wherein... (a) This anti-PD-1 V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 325; and (b) The anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

347.

45. The PD-1 binding protein of claim 44, wherein... (a) This anti-PD-1 V H It has the amino acid sequence of SEQ ID NO: 325; and (b) The anti-PD-1 V L It has the amino acid sequence of SEQ ID NO:

347.

46. ​​A PD-1 binding protein, the PD-1 binding protein comprising (a) Anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 V) H ), the anti-PD-1 V H Contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 445, 456, and 457, respectively; (2) SEQ ID NO 451, 458, and 459, respectively; or (3) SEQ ID NO 443, 460, and 457, respectively; and (b) Anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 V) L ), the anti-PD-1 V L It contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) respectively, SEQ ID NO 448, 449, and 450; (2) respectively, SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450.

47. The PD-1 binding protein of claim 46, wherein... (a) This anti-PD-1 V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 320; and (b) The anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

347.

48. The PD-1 binding protein of claim 47, wherein... (a) This anti-PD-1 V H It has the amino acid sequence of SEQ ID NO: 320; and (b) The anti-PD-1 V L It has the amino acid sequence of SEQ ID NO:

347.

49. A PD-1 binding protein, the PD-1 binding protein comprising (a) Anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 V) H ), the anti-PD-1 V H The device contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 445, 461, and 457, respectively; (2) SEQ ID NO 451, 462, and 459, respectively; or (3) SEQ ID NO 443, 463, and 457, respectively. (b) Anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 V) L ), the anti-PD-1 V L It contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) respectively, SEQ ID NO 448, 449, and 450; (2) respectively, SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450.

50. The PD-1 binding protein of claim 49, wherein... (a) This anti-PD-1 V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 324; and (b) The anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

347.

51. The PD-1 binding protein of claim 50, wherein... (a) This anti-PD-1 V H It has the amino acid sequence of SEQ ID NO: 324; and (b) The anti-PD-1 V L It has the amino acid sequence of SEQ ID NO:

347.

52. A PD-1 binding protein, the PD-1 binding protein comprising (a) Anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 V) H ), the anti-PD-1 V H The device contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 445, 461, and 447, respectively; (2) SEQ ID NO 451, 462, and 453, respectively; or (3) SEQ ID NO 443, 463, and 447, respectively. (b) Anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 V) L ), the anti-PD-1 V L It contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) respectively, SEQ ID NO 448, 449, and 450; (2) respectively, SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450.

53. The PD-1 binding protein of claim 52, wherein... (a) This anti-PD-1 V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 326; and (b) The anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

347.

54. The PD-1 binding protein of claim 53, wherein... (c) The anti-PD-1 V H It has the amino acid sequence of SEQ ID NO: 326; and (d) The anti-PD-1 V L It has the amino acid sequence of SEQ ID NO:

347.

55. A PD-1 binding protein, the PD-1 binding protein comprising (a) Anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 V) H ), the anti-PD-1 V H The device contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 445, 446, and 457, respectively; (2) SEQ ID NO 451, 452, and 459, respectively; or (3) SEQ ID NO 443, 455, and 457, respectively. (b) Anti-PD-1 immunoglobulin light chain variable domain (anti-PD-1 V) L ), the anti-PD-1 V L It contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) respectively, SEQ ID NO 448, 449, and 450; (2) respectively, SEQ ID NO: 454, LAS, and SEQ ID NO: 450; or (3) respectively, SEQ ID NO 448, 449, and 450.

56. The PD-1 binding protein of claim 55, wherein... (a) This anti-PD-1 V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 327; and (b) The anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

347.

57. The PD-1 binding protein of claim 56, wherein... (a) This anti-PD-1 V H It has the amino acid sequence of SEQ ID NO: 327; and (b) The anti-PD-1 V L It has the amino acid sequence of SEQ ID NO:

347.

58. A variable domain comprising an anti-PD1 immunoglobulin heavy chain (anti-PD-1 V) H ) and anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V) L The programmed death receptor 1 (PD-1) binding protein, wherein: (1) The anti-PD-1 V H Includes the following complementary determinant regions: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 445; CDR-H2 containing the amino acid sequence of SEQ ID NO: 446; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 447; (b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 451; CDR-H2 containing the amino acid sequence of SEQ ID NO: 452; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 453; or (c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 443; CDR-H2 containing the amino acid sequence of SEQ ID NO: 455; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 447; (2) The anti-PD-1 V L Includes the following complementary determinant regions: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; (b) CDR-L1 containing the amino acid sequence of SEQ ID NO: 454; CDR-L2, which contains the amino acid sequence of LAS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; or (c) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 contains the amino acid sequence of SEQ ID NO: 450, and in (i) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465; and this anti-PD-1V L It has a framework having the amino acid sequence of SEQ ID NO:

472. (ii) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465; and this anti-PD-1V L It has a framework having the amino acid sequence of SEQ ID NO:

471. (iii) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

473. (iv) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464; and this anti-PD-1V L It has a framework having the amino acid sequence of SEQ ID NO:

472. (v) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464; and this anti-PD-1V L It has a framework having the amino acid sequence of SEQ ID NO:

473. (vi) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464; and this anti-PD-1V L It has a framework having the amino acid sequence of SEQ ID NO:

474. (vii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

475. (viii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

471. (ix) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467; and this anti-PD-1V L It has a framework having the amino acid sequence of SEQ ID NO:

474. (x) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467; and this anti-PD-1V L It has a framework having the amino acid sequence of SEQ ID NO:

475. (xi) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468; and this anti-PD-1V L It has a framework having the amino acid sequence of SEQ ID NO:

471. (xii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

474. (xiii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

475. (xiv) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

475. (xv) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 466; and this anti-PD-1V L It has a framework having the amino acid sequence of SEQ ID NO: 471, or (xvi) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 470; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO:

474.

59. A variable domain comprising an anti-PD1 immunoglobulin heavy chain (anti-PD-1 V) H ) and anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V) L The programmed death receptor 1 (PD-1) binding protein, in which (1) The anti-PD-1 V H Includes the following complementary determinant regions: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 445; CDR-H2 containing the amino acid sequence of SEQ ID NO: 446; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 447; (b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 451; CDR-H2 containing the amino acid sequence of SEQ ID NO: 452; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 453; or (c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 443; CDR-H2 containing the amino acid sequence of SEQ ID NO: 455; and CDR-H3, containing the amino acid sequence of SEQ ID NO: 447, (2) The anti-PD-1 V L Includes the following complementary determinant regions: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; (b) CDR-L1 containing the amino acid sequence of SEQ ID NO: 454; CDR-L2, which contains the amino acid sequence of LAS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; or (c) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 contains the amino acid sequence of SEQ ID NO: 450, and in (i) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 472, except for one amino acid substitution. (ii) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution. (iii) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 465, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 473, except for one amino acid substitution. (iv) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 472, except for one amino acid substitution. (v) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 473, except for one amino acid substitution. (vi) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution. (vii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 464, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution. (viii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution. (ix) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution. (x) The anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 467, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution. (xi) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution. (xii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution. (xiii) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution. (xiv) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 468, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 475, except for one amino acid substitution. (xv) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 466, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 471, except for one amino acid substitution, or (xvi) This anti-PD-1 V H It has a framework having the amino acid sequence of SEQ ID NO: 470, except for one amino acid substitution; and this anti-PD-1 V L It has a framework having the amino acid sequence of SEQ ID NO: 474, except for one amino acid substitution. Among them, V H The substitution of one amino acid in V is from a non-cysteine ​​residue to a cysteine ​​residue, and V L The substitution of one amino acid in this is from a non-cysteine ​​residue to a cysteine ​​residue.

60. The PD-1 binding protein of claim 59, wherein the anti-PD-1 V H The amino acid substitution occurs at residue H44, and this anti-PD-1 V L The amino acid substitution is at residue L100, where the numbering is based on Kabat.

61. A variable domain comprising an anti-PD1 immunoglobulin heavy chain (anti-PD-1 V) H ) and anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V) L The programmed death receptor 1 (PD-1) binding protein, wherein: (1) The anti-PD-1 V H Includes the following complementary determinant regions: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 445; CDR-H2 containing the amino acid sequence of SEQ ID NO: 446; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 447; (b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 451; CDR-H2 containing the amino acid sequence of SEQ ID NO: 452; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 453; or (c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 443; CDR-H2 containing the amino acid sequence of SEQ ID NO: 455; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 447; (2) The anti-PD-1 V L Includes the following complementary determinant regions: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; (b) CDR-L1 containing the amino acid sequence of SEQ ID NO: 454; CDR-L2, which contains the amino acid sequence of LAS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 450; or (c) CDR-L1 containing the amino acid sequence of SEQ ID NO: 448; CDR-L2 containing the amino acid sequence of SEQ ID NO: 449; and CDR-L3 contains the amino acid sequence of SEQ ID NO: 450, and Among them, the anti-PD-1 V H and anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the following: (a) respectively, SEQ ID NO: 301 and 335; (b) respectively, SEQ ID NO: 301 and 336; (c) respectively, SEQ ID NO: 301 and 342; (d) respectively, SEQ ID NO: 301 and 343; (e) respectively, SEQ ID NO: 301 and 348; (f) respectively, SEQ ID NO: 305 and 337; (g) respectively, SEQ ID NO: 305 and 344; (h) respectively, SEQ ID NO: 305 and 345; (i) respectively, SEQ ID NO: 312 and 335; (j) respectively, SEQ ID NO: 312 and 336; (k) respectively, SEQ ID NO: 312 and 342; (l) respectively, SEQ ID NO: 312 and 343; (m) respectively, SEQ ID NO: 315 and 337; (n) respectively, SEQ ID NO: 315 and 344; (o) respectively, SEQ ID NO: 315 and 345; (p) respectively, SEQ ID NO: 322 and 346; (q) respectively, SEQ ID NO: 323 and 347; or (r) respectively, SEQ ID NO: 323 and 743.

62. The PD-1 binding protein of claim 61, wherein the anti-PD-1 V H and anti-PD-1 V L It has the following amino acid sequences: (a) respectively, SEQ ID NO: 301 and 335; (b) respectively, SEQ ID NO: 301 and 336; (c) respectively, SEQ ID NO: 301 and 342; (d) respectively, SEQ ID NO: 301 and 343; (e) respectively, SEQ ID NO: 301 and 348; (f) respectively, SEQ ID NO: 305 and 337; (g) respectively, SEQ ID NO: 305 and 344; (h) respectively, SEQ ID NO: 305 and 345; (i) respectively, SEQ ID NO: 312 and 335; (j) respectively, SEQ ID NO: 312 and 336; (k) respectively, SEQ ID NO: 312 and 342; (l) respectively, SEQ ID NO: 312 and 343; (m) respectively, SEQ ID NO: 315 and 337; (n) respectively, SEQ ID NO: 315 and 344; (o) respectively, SEQ ID NO: 315 and 345; (p) respectively, SEQ ID NO: 322 and 346; (q) respectively, SEQ ID NO: 323 and 347; or (r) respectively, SEQ ID NO: 323 and 743.

63. The PD-1 binding protein according to any one of claims 1-62, wherein the PD-1 binding protein further comprises an Fc region.

64. The PD-1 binding protein of claim 63, wherein the Fc region includes means for extending the half-life of the PD-1 binding protein.

65. The PD-1 binding protein of claim 64, wherein the means of extending the half-life includes Fc modification.

66. The PD-1 binding protein of claim 65, wherein the Fc modification is selected from the group consisting of: M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF) and L309D / Q311H / N434S (DHS).

67. The PD-1 binding protein of claim 66, wherein the Fc modification is M252Y / S254T / T256E (YTE) or M428L / N434S (LS).

68. The PD-1 binding protein of claim 67, wherein the Fc modification is M428L / N434S (LS).

69. The PD-1 binding protein according to any one of claims 63-68, wherein the Fc region is an IgG1, IgG2, or IgG4 Fc region.

70. The PD-1 binding protein according to any one of claims 1-69, wherein the PD-1 binding protein is an antibody or an antigen-binding fragment thereof.

71. The PD-1 binding protein of claim 70, wherein the PD-1 binding protein is a human or humanized antibody or an antigen-binding fragment thereof.

72. The PD-1 binding protein of claim 70 or 71, wherein the antigen-binding fragment is Fab, F(ab')2, Fab', single-chain Fv (scFv), Fv fragment, Fd fragment, or a biantibody.

73. A bispecific protein comprising the PD-1 binding protein as described in any one of claims 1-72, the bispecific protein further comprising a VEGF binding region, the VEGF binding region comprising an anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF V). H ), the anti-VEGF V H Includes the following complementary determinant regions: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 433; CDR-H2 containing the amino acid sequence of SEQ ID NO: 434; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 435; (b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 439; CDR-H2 containing the amino acid sequence of SEQ ID NO: 440; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 441; or (c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 443; CDR-H2 containing the amino acid sequence of SEQ ID NO: 444; and CDR-H3 containing the amino acid sequence of SEQ ID NO:

435.

74. The bispecific protein of claim 73, further comprising an anti-VEGF immunoglobulin light chain variable domain (anti-VEGF V). L This anti-VEGF VL contains the following complementary determinant regions: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 436; CDR-L2 containing the amino acid sequence of SEQ ID NO: 437; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 438; (b) CDR-L1 containing the amino acid sequence of SEQ ID NO: 442; CDR-L2 containing the amino acid sequence of FTS; and CDR-L3 containing the amino acid sequence of SEQ ID NO: 438; or (c) CDR-L1 containing the amino acid sequence of SEQ ID NO: 436; CDR-L2 containing the amino acid sequence of SEQ ID NO: 437; and CDR-L3 containing the amino acid sequence of SEQ ID NO:

438.

75. The bispecific protein of claim 73 or 74, wherein the anti-VEGF V... H It has an amino acid sequence that is at least 85% identical to that of SEQ ID NO: 431; and this anti-VEGF V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

432.

76. The bispecific protein of claim 75, wherein the anti-VEGF V... H It has the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L An amino acid containing SEQ ID NO:

432.

77. A bispecific protein comprising a PD-1 binding region and a VEGF binding region, wherein... (a) The PD-1 binding region contains: (i) Anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 V) H ), the anti-PD-1 V H The device contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 445, 456, and 447, respectively; (2) SEQ ID NO 451, 458, and 453, respectively; or (3) SEQ ID NO 443, 460, and 447, respectively. (ii) Anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V) L ), the anti-PD-1 V L It contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 448, 449, and 450, respectively; (2) SEQ ID NO: 454, LAS, and SEQ ID NO: 450, respectively; or (3) SEQ ID NO 448, 449, and 450, respectively; and (b) The VEGF binding region contains: (i) Anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF V) H The anti-VEGF VH contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 433, 434, and 435, respectively; (2) SEQ ID NO 439, 440, and 441, respectively; or (3) SEQ ID NO 443, 444, and 435, respectively. (ii) Anti-VEGF immunoglobulin light chain variable domain (anti-VEGF V) L The anti-VEGF VL contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 436, 437, and 438, respectively; (2) SEQ ID NO: 442, FTS, and SEQ ID NO: 438, respectively; or (3) SEQ ID NO: 436, 437, and 438, respectively.

78. The bispecific protein of claim 77, wherein... (a) This anti-PD-1 V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 325; and this anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347; and (b) The anti-VEGF V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

432.

79. The bispecific protein of claim 78, wherein... (a) This anti-PD-1 V H It has the amino acid sequence of SEQ ID NO: 325; and this anti-PD-1 V L It has the amino acid sequence of SEQ ID NO: 347; and (b) The anti-VEGF V H It has the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L It has the amino acid sequence SEQ ID NO:

432.

80. A bispecific protein comprising a PD-1 binding region and a VEGF binding region, wherein (a) The PD-1 binding region contains: (i) Anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 V) H ), the anti-PD-1 V H Contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 445, 456, and 457, respectively; (2) SEQ ID NO 451, 458, and 459, respectively; or (3) SEQ ID NO 443, 460, and 457, respectively; and (ii) Anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V) L ), the anti-PD-1 V L It contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 448, 449, and 450, respectively; (2) SEQ ID NO: 454, LAS, and SEQ ID NO: 450, respectively; or (3) SEQ ID NO 448, 449, and 450, respectively; and (b) The VEGF binding region contains: (i) Anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF V) H The anti-VEGF VH contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 433, 434, and 435, respectively; (2) SEQ ID NO 439, 440, and 441, respectively; or (3) SEQ ID NO 443, 444, and 435, respectively. (ii) Anti-VEGF immunoglobulin light chain variable domain (anti-VEGF V) L The anti-VEGF VL contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 436, 437, and 438, respectively; (2) SEQ ID NO: 442, FTS, and SEQ ID NO: 438, respectively; or (3) SEQ ID NO: 436, 437, and 438, respectively.

81. The bispecific protein of claim 80, wherein... (a) This anti-PD-1 V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 320; and this anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347; and (b) The anti-VEGF V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

432.

82. The bispecific protein of claim 81, wherein... (a) This anti-PD-1 V H It has the amino acid sequence of SEQ ID NO: 320; and this anti-PD-1 V L It has the amino acid sequence of SEQ ID NO: 347; and (b) The anti-VEGF V H It has the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L It has the amino acid sequence SEQ ID NO:

432.

83. A bispecific protein comprising a PD-1 binding region and a VEGF binding region, wherein... (a) The PD-1 binding region contains: (i) Anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 V) H ), the anti-PD-1 V H The device contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 445, 461, and 457, respectively; (2) SEQ ID NO 451, 462, and 459, respectively; or (3) SEQ ID NO 443, 463, and 457, respectively. (ii) Anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V) L ), the anti-PD-1 V L It contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 448, 449, and 450, respectively; (2) SEQ ID NO: 454, LAS, and SEQ ID NO: 450, respectively; or (3) SEQ ID NO 448, 449, and 450, respectively; and (b) The VEGF binding region contains: (i) Anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF V) H The anti-VEGF VH contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 433, 434, and 435, respectively; (2) SEQ ID NO 439, 440, and 441, respectively; or (3) SEQ ID NO 443, 444, and 435, respectively. (ii) Anti-VEGF immunoglobulin light chain variable domain (anti-VEGF V) L The anti-VEGF VL contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 436, 437, and 438, respectively; (2) SEQ ID NO: 442, FTS, and SEQ ID NO: 438, respectively; or (3) SEQ ID NO: 436, 437, and 438, respectively.

84. The bispecific protein of claim 83, wherein... (a) This anti-PD-1 V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 324; and this anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347; and (b) The anti-VEGF V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

432.

85. The bispecific protein of claim 84, wherein... (a) This anti-PD-1 V H It has the amino acid sequence of SEQ ID NO: 324; and this anti-PD-1 V L It has the amino acid sequence of SEQ ID NO: 347; and (b) The anti-VEGF V H It has the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L It has the amino acid sequence SEQ ID NO:

432.

86. A bispecific protein comprising a PD-1 binding region and a VEGF binding region, wherein... (a) The PD-1 binding region contains: (i) Anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 V) H ), the anti-PD-1 V H The device contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 445, 461, and 447, respectively; (2) SEQ ID NO 451, 462, and 453, respectively; or (3) SEQ ID NO 443, 463, and 447, respectively. (ii) Anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V) L ), the anti-PD-1 V L It contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 448, 449, and 450, respectively; (2) SEQ ID NO: 454, LAS, and SEQ ID NO: 450, respectively; or (3) SEQ ID NO 448, 449, and 450, respectively; and (b) The VEGF binding region contains: (i) Anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF V) H The anti-VEGF VH contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 433, 434, and 435, respectively; (2) SEQ ID NO 439, 440, and 441, respectively; or (3) SEQ ID NO 443, 444, and 435, respectively. (ii) Anti-VEGF immunoglobulin light chain variable domain (anti-VEGF V) L The anti-VEGF VL contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 436, 437, and 438, respectively; (2) SEQ ID NO: 442, FTS, and SEQ ID NO: 438, respectively; or (3) SEQ ID NO: 436, 437, and 438, respectively.

87. The bispecific protein of claim 86, wherein... (a) This anti-PD-1 V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 326; and this anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347; and (b) The anti-VEGF V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

432.

88. The bispecific protein of claim 87, wherein... (a) This anti-PD-1 V H It has the amino acid sequence of SEQ ID NO: 326; and this anti-PD-1 V L It has the amino acid sequence of SEQ ID NO: 347; and (b) The anti-VEGF V H It has the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L It has the amino acid sequence SEQ ID NO:

432.

89. A bispecific protein comprising a PD-1 binding region and a VEGF binding region, wherein... (a) The PD-1 binding region contains: (i) Anti-PD-1 immunoglobulin heavy chain variable domain (anti-PD-1 V) H ), the anti-PD-1 V H The device contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 445, 446, and 457, respectively; (2) SEQ ID NO 451, 452, and 459, respectively; or (3) SEQ ID NO 443, 455, and 457, respectively. (ii) Anti-PD1 immunoglobulin light chain variable domain (anti-PD-1 V) L ), the anti-PD-1 V L It contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 448, 449, and 450, respectively; (2) SEQ ID NO: 454, LAS, and SEQ ID NO: 450, respectively; or (3) SEQ ID NO 448, 449, and 450, respectively; and (b) The VEGF binding region contains: (i) Anti-VEGF immunoglobulin heavy chain variable domain (anti-VEGF V) H The anti-VEGF VH contains complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, which contain the amino acid sequences of: (1) SEQ ID NO 433, 434, and 435, respectively; (2) SEQ ID NO 439, 440, and 441, respectively; or (3) SEQ ID NO 443, 444, and 435, respectively. (ii) Anti-VEGF immunoglobulin light chain variable domain (anti-VEGF V) L The anti-VEGF VL contains complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, which contain the amino acid sequences of: (1) SEQ ID NO 436, 437, and 438, respectively; (2) SEQ ID NO: 442, FTS, and SEQ ID NO: 438, respectively; or (3) SEQ ID NO: 436, 437, and 438, respectively.

90. The bispecific protein of claim 89, wherein... (a) This anti-PD-1 V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 327; and this anti-PD-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 347; and (b) The anti-VEGF V H It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L It has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

432.

91. The bispecific protein of claim 90, wherein... (a) This anti-PD-1 V H It has the amino acid sequence of SEQ ID NO: 327; and this anti-PD-1 V L It has the amino acid sequence of SEQ ID NO: 347; and (b) The anti-VEGF V H It has the amino acid sequence of SEQ ID NO: 431; and this anti-VEGF-1 V L It has the amino acid sequence SEQ ID NO:

432.

92. The bispecific protein according to any one of claims 73-91, wherein the bispecific protein further comprises an Fc region.

93. The bispecific protein of claim 92, wherein the Fc region includes means for extending the half-life of the PD-1 binding protein.

94. The bispecific protein of claim 93, wherein the means of extending the half-life includes Fc modification.

95. The bispecific protein of claim 94, wherein the Fc modification is selected from the group consisting of: M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF) and L309D / Q311H / N434S (DHS).

96. The bispecific protein of claim 95, wherein the Fc modification is M252Y / S254T / T256E (YTE) or M428L / N434S (LS).

97. The bispecific protein of claim 96, wherein the Fc modification is M428L / N434S (LS).

98. The bispecific protein according to any one of claims 92-97, wherein the Fc region is an IgG1, IgG2, or IgG4 Fc region.

99. The bispecific protein of any one of claims 73-98, wherein the bispecific protein is an antibody or an antigen-binding fragment thereof.

100. The bispecific protein of claim 99, wherein the bispecific protein is a human or humanized antibody or an antigen-binding fragment thereof.

101. The bispecific protein of claim 99 or 100, wherein the antigen-binding fragment is Fab, F(ab')2, Fab', single-chain Fv (scFv), Fv fragment, Fd fragment, or a biantibody.

102. The bispecific protein according to any one of claims 73-101, wherein the bispecific protein comprises two PD-1 binding regions and two VEGF binding regions.

103. The bispecific protein according to any one of claims 73-101, wherein the bispecific protein comprises two PD-1 binding regions and one VEGF binding region.

104. The bispecific protein according to any one of claims 73-101, wherein the bispecific protein comprises a PD-1 binding region and two VEGF binding regions.

105. The bispecific protein of any one of claims 102-104, wherein each PD-1 binding region is scFv.

106. The bispecific protein of claim 105, wherein each VEGF-binding region comprises an anti-VEGF V protein on a first polypeptide chain. H Anti-VEGF V on the second polypeptide chain L Anti-VEGF V in Fab H and anti-VEGF V L or anti-VEGF V in CrossFab H and anti-VEGF V L .

107. The bispecific protein of any one of claims 102-104, wherein each VEGF binding region is scFv.

108. The bispecific protein of claim 107, wherein each PD-1 binding region comprises an anti-PD1 V antibody on a first polypeptide chain. H Anti-PD1 V on the second polypeptide chain L Anti-PD1 V in the Fab H and anti-PD1 V L or anti-PD1V within CrossFab H and anti-PD1 V L .

109. The bispecific protein of any one of claims 102-104, wherein each PD-1 binding region is VHH.

110. The bispecific protein of claim 109, wherein each VEGF-binding region comprises an anti-VEGF V protein on a first polypeptide chain. H Anti-VEGF V on the second polypeptide chain L Anti-VEGF V in Fab H and anti-VEGF V L or anti-VEGF V in CrossFab H and anti-VEGF V L .

111. A bispecific protein comprising at least one PD-1 binding region and at least one VEGF binding region, the bispecific protein comprising a first polypeptide chain having the sequence of SEQ ID NO: 92 and a second polypeptide chain having the sequence of SEQ ID NO:

239.

112. A bispecific protein comprising at least one PD-1 binding region and at least one VEGF binding region, the bispecific protein comprising a first polypeptide chain having the sequence of SEQ ID NO: 73 and a second polypeptide chain having the sequence of SEQ ID NO:

220.

113. A bispecific protein comprising at least one PD-1 binding region and at least one VEGF binding region, the bispecific protein comprising a first polypeptide chain having the sequence of SEQ ID NO: 91 and a second polypeptide chain having the sequence of SEQ ID NO:

238.

114. A bispecific protein comprising at least one PD-1 binding region and at least one VEGF binding region, the bispecific protein comprising a first polypeptide chain having the sequence of SEQ ID NO: 94 and a second polypeptide chain having the sequence of SEQ ID NO:

241.

115. A bispecific protein comprising at least one PD-1 binding region and at least one VEGF binding region, the bispecific protein comprising a first polypeptide chain having the sequence of SEQ ID NO: 96 and a second polypeptide chain having the sequence of SEQ ID NO:

243.

116. A dimer of a bispecific protein as described in any one of claims 111-115.

117. An isolated nucleic acid encoding one or more strands of a PD-1 binding protein as described in any one of claims 1-72 or a bispecific protein as described in any one of claims 73-116.

118. An expression vector comprising the isolated nucleic acid as described in claim 117.

119. A host cell comprising the isolated nucleic acid as described in claim 117 or the expression vector as described in claim 118.

120. A group of isolated nucleic acids that collectively encode a PD-1 binding protein as described in any one of claims 1-72 or a bispecific protein as described in any one of claims 73-116.

121. A set of expression vectors that collectively comprise the isolated nucleic acid genome as described in claim 120.

122. A host cell comprising the isolated nucleic acid set as described in claim 120 or the expression vector set as described in claim 121.

123. A pharmaceutical composition comprising the PD-1 binding protein as claimed in any one of claims 1-72 or the bispecific protein as claimed in any one of claims 73-116, and a pharmaceutically acceptable carrier.

124. A method comprising administering to a subject in need an effective amount of the PD-1 binding protein as described in any one of claims 1-72, the bispecific protein as described in any one of claims 73-116, or the pharmaceutical composition as described in claim 123.

125. The method of claim 124, wherein the subject has a disease or condition associated with abnormal PD-1 and / or VEGF expression or signaling or is at risk of having such a disease or condition.

126. The method of claim 125, wherein the disease or symptom is cancer.

127. The method of claim 126, wherein the cancer is lung cancer.

128. The method of claim 127, wherein the lung cancer is non-small cell lung cancer.

129. The method of claim 126, wherein the cancer is gastrointestinal cancer.

130. The method of claim 129, wherein the gastrointestinal cancer is colorectal cancer, bile duct cancer, gastric cancer, or hepatocellular carcinoma.

131. The method of claim 126, wherein the cancer is a reproductive cancer.

132. The method of claim 131, wherein the reproductive cancer is cervical cancer, endometrial cancer, or ovarian cancer.

133. The method of any one of claims 124-132, wherein the administration step comprises systemic administration of the PD-1 binding protein, the bispecific protein, or the pharmaceutical composition.

134. The method of claim 133, wherein systemic administration comprises intravenous administration of the PD-1 binding protein, the bispecific protein, or the pharmaceutical composition.

135. The method of claim 133, wherein systemic administration comprises subcutaneous administration of the PD-1 binding protein, the bispecific protein, or the pharmaceutical composition.

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