Anti-pd-1 monoclonal antibody and methods of preparation thereof

Abstract

The invention discloses an anti-human PD-1 monoclonal antibody and a preparation method thereof. The anti-human PD-1 monoclonal antibody can bind to an extracellular region of a human PD-1 protein receptor, and can block a PD-1 protein at a protein level and a cellular level and block binding of the PD-1 protein, thereby enhancing immunity. The monoclonal antibodies can be administered independently, in combination with other drugs, and / or independently or in combination in any given order to treat an individual in need thereof.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480075552.0 (22) Application Date 2024.11.13 (30) Priority Data 63 / 610,577 2023.12.15 US (85) PCT International Application Entering National Phase Date 2026.05.28 (86) PCT International Application Application Data PCT / IB2024 / 000811 2024.11.13 (87) PCT International Application Publication Data WO2025 / 128263 EN 2025.06.19 (71) Applicant: Yaohua Pharmaceutical Co., Ltd. Address: No. 3, Yuanqu Street, Nangang District, Taipei City, Taiwan, China Building (72) Inventors You Yide Yan Shilong Xu Xu Chen Mingtang Lin Guozhong (74) Patent Agency Beijing Lvmeng Intellectual Property Agency Co., Ltd. 11287 Patent Attorney Zhang Xiaoyuan (51) Int.Cl. C07K 16 / 28 (2006.01) A61K 39 / 395 (2006.01) A61P 35 / 00 (2006.01) C07K 14 / 705 (2006.01) C12N 15 / 64 (2006.01) C12N 5 / 0783 (2006.01) (54) Invention Title Anti-PD-1 Monoclonal Antibody and its Preparation Method (57) Abstract This paper discloses an anti-human PD-1 monoclonal antibody and its preparation method. The anti-human PD-1 monoclonal antibody can bind to the extracellular domain of the human PD-1 protein receptor and can block the PD-1 protein and its binding at both the protein and cellular levels, thereby enhancing immunity. The monoclonal antibody can be administered independently, in combination with other drugs, and / or independently or in any given order, to treat individuals in need. Claims (5 pages), Description (54 pages), Sequence Listing (electronic publication), Drawings (18 pages), CN 122270481 A, 2026.06.23, CN 1 22 27 04 81 A. 1. An isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain complementarity determining region 1 (VHCDR1) of the amino acid sequence listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 21, and a heavy chain complementarity determining region 2 (VHCDR1) of the amino acid sequence listed in SEQ ID NO: 10.The region includes the heavy chain complementarity determining region 3 (VHCDR2), comprising the amino acid sequence listed in SEQ ID NO: 11, SEQ ID NO: 20, or SEQ ID NO: 23; the light chain complementarity determining region 1 (VLCDR1), comprising the amino acid sequence listed in SEQ ID NO: 12; the light chain complementarity determining region 2 (VLCDR2), comprising the amino acid sequence listed in SEQ ID NO: 13; and / or the light chain complementarity determining region 3 (VLCDR3), comprising the amino acid sequence listed in SEQ ID NO: 14. 2. An isolated antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising one or more paratopes, said one or more paratopes comprising: at least one of the following residues: Phe2, Ser5, Ser6 and Tyr7 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 19; at least one of the following residues: Phe2, Ser5 and Tyr6 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 21; at least one of the following residues: Thr13, Gly14, Gly15, Gly16, Ser17 and Tyr18 of the heavy chain variable sequence (VHCDR2) of SEQ ID NO: 10; at least one of the following residues: Tyr33, Leu34, Asn35, Asp38 and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 11; or at least one of the following residues: SEQ ID NO: 19; 3. A separated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising one or more complementary sites, said one or more complementary sites comprising: at least one of the following residues: Asn9 of the light chain variable sequence (VLCDR1) of SEQ ID NO: 12; at least one of the following residues: SEQ ID NO:Tyr12 and Ser18 of the heavy chain variable sequence (VLCDR2) of SEQ ID NO: 13; or at least one of the following residues: Ser22, Asn23 and Trp25 of the heavy chain variable sequence (VLCDR3) of SEQ ID NO: 14. 4. An isolated antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising one or more complementary sites, said one or more complementary sites comprising: at least one of the following residues: Phe2, Ser5, Ser6 and Tyr7 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 19; at least one of the following residues: Phe2, Ser5 and Tyr6 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 21; at least one of the following residues: Thr13, Gly14, Gly15, Gly16, Ser17 and Tyr18 of the heavy chain variable sequence (VHCDR2) of SEQ ID NO: 10; at least one of the following residues: Tyr33, Leu34 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 11. (Claims 1 / 5 page 2 CN 122270481 A) Asn35, Asp38, and Tyr39; at least one of the following residues: Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 20; at least one of the following residues: Asn9 of the light chain variable sequence (VLCDR1) of SEQ ID NO: 12; at least one of the following residues: Tyr12 and Ser18 of the heavy chain variable sequence (VLCDR2) of SEQ ID NO: 13; or at least one of the following residues: Ser22, Asn23, and Trp25 of the heavy chain variable sequence (VLCDR3) of SEQ ID NO: 14. 5. An isolated antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 9 and a light chain variable region (VL) comprising SEQ ID NO: 6; or (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 18 and a light chain variable region (VL) comprising SEQ ID NO: 6; or (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 19 and a light chain variable region (VL) comprising SEQ ID NO: 6; or (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 21 and a light chain variable region (VL) comprising SEQ ID NO: 6. 6. An isolated antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising (i) a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region (VL) comprising SEQ ID NO: 9.5. The heavy chain variable region (VH) of SEQ ID NO: 22 or the heavy chain variable region (VH) of SEQ ID NO: 22; and (ii) the light chain variable region (VL) of SEQ ID NO: 6. 7. An isolated antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising: heavy chain complementarity-determining region 1 (VHCDR1), heavy chain complementarity-determining region 2 (VHCDR2) and / or heavy chain complementarity-determining region 3 (VHCDR3) derived from the amino acid sequence of the heavy chain variable region listed in SEQ ID NO: 5; and light chain complementarity-determining region 1 (VLCDR1), light chain complementarity-determining region 2 (VLCDR2) and / or light chain complementarity-determining region 3 (VLCDR3) derived from the amino acid sequence of the light chain variable region listed in SEQ ID NO: 6. 8. The antibody or antigen-binding portion thereof according to any one of claims 1 to 7, further comprising a heavy chain constant region (HCCR), said heavy chain constant region comprising the amino acid sequence listed in SEQ ID NO: 7; wherein said HCCR optionally comprises one, two, or three conserved modified amino acid substitutions. 9. The antibody or antigen-binding portion thereof according to any one of claims 1 to 7, further comprising a light chain constant region (LCCR), said light chain constant region comprising the amino acid sequence of SEQ ID NO: 8; wherein said LCCR optionally comprises one, two, or three conserved modified amino acid substitutions. 10. The antibody or antigen-binding portion thereof according to any one of claims 1 to 7, further comprising: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally comprises one, two, or three conserved modified amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two, or three conserved modified amino acid substitutions. 11. The antibody or antigen-binding portion thereof according to claim 10, further comprising a heavy chain signal peptide (HCSP) at the N-terminus of the VH, wherein the heavy chain signal peptide comprises the amino acid listed in SEQ ID NO: 15. 12. The antibody or antigen-binding portion thereof according to claim 10, further comprising a light chain signal peptide (LCSP) at the N-terminus of the VL, wherein the light chain signal peptide comprises the amino acid listed in SEQ ID NO: 8.13. An antibody or antigen-binding portion thereof according to any one of claims 1 to 7, wherein the antibody is a human or humanized antibody. 14. An antibody or antigen-binding portion thereof according to any one of claims 1 to 7, wherein the antibody is an IgG4 isotype. 15. A pharmaceutical composition for treating, inhibiting, and / or suppressing solid tumors, comprising (i) an antibody or antigen-binding portion thereof according to any one of claims 1 to 7; and (ii) a pharmaceutically acceptable carrier. 16. A polynucleotide molecule encoding an antibody or antigen-binding portion according to any one of claims 1 to 7. 17. An expression vector comprising the polynucleotide molecule according to claim 16, wherein the expression vector encoding LCSP does not contain introns. 18. A host cell comprising the expression vector according to claim 17. 19. A method for preventing, treating, and / or inhibiting tumors in an individual, comprising the step of: administering an effective amount of the antibody or antigen-binding portion according to claims 1 to 7 to the individual. 20. The method of claim 19, wherein the tumor comprises hepatocellular carcinoma, or a solid tumor comprising melanoma and / or renal cell carcinoma (RCC). 21. A method for regulating the activity of one or more T cells in an individual in vitro, ex vivo, and / or in vivo, the method comprising the steps of: contacting the T cells with an antibody or its antigen-binding moiety that preferentially binds to human programmed death receptor 1 (hPD-1), wherein the antibody blocks and / or regulates the binding of human PD-L1 and / or human PD-L2 to hPD-1, and wherein the antibody or its antigen-binding moiety comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21; a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10; and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11, SEQ ID NO: 20, or SEQ ID NO: 23; and a light chain complementarity-determining region 1 comprising the amino acid sequence listed in SEQ ID NO: 12. (VLCDR1), including light chain complementarity-determining region 2 (VLCDR2) of the amino acid sequence listed in SEQ ID NO: 13, and / or including light chain complementarity-determining region 3 (VLCDR3) of the amino acid sequence listed in SEQ ID NO: 14. 22.23. The method of claim 21, wherein the antibody or antigen-binding portion thereof further comprises a heavy chain constant region (HCCR), the heavy chain constant region comprising the amino acid sequence listed in SEQ ID NO: 7, and the HCCR optionally comprising one, two, or three conserved modified amino acid substitutions. 24. The method of claim 21, wherein the antibody or antigen-binding portion thereof further comprises a light chain constant region (LCCR), the light chain constant region comprising the amino acid sequence of SEQ ID NO: 8, and wherein the LCCR optionally comprises one, two, or three conserved modified amino acid substitutions. 25. The method of claim 21, wherein the one or more T cells comprise one or more natural killer cells, one or more cytotoxic T cells, one or more helper T cells, one or more monocytes, one or more memory T cells, and / or one or more regulatory T cells. 25. The method of claim 21, wherein the antibody or antigen-binding moiety blocks the binding of human PD-L1 and human PD-L2 to human PD-1 at an IC50 of about 1 nM or lower. 26. A method for generating an antibody or antigen-binding moiety vector construct, comprising culturing host cells according to claim 18 under conditions allowing expression of the antibody or antigen-binding moiety construct; and purifying and / or recovering the resulting antibody or antigen-binding moiety vector construct from the culture. 27. An antibody or antigen-binding moiety thereof for treating solid tumors, comprising: a. a heavy chain variable region (VH) of SEQ ID NO: 5 or a heavy chain variable region (VH) of SEQ ID NO: 22; b. a light chain variable region (VL) of SEQ ID NO: 6; and optionally comprising another therapeutic agent. 28. The antibody of claim 27 or its antigen-binding portion, further comprising: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally comprises one, two, or three conserved modified amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two, or three conserved modified amino acid substitutions; and optionally comprising another therapeutic agent. 29. The antibody of claim 27 or its antigen-binding portion, wherein the other therapeutic agent comprises P1101, P1801, and / or another anti-human PD1 antibody. 30. The antibody of claim 28 or its antigen-binding portion, wherein the other therapeutic agent comprises P1101, P1801, and / or another anti-human PD1 monoclonal antibody. 31.32. An antibody or antigen-binding portion thereof according to any one of claims 28 to 30, wherein the solid tumor comprises hepatocellular carcinoma, renal cell carcinoma, and / or melanoma. 33. A combination for treating, inhibiting, preventing, and / or suppressing a solid tumor in an individual, wherein the combination comprises an antibody or antigen-binding portion thereof; and another therapeutic agent, wherein the antibody or antigen-binding portion thereof comprises: a. a heavy chain variable region (VH) comprising the heavy chain variable region (VH) of SEQ ID NO: 5 or the heavy chain variable region (VH) comprising the heavy chain variable region (VH) of SEQ ID NO: 22; and b. a light chain variable region (VL) comprising the light chain variable region (VL) of SEQ ID NO: 6. 34. The combination according to claim 32, wherein the antibody or antigen-binding portion thereof further comprises: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally comprises one, two, or three conserved amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions. 34. The combination of any one of claims 32 to 33, wherein the therapeutic agent comprises P1101, P1801, and / or another anti-human PD1 monoclonal antibody. 35. The combination of claim 34, wherein the P1101, P1801, and / or another anti-human PD1 monoclonal antibody are administered sequentially and / or approximately simultaneously. 36. The combination of claim 35, wherein the P1101 and / or P1801 are administered prior to another anti-human PD1 monoclonal antibody. 37. The combination of claim 32 or 33, wherein the antibody or its antigen-binding portion and the other therapeutic agent are administered as a single composition or as separate compositions. 38. The combination of any one of claims 32 to 37, wherein the solid tumor comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma. 39. A recombinant protein comprising (i) an antibody or antigen-binding moiety according to claim 1; and (ii) an optional tag sequence for expression for detection and / or purification. 40. A method of treating cancer with an isolated antibody or antigen-binding moiety according to any one of claims 1 to 2, 5 to 7, or 27, wherein the method comprises administering the isolated antibody or antigen-binding moiety to an individual. 41. The isolated antibody or antigen-binding moiety according to claim 40, wherein the cancer comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma. 42. An isolated antibody or antigen-binding moiety according to any one of claims 1 to 2, 5 to 7, or 27.Use of the combined portion for manufacturing a pharmaceutical agent for therapeutic and / or preventive treatment of cancer. 43. Use of the isolated antibody or its antigen-binding portion according to claim 42, wherein the cancer comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma. Claims 5 / 5 Page 6 CN 122270481 A Anti-PD-1 monoclonal antibody and method for preparation thereof

[0001] Sequence Listing This application is filed electronically via EFS-Web and includes a sequence listing submitted electronically in ST.26 format in .xml format. The .xml file contains a sequence listing named “025-PCT-SEQ-Listing.xml” generated on November 8, 2024, and having a size of 23,068 bytes and 24,576 bytes on disk. The sequence listing contained in the .xml file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to the biomedical field, and more particularly to anti-PD-1 monoclonal antibodies optionally combined with another therapeutic agent (e.g., pegylated human interferon α2b), as well as methods for their preparation and applications. Background Art

[0003] Programmed protein death 1 (PD-1) is known in the art. Simply put, it is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, macrophages, and myeloid cells. The initial members of this family, CD28 and ICOS, were discovered due to their functional role in enhancing T cell proliferation upon the addition of monoclonal antibodies.

[0004] The PD-1 gene is a 50-55 kDa type I transmembrane protein and is part of the Ig gene superfamily. PD-1 contains a proximal immunoreceptor tyrosine inhibitory motif (ITIM) and a distal tyrosine-based switch motif (ITSM). Although structurally similar to CTLA-4, PD-1 lacks the MYPPPY motif, which is crucial for the binding of B7-1 and B7-2. Two ligands for PD-1 have been identified. PD-L1 and PD-L2 have been shown to downregulate T cell activation upon binding to PD-1. Both PD-L1 and PD-L2 are B7 homologs; they bind to PD-1 but not to other CD28 family members. PD-L1, a ligand of PD-1, is abundant in various human cancers. The interaction between PD-1 and PD-L1 leads to a reduction in tumor-infiltrating lymphocytes, T cell receptor-mediated proliferation, and immune evasion by cancer cells. Inhibition of the local interaction between PD-1 and PD-L1 can reverse immunosuppression, and when…When the interaction between PD-1 and PD-L2 is blocked simultaneously, the effects are additive. PD-1 has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes, and rheumatoid arthritis.

[0005] In a first aspect, this disclosure relates to an isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 21, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11 or SEQ ID NO: 20; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0006] In a second aspect, this disclosure relates to an isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 5 or SEQ ID NO: 22; and a light chain variable region (VL) comprising SEQ ID NO: 6.

[0007] In another aspect, this disclosure relates to an isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain complementarity-determining region 1 (VHCDR1), a heavy chain complementarity-determining region 2 (VHCDR2), and / or a heavy chain complementarity-determining region 3 (VHCDR3) of or derived from the heavy chain variable region amino acid sequence listed in SEQ ID NO: 5 (e.g., determined, derived, or copied therefrom); and a light chain complementarity-determining region 1 (VLCDR1), a light chain complementarity-determining region 2 (VLCDR2), and / or a light chain complementarity-determining region 3 (VLCDR3) of (e.g., determined, derived, or copied therefrom) the light chain variable region amino acid sequence listed in SEQ ID NO: 6.

[0008] Other aspects include the fact that the isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1 further comprises a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7; andThe HCCR optionally includes one, two, or three conserved amino acid substitutions.

[0009] An additional aspect includes the isolated antibody or antigen-binding portion thereof of aspect 2, further comprising a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8; and wherein the LCCR optionally includes one, two, or three conserved amino acid substitutions.

[0010] Other aspects of aspect 2 include the isolated antibody or antigen-binding portion thereof of aspect 2, further comprising a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally includes one, two, or three conserved amino acid substitutions; and wherein the isolated antibody or antigen-binding portion thereof further comprises a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally includes one, two, or three conserved amino acid substitutions.

[0011] In some aspects, the VH of the antibody or antigen-binding moiety further comprises a heavy chain signal peptide (HCSP) at the N-terminus of the VH, the heavy chain signal peptide comprising the amino acids listed in SEQ ID NO: 15.

[0012] In another aspect, the antibody or antigen-binding moiety of the present disclosure comprises a VL, wherein the VL further comprises a light chain signal peptide (LCSP) at the N-terminus of the VL, the light chain signal peptide comprising the amino acids listed in SEQ ID NO: 16.

[0013] In one aspect, the present disclosure provides an antibody or antigen-binding moiety of human form or humanized from a different species, and may be IgG1, IgG2, IgG3, or IgG4.

[0014] In another aspect, the present disclosure includes a pharmaceutical composition for treating, inhibiting, and / or suppressing solid tumors, comprising (i) an antibody or antigen-binding moiety disclosed herein; and (ii) a pharmaceutically acceptable carrier.

[0015] In another aspect, a polynucleotide molecule encoding the antibody or antigen-binding moiety of this disclosure is considered and prepared, which in one embodiment may be an expression vector. In one embodiment, such an expression vector encodes a light chain signal peptide (LCSP) and / or a heavy chain signal peptide (HCSP), but does not contain introns. The expression vector can be used in host cells, such as Escherichia coli (E. coli).

[0016] In one aspect, this disclosure relates to a method of preventing, treating, and / or suppressing tumors in an individual, comprising administering an effective amount of an antibody or antigen-binding moiety to the individual. These tumors may include hepatocellular carcinoma, or solid tumors comprising melanoma and / or renal cell carcinoma (RCC).

[0017] In another aspect, this disclosure relates to regulating one or more T cells in an individual in vitro, ex vivo, and / or in vivo.A method for activating the activity of a T cell, the method comprising: contacting a T cell with an antibody or an antigen-binding portion thereof that preferentially binds to human programmed death receptor 1 (hPD-1), wherein the antibody blocks and / or modulates the binding of human PD-L1 and / or human PD-L2 to hPD-1, and wherein the antibody or the antigen-binding portion thereof comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 5 or SEQ ID NO: 22; and a light chain variable region (VL) comprising SEQ ID NO: 6. In another aspect, the antibody or its antigen-binding portion may further comprise a heavy chain constant region (HCCR), said heavy chain constant region comprising the amino acid sequence listed in SEQ ID NO: 7, page 2 / 54, CN 122270481 A, and said HCCR optionally comprising one, two or three conserved modified amino acid substitutions, and / or wherein the antibody or its antigen-binding portion further comprises a light chain constant region (LCCR), said light chain constant region comprising the amino acid sequence of SEQ ID NO: 8, and wherein said LCCR optionally comprises one, two or three conserved modified amino acid substitutions.

[0018] In another aspect, the methods disclosed herein can regulate T cells, such as one or more natural killer cells, one or more cytotoxic T cells, one or more helper T cells, one or more monocytes, one or more memory T cells and / or one or more regulatory T cells.

[0019] In one aspect, the antibody or antigen-binding moiety disclosed herein blocks human PD-L1 and human PD-L2 with an IC50 of about 1 nM or lower.

[0020] Another aspect of this disclosure includes a method for producing an antibody or antigen-binding moiety vector construct comprising culturing host cells containing an expression vector of interest under conditions allowing expression of the antibody or antigen-binding moiety construct; and purifying and / or recovering the resulting antibody or antigen-binding moiety vector construct from the culture.

[0021] In one aspect, an antibody or its antigen-binding moiety for treating solid tumors may optionally contain another therapeutic agent. Such optional agents may be P1101 and / or another anti-human PD1 monoclonal antibody for treating solid tumors (including, but not limited to, RCC and / or melanoma).

[0022] This disclosure includes combinations for treating, inhibiting, preventing, and / or suppressing solid tumors in an individual, wherein the combination comprises an antibody or an antigen-binding portion thereof; and another therapeutic agent, wherein the antibody or antigen-binding portion thereof comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 5 or SEQ ID NO: 22; and a light chain variable region (VL) comprising SEQ ID NO: 6.

[0023] Additionally, in the above combinations, the antibody or antigen-binding portion thereof further comprises a heavy chain constant region (HCCR).The heavy chain constant region comprises the amino acid sequence listed in SEQ ID NO: 7, and the HCCR optionally comprises one, two, or three conserved modified amino acid substitutions; and the antibody or its antigen-binding portion further comprises a light chain constant region (LCCR), the light chain constant region comprising the amino acid sequence of SEQ ID NO: 8, and the LCCR optionally comprises one, two, or three conserved modified amino acid substitutions.

[0024] In another aspect, this disclosure relates to a method for preventing, treating, and / or inhibiting solid tumors in an individual (e.g., a human), comprising administering effective amounts of P1101 and P1801 to the individual in a continuous or combined manner. The administration system can be in any continuous and / or sequential manner. In some aspects, P1101 is administered before P1801, or vice versa, and administration can be performed for various durations and combinations. The solid tumors treated include hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

[0025] In one embodiment, the present disclosure provides an isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 21, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11, SEQ ID NO: 20 or SEQ ID NO: 23; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0026] In another embodiment, this disclosure provides a segregated antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising one or more paratopes, said one or more paratopes comprising: at least one of the following residues: Phe2, Ser5, Ser6 and Tyr7 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 19;

[0027] at least one of the following residues: Phe2, Ser5 and Tyr6 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 21; at least one of the following residues: SEQ ID NO:Thr13, Gly14, Gly15, Gly16, Ser17, and Tyr18 of the heavy chain variable sequence (VHCDR2) of SEQ ID NO: 10; at least one of the following residues: Tyr33, Leu34, Asn35, Asp38, and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 11; or at least one of the following residues: Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 20.

[0028] In one embodiment, the present disclosure provides a segregated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising one or more complementary sites, said one or more complementary sites comprising: at least one of the following residues: Asn9 of the light chain variable sequence (VLCDR1) of SEQ ID NO: 12; at least one of the following residues: Tyr12 and Ser18 of the heavy chain variable sequence (VLCDR2) of SEQ ID NO: 13; or at least one of the following residues: Ser22, Asn23 and Trp25 of the heavy chain variable sequence (VLCDR3) of SEQ ID NO: 14.

[0029] Another embodiment of this disclosure provides a segregated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising one or more complementary sites, said one or more complementary sites comprising: at least one of the following residues: Phe2, Ser5, Ser6 and Tyr7 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 19; at least one of the following residues: Phe2, Ser5 and Tyr6 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 21; at least one of the following residues: Thr13, Gly14, Gly15, Gly16, Ser17 and Tyr18 of the heavy chain variable sequence (VHCDR2) of SEQ ID NO: 10; at least one of the following residues: Tyr33, Leu34, Asn35, Asp38 and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 11; at least one of the following residues: SEQ ID NO: Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 20; at least one of the following residues: Asn9 of the light chain variable sequence (VLCDR1) of SEQ ID NO: 12; at least one of the following residues: Tyr12 and Ser18 of the heavy chain variable sequence (VLCDR2) of SEQ ID NO: 13; or at least one of the following residues: Ser22, Asn23, and...Trp25.

[0030] In one embodiment, this disclosure provides an isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 9 and a light chain variable region (VL) comprising SEQ ID NO: 6; or a heavy chain variable region (VH) comprising SEQ ID NO: 18 and a light chain variable region (VL) comprising SEQ ID NO: 6; or a heavy chain variable region (VH) comprising SEQ ID NO: 19 and a light chain variable region (VL) comprising SEQ ID NO: 6; or a heavy chain variable region (VH) comprising SEQ ID NO: 21 and a light chain variable region (VL) comprising SEQ ID NO: 6.

[0031] In one embodiment, this disclosure provides a segregated antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 5 or a heavy chain variable region (VH) comprising SEQ ID NO: 22; and a light chain variable region (VL) comprising SEQ ID NO: 6.

[0032] In any embodiment including an antibody or antigen-binding moiety thereof, the antibody or antigen-binding moiety thereof may further comprise a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7; and wherein the HCCR optionally comprises one, two, or three conserved amino acid substitutions.

[0033] In any embodiment including an antibody or antigen-binding moiety thereof, the antibody or antigen-binding moiety thereof may further comprise a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8; and wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions.

[0034] In any of the embodiments comprising an antibody or its antigen-binding portion, the antibody or its antigen-binding portion may further comprise: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally comprises one, two, or three conserved amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions. Specification 4 / 54 pages 10 CN 122270481 A

[0035] In any of the embodiments comprising an antibody or its antigen-binding portion, the VH may further comprise a heavy chain signal peptide (HCSP) at the N-terminus of the VH, the heavy chain signal peptide comprising the amino acids listed in SEQ ID NO: 15.In any of the embodiments including an antibody or its antigen-binding portion, the VL may further include a light chain signal peptide (LCSP) at the N-terminus of the VL, the light chain signal peptide comprising the amino acids listed in SEQ ID NO: 16. In any of the embodiments including an antibody or its antigen-binding portion, the antibody may be a human or humanized antibody. In any of the embodiments including an antibody or its antigen-binding portion, the antibody may be an IgG4 isotype.

[0036] In one aspect, this disclosure provides a pharmaceutical composition for treating, inhibiting, and / or suppressing solid tumors, comprising (i) an antibody or its antigen-binding portion disclosed in any of the embodiments listed herein; and (ii) a pharmaceutically acceptable carrier.

[0037] In one embodiment, this disclosure provides a polynucleotide molecule encoding an antibody or antigen-binding portion in any of the embodiments listed herein.

[0038] In one embodiment, this disclosure provides an expression vector comprising a polynucleotide molecule encoding an antibody or antigen-binding moiety disclosed in any of the embodiments listed herein, wherein the expression vector encoding LCSP and / or HCSP does not contain introns. In one embodiment, this disclosure provides a host cell comprising the expression vector.

[0039] In one embodiment, this disclosure provides a method for preventing, treating, and / or suppressing a tumor in an individual, comprising the step of administering to the individual an effective amount of an antibody or antigen-binding moiety disclosed in any of the embodiments listed herein. The tumor includes hepatocellular carcinoma, or a solid tumor comprising melanoma and / or renal cell carcinoma (RCC).

[0040] In one embodiment, this disclosure provides a method for regulating the activity of one or more T cells of an individual in vitro, ex vivo, and / or in vivo, the method comprising the steps of: contacting the T cells with an antibody or its antigen-binding portion that preferentially binds to human programmed death receptor 1 (hPD-1), wherein the antibody blocks and / or regulates the binding of human PD-L1 and / or human PD-L2 to hPD-1, and wherein the antibody or its antigen-binding portion comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21; a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10; and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11, SEQ ID NO: 20, or SEQ ID NO: 23; and a light chain complementarity-determining region 1 comprising the amino acid sequence listed in SEQ ID NO: 12. (VLCDR1), including SEQ ID NO:The light chain complementarity-determining region 2 (VLCDR2) of the amino acid sequence listed in SEQ ID NO: 13, and / or the light chain complementarity-determining region 3 (VLCDR3) of the amino acid sequence listed in SEQ ID NO: 14.

[0041] In one embodiment of a method for regulating the activity of one or more T cells of an individual in vitro, ex vivo, and / or in vivo, the antibody or its antigen-binding portion further comprises a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, and the HCCR optionally comprises one, two, or three conserved amino acid substitutions. In the method, the antibody or its antigen-binding portion may further comprise a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions. In the method, the one or more T cells may comprise one or more natural killer cells, one or more cytotoxic T cells, one or more helper T cells, one or more monocytes, one or more memory T cells, and / or one or more regulatory T cells. In the method, the antibody or antigen-binding moiety may block the binding of human PD-L1 and human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

[0042] In one aspect, this disclosure provides a method for generating an antibody or antigen-binding moiety vector construct comprising culturing host cells disclosed in any of the examples listed herein (pages 5 / 54, CN 122270481 A) under conditions allowing expression of the antibody or antigen-binding moiety construct; and purifying and / or recovering the resulting antibody or antigen-binding moiety vector construct from the culture.

[0043] In another aspect, this disclosure provides an antibody or antigen-binding portion thereof for treating solid tumors, comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 5 or a heavy chain variable region (VH) comprising SEQ ID NO: 22; a light chain variable region (VL) comprising SEQ ID NO: 6; and optionally comprising another therapeutic agent. The antibody or antigen-binding portion thereof may further comprise: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally comprises one, two, or three conserved amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions; and optionally comprising another therapeutic agent. The other therapeutic agent may comprise P1101, P1801, and / or another anti-human PD1 monoclonal antibody. In the antibody or antigen-binding portion thereof for use, the solid tumor may comprise hepatocellular carcinoma,Renal cell carcinoma and / or melanoma.

[0044] In one embodiment, this disclosure provides a combination for treating, inhibiting, preventing, and / or suppressing a solid tumor in an individual, wherein the combination comprises an antibody or an antigen-binding portion thereof; and another therapeutic agent, wherein the antibody or the antigen-binding portion thereof comprises: a heavy chain variable region (VH) comprising the heavy chain variable region (VH) of SEQ ID NO: 5 or the heavy chain variable region (VH) comprising the heavy chain variable region (VL) comprising the light ... In the combination, the P1101, P1801, and / or another anti-human PD1 monoclonal antibody may be administered sequentially. In the combination, the P1101 and / or P1801 is administered prior to another anti-human PD1 monoclonal antibody. In the combination, the solid tumor may include hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

[0045] In one embodiment, this disclosure provides a recombinant protein comprising (i) an antibody or antigen-binding moiety disclosed herein; and (ii) an optional tag sequence for expression for detection and / or purification.

[0046] In another embodiment, any of the embodiments including an antibody or its antigen-binding moiety may be used in a method of treating cancer, wherein the method comprises administering an isolated antibody or its antigen-binding moiety to an individual. In one embodiment, the cancer comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

[0047] In one embodiment, any of the embodiments including an antibody or its antigen-binding moiety may be used to manufacture an agent for therapeutic and / or prophylactic treatment of cancer. In one embodiment, the cancer comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma. Brief Description of the Drawings

[0048] To more fully understand the features and advantages of this disclosure, reference is now made to the detailed description of this disclosure and the accompanying drawings, wherein:

[0049] Figures 1(a) to 1(r) represent the amino acid sequence of P1801. Figure 1(a) represents the complete heavy chain of P1801 comprising three CDRs and a variable domain plus a constant region. It also includes a heavy chain signal peptide that will eventually be cleaved by the host cell. The complete heavy chain sequence is represented as SEQ ID NO: 1. Figure 1(b) represents the complete light chain of P1801 comprising three CDRs and a variable domain plus a constant region.The chain also includes a light chain signal peptide that will eventually be cleaved by the host cell. The complete light chain sequence is represented by SEQ ID NO: 2. Figure 1(c) shows the heavy chain of P1801 without the heavy chain signal peptide (SEQ ID NO: 3). Figure 1(d) shows the light chain of P1801 without the light chain signal peptide (SEQ ID NO: 4). Figure 1(f) shows the complete heavy chain variable region of P1801 (SEQ ID NO: 5). Figure 1(g) shows the complete light chain variable region of P1801 (SEQ ID NO: 6). Figure 1(h) shows the heavy chain constant region of P1801 (SEQ ID NO: 7). Figure 1(i) shows the light chain constant region of P1801 (SEQ ID NO: 8). Figure 1(j) shows the heavy chain CDR1 of P1801 (SEQ ID NO: 9). Figure 1(k) shows the heavy chain CDR2 of P1801 (SEQ ID NO: 10). Figure 1(l) shows the heavy chain CDR3 of P1801 (SEQ ID NO: 11). Figure 1(m) shows the light chain CDR1 of P1801 (SEQ ID NO: 12). Figure 1(n) shows the light chain CDR2 of P1801 (SEQ ID NO: 13). Figure 1(o) shows the light chain CDR3 of P1801 (SEQ ID NO: 14). Figure 1(p) shows the heavy chain signal peptide of P1801 (SEQ ID NO: 15). Figure 1(q) shows the light chain signal peptide of P1801 (SEQ ID NO: 16). Figure 1(r) shows the hinge sequence located in the heavy chain (SEQ ID NO: 17). Figure 1(s) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 18). Figure 1(t) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 19). Figure 1(u) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 20). Figure 1(v) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 21). Figure 1(w) shows the alternative intact heavy chain variable region of P1801 (SEQ ID NO: 22). Figure 1(x) shows the alternative heavy chain CDR3 of P1801 (SEQ ID NO: 23).

[0050] Figure 2 shows the chemical structure of P1101. IFN represents human interferon α2b. mPEG represents a polyethylene glycol polymer, and each mPEG has a molecular weight of about 10 kD to 30 kD, and / or each mPEG has a molecular weight of about 20 kD.

[0051] Figure 3 shows the comparison of the selected rodent anti-hPD-1 mAb (mP1801) with commercially known anti-PD-1.Competitive ELISA of the interference ability of mAb.

[0052] Figures 4(a) to 4(c) illustrate different embodiments of administration of combinations of molecules of the present disclosure (e.g., P1101 and anti-PD1 monoclonal antibody, e.g., P1801). Solid and dashed arrows may be molecule 1, e.g., P1101; and molecule 2, e.g., anti-PD1 monoclonal antibody, or vice versa.

[0053] Figure 5 illustrates another different embodiment of administration of combinations of molecules of the present disclosure. For example, molecule 1 may be P1101 or anti-PD1 monoclonal antibody, e.g., P1801. Molecule 2 may be P1101 or anti-PD1 monoclonal antibody, e.g., P1801, or vice versa.

[0054] Figure 6 illustrates yet another different embodiment of administration of combinations of molecules disclosed in the present disclosure. For example, molecule 1 may be P1101 or anti-PD1 monoclonal antibody, e.g., P1801. Molecular 2 may be P1101 or an anti-PD1 monoclonal antibody, such as P1801.

[0055] Figure 7 illustrates another embodiment of the administration regimen of the combination of molecules disclosed in this disclosure. For example, Figure 7(a) shows that molecule 1 may be hIgG4 (as an isotope control), P1101, or an anti-PD1 monoclonal antibody, such as P1801, cimiplimab, pembrolizumab, or novolumab administered alone. Figure 7(b) shows an alternation pattern between molecules 1 and 2, wherein molecule 1 may be mP1101, molecule 2 may be P1801, and vice versa.

[0056] Figures 8(a) and 8(b) show the binding assay of P1801 compared to various commercially available anti-PD1 mAbs against the PD1 antigen. The Y-axis is wavelength, and the x-axis is time in seconds. As shown in Figure 8(a), the second line from the top is P1801 and is marked with an arrow. As shown in Figure 8(b), the third line from the top is P1801 and is marked with an arrow.

[0057] Figure 9 shows the binding specificity of P1801. P1801 specifically binds to PD-1, but not to PD-L1, PD-L2, CTLA4, or CD28 because they are flat and undetectable.

[0058] Figure 10 shows the average RCC tumor volume from the seven different groups described in the protocol of Example 9.

[0059] Figure 11 shows the actual RCC tumor measurement from Group 1 of Example 9.

[0060] Figure 12 shows the actual RCC tumor measurement from Group 6 of Example 9.

[0061] Figure 13 shows five different groups used for individuals to determine the effective dose of P1101 + P1801. Specification 7 / 54 pages 13 CN 122270481 A

[0062] Figure 14 shows the binding specificity of P1101 + P1801.The effective dose of P1801 is further optimized for the dose group in Figure 13. The top ring is for RCC and the bottom ring is for melanoma.

[0063] Figure 15 is another example of another group.

[0064] Figure 16 is an image generated by the final 3D reconstruction of PD1-AcroBio + PS 00066 + PS00067 at a nominal resolution of 2.9A using ChimeraX.

[0065] Figure 17 is an image of the final 2.9A map generated using ChimeraX, colored with local resolution values ​​calculated by cryoSPARC 4.4.

[0066] Figure 18 is a structural image of the P1801 / PD1 complex resolved by cryo-electron microscopy.

[0067] Figure 19 is a structural image of the P1801 / PL1 / PD1 complex resolved by cryo-electron microscopy, showing the overlap of P1801 / PD1 with PD-L1 / PD1.

[0068] Figure 20 is an image showing the epitope localization of the P1801 epitope.

[0069] Figure 21 is an image showing the epitope localization of the PD-L1 epitope.

[0070] Figure 22 is a model of the P1801 CDR at the PD-1 binding interface.

[0071] Figure 23 is a graph showing the binding of the epitope to the anti-PD-1 antibody.

[0072] Although various embodiments of the present disclosure are described in detail below, it should be understood that the present disclosure provides numerous applicable inventive concepts that can be practiced in a wide variety of specific situations. The specific embodiments discussed herein are for illustrative purposes only and do not limit the scope of the present disclosure.

[0073] To aid in the understanding of the present disclosure, several terms are defined below. The terms defined herein have meanings as commonly understood by those skilled in the art related to the present disclosure. For example, the terms “a / an” and “the” are not intended to refer only to singular entities, but rather to include general categories that can be used to describe specific instances. The terms used herein are used to describe specific embodiments of this disclosure, but their use does not limit this disclosure except as set forth in the claims.

[0074] i. Definitions

[0075] As used herein, the term “isolated molecule” refers to a molecule (where the molecule is, for example, a polypeptide, polynucleotide, or antibody) that, due to its origin or derivative, (1) does not associate with the naturally associated components that accompany it in its natural state; (2) is substantially free of other molecules from the same source (e.g., species, cells expressing it, libraries, etc.); (3) is expressed by cells from a different species; or (4) is not found in nature. Thus, a chemically synthesized molecule or expressed in a cell system different from the system of its natural origin will be “isolated” from its naturally associated components. Isolation can also render the molecule substantially free of its naturally associated components using purification techniques well known in the art. It can be determined by a variety of methods known in the art.Molecular purity or homogeneity. For example, the purity of a peptide sample can be determined using polyacrylamide gel electrophoresis and gel staining to visualize the peptide using techniques well-known in the art. For certain purposes, higher resolution can be provided by using HPLC or other well-known methods for purification in the art.

[0076] An “antibody” is an immunoglobulin molecule capable of specifically binding to targets such as carbohydrates, polynucleotides, lipids, peptides, etc., through at least one antigen recognition site located in a variable region of an immunoglobulin molecule. As used herein, the term covers not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen-binding portion thereof that competes for specific binding with an intact antibody, and any other modified configuration of an immunoglobulin molecule containing an antigen recognition site. Antigen-binding moieties include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAb, such as shark and camel antibodies), fragments including complementarity-determining regions (CDRs), single-chain variable fragment antibodies (scFv), macrobody, minibody, intrabody, diabody, triabody, tetrabody, v-NAR, and bi-scFv, as well as polypeptides containing at least a portion of an immunoglobulin sufficient to cause specific antigen binding. Antibodies include any class of antibodies, such as IgG, IgA, or IgM (or their subclasses), and antibodies need not be of any particular class. Immunoglobulins can be classified into different classes based on the antibody amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are known in the art.

[0077] The “variable region” of an antibody refers to the antibody light chain variable region or antibody heavy chain variable region, either alone or in combination. As is known in the art, both the heavy chain variable region and the light chain variable region consist of four frame regions (FRs), which are connected by three complementarity-determining regions (CDRs), also known as hypervariable regions, and the heavy chain variable region and the light chain variable region promote the formation of the antibody’s antigen-binding site. If a variant of the target variable region is needed, especially a variant with substitutions in amino acid residues outside the CDR region (i.e., within the frame region), then this can be achieved by associating the target variable region with a variant containing amino acid residues belonging to the same classical class as the target variable region.The appropriate amino acid substitutions, preferably conserved amino acid substitutions, are identified by comparing the variable regions of other antibodies with other CDR1 and CDR2 sequences.

[0078] In some embodiments, the CDR is clearly delineated and the residues containing the antibody binding site are identified by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. In some embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, various analytical methods can be used to identify or estimate the CDR region. In some embodiments, various analytical methods can be used to identify or estimate the CDR region. Examples of these methods include, but are not limited to, Kabat definition, Chothia definition, AbM definition, contact definition, and conformation definition. In one embodiment, the CDR of this disclosure is determined by the Kabat definition. In another embodiment, the CDR is determined by the Chothia definition.

[0079] As used herein, the “EU index” or “EU number” system is based on the sequence number of the first sequenced human IgG (EU antibody). Because the most commonly used reference for this convention is the Kabat sequence manual, the EU index is sometimes mistakenly confused with the Kabat index. The EU index does not provide for insertions and deletions, therefore, in some cases, comparisons of IgG positions between IgG subclasses and species may be ambiguous, especially in hinge regions. Nevertheless, this convention is sufficient for simple comparisons of Fc regions in many Fc structure-function studies.

[0080] As used herein, a “configurational definition” of a CDR refers to a CDR position that is identifiable as a residue contributing enthalpy to antigen binding.

[0081] As used herein, a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, the CDR can be defined according to Kabat's definition (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), IMGT's definition, Chothia's definition (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), Martin's definition, extended definition, and AbM definition (using Oxford Molecular Biology).The CDR is defined using any of the following methods: Molecular AbM antibody modeling software, contact definition, conformation definition, and / or any CDR determination method well-known in the field.

[0082] A CDR is generally considered to be a structured loop that participates in antigen binding and exhibits a highly variable amino acid composition. Different numbering schemes or different definitions of CDR length can be used to define CDRs based on antibody amino acid sequences. For example, the Kabat (and IMGT) CDR definition is based on sequence alignment, while the Chothia CDR definition better reflects the loop structure in the antibody 3D architecture. The Kabat numbering scheme is generally considered to be the widely adopted standard for antibody residue numbering. The Chothia numbering scheme is based on the alignment of antibody structures and ensures a better correspondence with structural loops. The Chothia numbering scheme has the following characteristics: topologically aligned residues from different antibodies are numbered at the same position, and the Chothia CDR definition corresponds to the structural antigen-binding loop in most antibody sequences. According to the nomenclature of Kabat and based on Chothia, there are some CDR positional shifts and / or deviations in the length of the loops defined on highly variable amino acids in the CDRs.

[0083] The Martin numbering scheme focuses on the structural alignment of different frame regions of unconventional length. The Martin numbering scheme highlights residues that are not present in most sequences and structures, thus defining these residues as deletion sites and suggesting corrections for insertion points within frame region 3 of the heavy chain domain. The Martin numbering scheme uses the numbering software ABnum and provides a numbering scheme consisting of the Chothia numbering system corrected by ABnum software, which integrates sequences from the Kabat, IMGT, and PDB databases. The IMGT numbering scheme is a reference in immunogenetics and immunoinformatics and provides a standardized numbering system for all protein sequences of the immunoglobulin superfamily, including variable domains from antibody light and heavy chains and T cell receptor chains from different species. The IMGT numbering scheme is based on the amino acid sequence alignment of germline V genes. The IMGT numbering scheme covers all variable domains and various tools have been developed to analyze full-length sequences. Alternatively, any other novel method not mentioned in this disclosure may be used to determine the CDR.

[0084] As is known in the art, the “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0085] As used herein, a “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies constituting the population are identical except for the possible presence of a small number of naturally occurring mutations. Monoclonal antibodyHighly specific for a single antigenic site. Furthermore, in contrast to polyclonal antibody formulations that typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier "monoclonal" indicates that the antibody is obtained from a largely homogeneous population of antibodies and should not be construed as requiring any special method to produce the antibody. For example, the monoclonal antibodies used according to this disclosure can be prepared by the hybridoma method first described in Kohler and Milstein, 1975, Nature 256:495, or by the recombinant DNA method described, for example, in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries produced using techniques known in the art.

[0086] As used herein, a “humanized” antibody refers to a form of non-human (e.g., murine) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of an antibody) containing a minimal sequence derived from a non-human immunoglobulin. A humanized antibody is a human immunoglobulin (recipient antibody) in which residues of a recipient’s CDR are replaced by residues of a CDR from a non-human species (donor antibody) having the desired specificity, affinity, and ability. Humanized antibodies may contain residues that are not present in the recipient antibody or in the introduced CDR or frame sequence, but may include such residues to further improve and optimize antibody performance.

[0087] Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” all refer to both of the following: (1) a treatment measure that cures, alleviates, or reduces the symptoms of a diagnosed pathological condition or disease and / or prevents its progression, and / or (2) a preventive or preventative measure that prevents and / or alleviates the development of a targeted pathological condition or disease. Therefore, those who need treatment include: people who already have the disease; people who are susceptible to the disease; and people who need to prevent the disease. In some aspects, according to the methods of this disclosure, if a patient shows, for example, overall, partial, or temporary remission of a certain type of cancer, then the individual's cancer is successfully “treated”.

[0088] A “human antibody” is an antibody whose amino acid sequence corresponds to the amino acid sequence of a human-produced antibody and / or is prepared using any of the human antibody preparation techniques disclosed herein. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues (see page 10 / 54 of CN 122270481 A).

[0089] The term "chimeric antibody" refers to an antibody whose variable region sequence originates from one species and whose constant region sequence originates from another species, such as an antibody whose variable region sequence originates from a mouse antibody and whose constant region sequence originates from a human antibody.

[0090] As used herein, the terms “inhibition” and / or “modulation” of PDL1 and / or PDL2 activity refer to the ability of an anti-PD1 binding molecule (e.g., an antibody or its antigen-binding fragment) to bind to PD1, and subsequently, in some aspects, to inhibit PDL1 and / or PDL2 activity by reducing it by at least about 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or 100%.

[0091] The term “epitope” refers to a portion of a molecule that can be recognized by an antibody and bind to one or more of its antigen-binding regions. Epitopes are typically composed of surface clusters of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. In some embodiments, an epitope may be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all interaction sites between the protein and the interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the protein. A “nonlinear epitope” or “conformational epitope” comprises a discontinuous polypeptide (or amino acid) within an antigen protein to which an antibody specific for the epitope binds.

[0092] As used herein, the term “antigenic epitope” is defined as a portion of an antigen that can specifically bind to an antibody, which can be determined by any method well known in the art (e.g., by routine immunoassay). Once the desired epitope on the antigen is determined, an antibody against that epitope can be generated. Alternatively, during the exploration process, the generation and characterization of antibodies can elucidate information about the desired epitope. Antibodies that bind to the same epitope can be competitively screened. This is achieved by conducting competitive and cross-competitive studies to identify antibodies that compete or cross-competitively bind to PD-1, for example, antibodies that compete to bind to the antigen.

[0093] The antibody or its antigen-binding fragment contains "complementary" and "non-complementary" amino acid residues. The "complementary" amino acid of the antibody or its antigen-binding fragment may have a nucleus, for example, within about 4 Å of the nucleus of an antibody or its antigen-binding fragment that preferentially binds to the PD-1 isoform. Since each isoform forms a structurally different complex with the antibody or its antigen-binding fragment, the complementary residues of each isoform may be different. "Antibodies or their antigen-binding fragments that preferentially bind to the PD-1 complementary residues..."The term "complementary part" may have a nucleus within approximately 4 Å of the nucleus of a human antibody or antigen-binding part that preferentially binds to PD-1. An antibody or antigen-binding part that preferentially binds to PD-1 complementary residues may have a nucleus within approximately 4 Å of the nucleus of any isoform.

[0094] Residues may be named complementary residues regardless of their position within the CDR or FW (framework) region, as defined by Kabat nomenclature. For example, many complementary residues are located within the CDR region, as shown in Table 15. However, some complementary residues are located within the FW region. A "non-complementary" amino acid is any amino acid of the antibody or its antigen-binding fragment that is not a "complementary" amino acid, regardless of whether the residue is located within the CDR or FW region.

[0095] The terms "programmed cell death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PD1," and "PD-1" are used interchangeably. “PDCD1,” “hPD-1,” and “hPD-I” are all used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs having at least one epitope shared with PD-1. The complete PD-1 sequence is available at GenBank accession number U64863 and is incorporated herein by reference.

[0096] The term “agonist” refers to a substance that promotes (i.e., induces, induces, enhances, or increases) the biological activity or action of another molecule. The term agonist encompasses substances that bind to receptors (e.g., antibodies) and substances that promote receptor function without binding to them (e.g., by activating related proteins, as described on page 11 / 54 of the specification, CN 122270481 A).

[0097] The terms “antagonist” or “inhibitor” refer to substances that prevent, block, inhibit, neutralize, or reduce the biological activity or action of another molecule (e.g., a receptor).

[0098] As used herein, “anti-PD-1 antibody” refers to an antibody capable of inhibiting PD-1 biological activity and / or one or more downstream events mediated by PD-1. Anti-PD-1 antibody encompasses antibodies that block, antagonize, inhibit, or reduce (to any extent, including significantly) PD-1 biological activity, including PD-1-mediated downstream events such as PD-L1 binding and downstream signaling, PD-L2 binding and downstream signaling, T cell proliferation inhibition, T cell activation inhibition, IFN secretion inhibition, IL-2 secretion inhibition, TNF secretion inhibition, IL-10 induction, and suppression of anti-tumor immune responses. For the purposes of this disclosure, it should be understood that the term “anti-PD-1 antibody” (interchangeably referred to as “PD-1 antibody”) encompasses all previously identified terms, names, and functional states and properties that substantially invalidate, reduce, or neutralize PD-1 itself, PD-1 biological activity, or the results of such biological activity to any extent. In some embodiments, anti-PD-1 antibodies bind to PD-1 and upregulate anti-tumor immune responses. This document provides anti-PD-1 antibodies...Examples of PD-1 antibodies include P1801. In one embodiment, P1801 is first produced in a mouse form and subsequently humanized for human use. In some embodiments, known forms of mouse anti-PD1 monoclonal antibodies, RMP 1-14, can be used to demonstrate efficacy in mouse models.

[0099] The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” are used interchangeably herein to refer to an amino acid chain of any length. The chain may be linear or branched and may contain modified amino acids and / or may be interspersed with non-amino acids. The term also covers amino acid chains that have been modified naturally or by intervention; said intervention is, for example, the formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as binding to a labeled component. This definition also includes, for example, one or more analogs of amino acids (including, for example, non-natural amino acids) and other modified polypeptides known in the art. It should be understood that polypeptides may exist as a single chain or related chains.

[0100] As is known in the art, the terms “polynucleotide” or “nucleic acid” as used interchangeably herein refer to a nucleotide chain of any length and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerases. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and analogs thereof. If modifications are present, the nucleotide structure may be modified before or after chain assembly. The nucleotide sequence may be interspersed with non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by binding to a labeled component. Other types of modifications include, for example, "caps"; substitution of one or more naturally occurring nucleotides with analogs; internucleotide modifications, such as those with non-electrolyte bonds (e.g., methyl phosphonate, triphosphate, aminophosphate, carbamate, etc.) and those with charged bonds (e.g., thiophosphate, dithiophosphate, etc.); modifications containing side groups such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); modifications containing intercalating agents (e.g., acridine, psoralen, etc.); modifications containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.); modifications containing alkylating agents; modifications with modified bonds (e.g., α-mutated isomers of nucleic acids); and unmodified forms of polynucleotides. Furthermore, any hydroxyl group generally present in sugars can be, for example, replaced by a phosphonate group or a phosphate group, protected by a standard protecting group, or activated to prepare for forming additional bonds with other nucleotides, or can be bound to a solid carrier. The 5' and 3' OH groups can be phosphorylated or partially substituted with amines or organic end-capping groups of 1 to 20 carbon atoms. Other hydroxyl groups can also be derived as standard protecting groups. Polynucleotides may also contain substances generally known in the field.Similar forms of ribose or deoxyribose include, for example, 2'-O-methylribose, 2'-O-allylribose, 2'-fluororibose or 2'-azidoribose, carbocyclic sugar analogs, α-mutantose or β-mutantose, epimeric sugars (e.g., arabinose, xylose or lyxose); pyranose, furanose, sedoheptulose, acyclic analogs and non-basic nucleoside analogs (e.g., methylriboside). One or more phosphodiester specifications 12 / 54 pages 18 CN 122270481 A The bond may be replaced by an alternative linking group. These alternative linker groups include, but are not limited to, embodiments in which the phosphate ester group is replaced by P(O)S (“thioester”), P(S)S (“dithioester”), (O)NR2 (“amide ester”), P(O)R, P(O)OR', CO, or CH2 (“methyl acetal”), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1 to 20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aromatic aldehyde group optionally containing an ether (-O-) bond. Not all bonds in a polynucleotide need to be uniform. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0101] Antibodies that “preferentially bind” or “specifically bind” (used interchangeably herein) to an epitope are terms understood in the art, and methods for determining such specific or preferential binding are also known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if its reaction or association with a specific cell or substance is more frequent, faster, longer-lasting, and / or has a greater affinity than its association with alternative cells or substances. An antibody is said to "specifically bind" or "preferentially bind" to a target if its binding to a target has a greater affinity, is easier, and / or lasts longer than its binding to other substances. For example, an antibody that specifically or preferentially binds to a PD-1 epitope is an antibody that binds to that epitope with a greater affinity, is easier, and / or lasts longer than its binding to other PD-1 epitopes or non-PD-1 epitopes. Reading this definition should also help to understand that, for example, an antibody (or part or epitope) that specifically or preferentially binds to a first target may specifically or preferentially bind to a second target, or may not specifically or preferentially bind to a second target. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, the mention of binding refers to preferential binding.

[0102] As used herein, “substantially pure” means a material that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0103] "Host cell" includes individual cells or cell cultures that may be, or have been, a recipient of a vector for incorporating polynucleotide intercalations. Host cell includes progeny of a single host cell, and progeny may not necessarily be identical to the original parent cell (in terms of morphology or genomic DNA complement) due to natural, accidental, or intentional mutations. Host cell includes cells in vivo transfected with one or more polynucleotides disclosed herein. An example is *Escherichia coli*.

[0104] Unless otherwise indicated, as used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range and, where appropriate, to include fractions (e.g., tenths and hundredths of an integer).

[0105] As used in the art, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. Preferably, the FcR is a naturally occurring human FcR sequence. Furthermore, preferred FcRs are FcRs (γ receptors) that bind IgG antibodies and include receptors of the FcγRI, FcγRII, and FcγRIIII subclasses, including allelic variants and alternating splice forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”), both with similar amino acid sequences, differing primarily in their cytoplasmic domains. FcRs are reviewed and known in the field. “FcR” also includes neonatal receptors FcRn responsible for transferring maternal IgG to the fetus.

[0106] As used herein with respect to antibodies, the term “competition” means that the binding of a first antibody or its antigen-binding moiety to an epitope is sufficiently similar to the binding of a second antibody or its antigen-binding moiety to such that the binding of the first antibody to its homologous epitope in the presence of the second antibody results in a detectable reduction compared to the binding of the first antibody in the absence of the second antibody. Another scenario is that the binding of the second antibody to its epitope may be detectably reduced in the presence of the first antibody, but this is not always the case. That is, the first antibody may inhibit the binding of the second antibody to its epitope, while the second antibody may not inhibit the binding of the first antibody to its respective epitope. However, when each antibody can detectably inhibit the binding of another antibody to its homologous epitope or ligand, regardless of the degree of inhibition being the same, greater, or less, these antibodies are said to "cross-compete" with each other for binding to their respective epitopes. This disclosure covers both competitive and cross-compete antibodies. Regardless of the mechanism by which such competition or cross-compete occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or a portion thereof), those skilled in the art will understand, based on the teachings provided herein, that such competitive and / or cross-compete antibodies are covered and applicable to the methods disclosed herein.

[0107] The “functional Fc region” possesses at least one effector function of the natural sequence Fc region. Exemplary “effector functions” include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions typically require the combination of the Fc region with a binding domain (e.g., antibody variable domain), and such antibody effector functions can be assessed using various assays known in the art.

[0108] The “natural sequence Fc region” contains an amino acid sequence consistent with the amino acid sequence of Fc regions found in nature. The “variant Fc region” contains an amino acid sequence that differs from the amino acid sequence of the natural sequence Fc region due to at least one amino acid modification, but retains at least one effector function of the natural sequence Fc region. Preferably, compared to the natural sequence Fc region or the Fc region of the parent peptide, the variant Fc region has at least one amino acid substitution in the natural sequence Fc region or the Fc region of the parent peptide, for example, about one to about ten amino acid substitutions, or about one to about five amino acid substitutions. The variant Fc region described herein has at least about 80% sequence identity with the natural sequence Fc region and / or the Fc region of the parent polypeptide, at least about 90% sequence identity with it, and at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least 99.5% sequence identity with it.

[0109] As used herein, the term “dose” refers to the amount of one or more active ingredients intended to be administered to an individual each time. The term “dosage” sometimes refers to the plural form of the term dose. Each dose and dosage may contain a constant and / or variable amount of one or more active ingredients, and the amount of active ingredient in each dose or dosage may vary, as appropriate. A unit dose or dosage may be in the form of tablets, capsules, pouches, or liquids. For example, a dose (e.g., in the form of one or two separate tablets) may be 0.5 mg of compound A, or may include 0.5 mg of compound A and 0.1 mg of compound B. As appropriate, dosage and amount may be used interchangeably.

[0110] As used herein, an “effective amount,” “effective dose,” or “effective quantity” of a drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired outcomes. In a more specific aspect, an effective quantity prevents, alleviates, or improves symptoms of a disease, and / or prolongs the survival of an individual undergoing treatment. For preventative use, beneficial or desired outcomes include, but are not limited to: eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including the disease, its complications, and biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes that occur during disease development. For therapeutic use, beneficial or desired outcomes include, but are not limited to, clinical outcomes such as: reducing disease (e.g., cancer, including, but...)Effective dosage (not limited to solid tumors) can reduce the dosage of other agents required to treat the disease, enhance the effect of another drug treatment, and / or delay the progression of the patient's cancer, for one or more symptoms. An effective dosage may be administered in one or more doses. For the purposes of this disclosure, an effective dosage of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventative or therapeutic treatment. As understood in clinical contexts, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved when combined with another drug, compound, or pharmaceutical composition. Therefore, "effective dosage" or "effective amount" can be considered in the context of administering one or more therapeutic agents, and if the desired result is achieved or realized when combined with one or more other agents, then the single agent can be considered to be administered in an effective amount.

[0111] "Individual" or "subject" refers to a mammal, with a non-limiting example being a human. Mammals also include, but are not limited to, farm animals (e.g., cows, pigs, horses, chickens, etc.), racing animals, pets, primates, horses, dogs, cats, mice, and rats.

[0112] As used herein, a “vector” or “expression vector” means a construct capable of delivering and expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, particles, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0113] As used herein, an “expression control sequence” is a nucleic acid sequence that directs the transcription of nucleic acids. The expression control sequence may be a promoter, such as a constitutive or inducible promoter; or an enhancer. The expression control sequence is operatively linked to the nucleic acid sequence to be transcribed.

[0114] As used herein, a “pharmaceutically acceptable carrier” or “pharmaceuticalally acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity or stability and does not react with an individual’s immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate-buffered saline solutions, water, emulsions (e.g., oil / water emulsions), and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline (PBS) or physiological saline (0.9%). Compositions containing these carriers are formulated using known conventional methods.

[0115] As used herein, the term “kon” refers to the rate constant of antibody association to antigen. Specifically, the rate constants (kon and koff) and equilibrium dissociation constant are measured using full-length antibodies and / or Fab antibody fragments (i.e., monovalent) and PD-1.

[0116] As used herein, the term “koff” refers to the rate constant of antibody dissociation from the antibody / antigen complex.

[0117] The term “immunomodulator” refers to a substance capable of altering (e.g., inhibiting, reducing, enhancing, strengthening, or stimulating) an immune response (as defined herein) or processing any component of the innate, humoral, or cellular immune system of a host mammal. Thus, the term “immunomodulator” encompasses “immune effector cell enhancers” and “immunosuppressor cell inhibitors” as defined herein, as well as substances that affect other components of the mammalian immune system.

[0118] The term “immune response” refers to any detectable response of the host mammalian immune system to a specific substance (e.g., an antigen or immunogen), such as an innate immune response (e.g., activation of the dopamine receptor signaling cascade), a cell-mediated immune response (e.g., a response mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and a humoral immune response (e.g., a response mediated by B cells, such as the production and secretion of antibodies into plasma, lymph, and / or tissue fluid).

[0119] The term “immunogenicity” refers to the ability of a substance to elicit, trigger, stimulate, or induce an immune response or to improve, enhance, increase, or prolong a pre-existing immune response against a specific antigen, whether alone or in connection with a carrier, in the presence or absence of an adjuvant.

[0120] The term “immunosuppressive cell inhibitor” or “ISC inhibitor” refers to a substance capable of reducing or inhibiting the number or function of immunosuppressive cells in mammals. Examples of immunosuppressive cells include regulatory T cells (“Treg”), bone marrow-derived suppressor cells, and tumor-associated macrophages.

[0121] In the case of administration of a substance to mammals, including humans, the terms “intradermal administration,” “id,” or “administered intradermally” refer to the delivery of a substance to the dermis of the skin of a mammal. The skin of a mammal consists of the epidermis, dermis, and subcutaneous layer. The epidermis is the outermost layer of the skin. The dermis (which is the middle layer of the skin) contains nerve endings, sweat glands and sebaceous glands, hair follicles, and blood vessels. The subcutaneous layer consists of fat and connective tissue that houses larger blood vessels and nerves. In contrast to intradermal application, "subcutaneous application" refers to the application of a substance into the subcutaneous layer, and "local application" refers to the application of a substance onto the skin surface.

[0122] The term "preventing" or "prevent" means (a) preventing the onset of a disease, or (b) delaying the onset of a disease or the onset of its symptoms.

[0123] The term "tumor-associated antigen" or "TAA" refers to an antigen that is specifically expressed by tumor cells or that is expressed by tumor cells at a higher frequency or density compared to non-tumor cells of the same tissue type. Tumor-associated antigens may be antigens that are not normally expressed by the host; they may be mutations, truncations, misfoldings, or other aberrant expressions of molecules that are normally expressed by the host; they may be...The same as normally expressed molecules, but expressed at abnormally high levels; or it may be expressed under abnormal conditions or environments. Tumor Phase Specification 15 / 54 Page 21 CN 122270481 A The related antigen may be, for example, a protein or protein fragment, a complex carbohydrate, a ganglioside, a hapten, a nucleic acid, or any combination of these or other biomolecules.

[0124] The term “vaccine” refers to an immunogenic composition intended for administration to mammals to elicit an immune response against a specific antigen in mammals. Vaccines typically contain agents (known as “antigens” or “immunogens”) that are similar to or derived from targets of the immune response, such as pathogenic microorganisms or tumor cells. Vaccines intended for the treatment of tumors (e.g., cancer) typically contain antigens derived from TAAs found on the target tumor and are capable of inducing immunogenicity against TAAs on the target tumor.

[0125] The term “vaccine-based immunotherapy regimen” refers to a treatment regimen in which a vaccine is administered in combination with one or more immunomodulators. The vaccine and immunomodulators may be administered together in a single formulation or separately.

[0126] As used herein, the term “antigen-binding portion” (or simply “antibody portion”) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-1). Antigen-binding function of antibodies has been shown to be performed by fragments of full-length antibodies. Examples of binding fragments covered within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a divalent fragment comprising two Fab fragments linked by disulfide bonds in a hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) a dAb fragment consisting of a VH domain; and (vi) a separated complementarity-determining region (CDR). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be conjugated using recombinant methods by making them capable of being produced as a single protein chain via synthetic linkers, wherein the VL region pairs with the VH region to form a monovalent molecule (referred to as a single-chain Fv (scFv)). These single-chain antibodies are also intended to be covered within the term "antigen-binding portion" of the antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for utility in the same manner as intact antibodies.

[0127] As used herein, the term "human antibody" is intended to include antibodies in which both the framework region and the CDR region in the variable region are derived from human immunoglobulin sequences. Furthermore, if the antibody contains a constant region, then the constant region is also derived from a human immunoglobulin sequence. Human antibodies of this disclosure may include amino acid residues not encoded by human immunoglobulin sequences (e.g.,(Mutations introduced through random or site-directed mutagenesis in vitro, or through somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., a mouse) has been transplanted onto a human frame sequence.

[0128] The term "human monoclonal antibody" refers to an antibody exhibiting single binding specificity in which both the frame region and the CDR region in the variable region are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising B cells obtained from a transgenic nonhuman animal (e.g., a transgenic mouse) fused with immortalized cells, the transgenic nonhuman animal having a genome containing human heavy chain transgenes and light chain transgenes.

[0129] As used herein, the terms “humanized antibody” or “recombinant human antibody” include all human antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as (a) antibodies isolated from transgenic or transchromosomally transgenic animals (e.g., mice) or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transgenic to express human antibodies, such as from transfected tumors; (c) antibodies isolated from recombinant, combined human antibody libraries; and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. These recombinant human antibodies have variable regions in which the frame region and CDR region are derived from human germline immunoglobulin sequences. However, in some embodiments, these recombinant human antibodies may be mutagenized in vitro (or in vivo somatic cell mutagenized when using transgenic animals with human Ig sequences), so the amino acid sequences of the VH and VL regions of the recombinant antibody, although derived from and associated with human germline VH and VL sequences, may not be naturally present in the in vivo human antibody pedigree specification, page 16 / 54, 22 CN 122270481 A.

[0130] As used herein, “isotype” refers to an antibody class (e.g., IgM or IgG1) encoded by a heavy chain constant region gene.

[0131] The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably herein with the term “antibody that specifically binds to an antigen.”

[0132] The term “human antibody derivative” refers to any modified form of a human antibody, such as an antibody conjugate with another agent or antibody.

[0133] The term "chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, for example, an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody.

[0134] As used herein, the terms "Kassoc" or "Ka" refer to the association rate of a particular antibody-antigen interaction, and as...As used herein, the terms “Kdis” or “Kd” refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term “KD” refers to the dissociation constant, which is obtained by the ratio of Kd to KA (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods recognized in the art. Methods for determining the KD of an antibody include the use of surface plasmon resonance, for example, using a biosensor system such as the Biacore (trademark) system.

[0135] As used herein, the term “high affinity” for an IgG antibody means that the antibody has a KD of about 10⁻⁸ M or less, about 10⁻⁹ M or less, or about 10⁻¹⁰ M or less for the target antigen. However, “high affinity” binding can vary for other antibody isotypes. For example, “high affinity” binding for an IgM isotype means having a KD of about 10⁻⁷ M or less, about 10⁻⁸ M or less, or about 10⁻⁹ M or less.

[0136] As used herein, “about” means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, the range of error depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” may mean within one or more standard deviations. Alternatively, “about” may mean a range of up to ±5%, ±10%, ±15%, or ±20%. Furthermore, particularly in biological systems or processes, the term may mean up to one order of magnitude or up to ±3 times the value. When a particular value is provided in this application and the claims thereof, the meaning of “about” should be assumed to be within an acceptable range of error for that particular value, unless otherwise stated. References herein to “about” a value or parameter include (and describe) embodiments of that value or parameter itself. For example, a description of “about X” includes a description of “X”. Numerical ranges include numerical values ​​that define the range. Alternatively or additionally, throughout this disclosure, values ​​expressed in range format should be interpreted flexibly, including not only the numerical values ​​whose boundaries are explicitly listed, but also all individual numerical values ​​or subranges covered within that range, as if each numerical value and subrange were explicitly listed. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted as including not only about 0.1% to about 5%, but also the individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range, and such figures may be accurate to two decimal places. Unless otherwise indicated, the expression “about X to Y” has the same meaning as “about X to about Y”. Similarly, unless otherwise indicated, the expression “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z”.

[0137] As used herein, the term "conserved sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing an amino acid sequence. These conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this disclosure using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitution is the substitution of an amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include the following amino acids: those with basic side chains (e.g., lysine, arginine, histidine), those with acidic side chains (e.g., aspartic acid, glutamic acid), those with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), those with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), those with β-branched side chains (e.g., threonine, valine, isoleucine), and those with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibodies disclosed herein may be substituted with other amino acid residues from the same side chain family, and the altered antibodies may be tested using the functional assays described herein to determine the retained function (i.e., the functions shown in (c) to (I) above).

[0138] The term “treatment” or “therapeutic approach” means the application of an active pharmaceutical agent for the purpose of curing, healing, slowing, alleviating, altering, remedying, reducing, improving, or affecting a symptom (e.g., a disease), a symptom of a symptom, or preventing or delaying the onset of a symptom, complications, a biochemical marker of a disease, or otherwise statistically significantly inhibiting or suppressing the further development of a disease, symptom, or condition.

[0139] As used herein, an “adverse event” (AE) is any unfavorable and generally unexpected, or even undesirable, sign (including abnormal laboratory test results), symptom, or disease associated with the use of a medical treatment. For example, an adverse event may be associated with activation of the immune system in response to treatment or expansion of immune system cells (e.g., T cells). Medical treatment may result in one or more associated AEs, and each AE may have the same or different severity. A reference to a method capable of “modifying an adverse event” refers to a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0140] As used herein, the term "drug" refers to any compound having the desired biological activity and reactive functional groups that can be used to prepare the conjugates of this disclosure. Biological activity includes activities for diagnosing, curing, alleviating, treating, or preventing diseases in humans or other animals. Therefore, compounds covered by the term "drug" include those having the desired reactive functional groups.Drugs identified in official national pharmacopoeias and, for example, the U.S. Homeopathic Pharmacopeia or the National Formulary or any of their supplements. Exemplary drugs are described in the Physician's Desk Reference (PDR) and the Orange Book maintained by the U.S. Food and Drug Administration (FDA).

[0141] As used herein, the term "biologic drug" means any compound or protein having the desired biological activity and a reactive functional group that can be used to prepare the conjugates of this disclosure. Biological activity includes activity for the diagnosis, cure, amelioration, treatment, or prevention of disease in humans or other animals. Thus, compounds or proteins referred to by the term "biologic drug" include drugs identified in official national pharmacopoeias and, for example, the Purple Book maintained by the U.S. Food and Drug Administration (FDA), provided they have the desired reactive functional group.

[0142] As used herein, the term HCC refers to hepatocellular carcinoma, a cancer that originates in the liver. It differs from "secondary" liver cancer, which is the spread of cancer from other organs to the liver.

[0143] The term "cytotoxic agent" refers to a substance that inhibits or blocks cell expression activity, cell function, and / or causes cell damage. The term includes radioactive isotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzyme-active toxins (including fragments and / or variants) derived from bacteria, fungi, plants, or animals. Examples of cytotoxic agents include, but are not limited to: auristatin (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, metotanol, ricin, ricin A chain, cobustatin, dokamicin, dorastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, CC1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxyanthracnose.Diketone, Actinomycin, Diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, Abrin, Abrin A chain, Modeccin. (Instructions for use 18 / 54 pages, 24 CN 122270481) AA chain, α-Sarcina, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crocotin, calicheamicin, Sapaonaria officinalis inhibitor. Inhibitors), glucocorticoids and other chemotherapeutic agents, and radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or Bi213, P32 and Lu (including Lu177). Antibodies can also bind to an anticancer prodrug activating enzyme that converts the prodrug into its active form.

[0144] Figure 1(a) shows the complete heavy chain of P1801 comprising three CDRs and a variable domain plus a constant region. It also includes a heavy chain signal peptide that will eventually be cleaved by the host cell. The complete heavy chain sequence is represented as SEQ ID NO: 1. Figure 1(b) shows the complete light chain of P1801 comprising three CDRs and a variable domain plus a constant region. It also includes a light chain signal peptide that will eventually be cleaved by the host cell. The complete light chain sequence is represented as SEQ ID NO: 2. Figure 1(c) shows the heavy chain of P1801 without the heavy chain signal peptide (SEQ ID NO: 3). Figure 1(d) shows the light chain of P1801 without the light chain signal peptide (SEQ ID NO: 4). Figure 1(f) shows the complete heavy chain variable region of P1801 (SEQ ID NO: 5). Figure 1(g) shows the complete light chain variable region of P1801 (SEQ ID NO: 6). Figure 1(h) shows the heavy chain constant region of P1801 (SEQ ID NO: 7). Figure 1(i) shows the light chain constant region of P1801 (SEQ ID NO: 8). Figure 1(j) shows the heavy chain CDR1 of P1801 (SEQ ID NO: 9). Figure 1(k) shows the heavy chain CDR2 of P1801 (SEQ ID NO: 10). Figure 1(l) shows the heavy chain CDR3 of P1801 (SEQ ID NO: 11). Figure 1(m) shows the light chain CDR1 of P1801.(SEQ ID NO: 12). Figure 1(n) shows the light chain CDR2 of P1801 (SEQ ID NO: 13). Figure 1(o) shows the light chain CDR3 of P1801 (SEQ ID NO: 14). Figure 1(p) shows the heavy chain signal peptide of P1801 (SEQ ID NO: 15). Figure 1(q) shows the light chain signal peptide of P1801 (SEQ ID NO: 16). Figure 1(r) shows the hinge sequence located in the heavy chain (SEQ ID NO: 17). Figure 1(s) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 18). Figure 1(t) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 19). Figure 1(u) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 20). Figure 1(v) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 21). Figure 1(w) shows the alternative heavy chain CDR2 (SEQ ID NO: 10) of P1801. Figure 1(x) shows the alternative heavy chain CDR3 (SEQ ID NO: 23) of P1801.

[0145] As used herein, the term “P1801” refers to the anti-PD-1 monoclonal antibody of this disclosure shown in Figure 1. For example, detailed amino sequences of the heavy chain / light chain and all CDRs or alternative CDRs are shown in Figures 1(a) to 1(o) and Figures 1(a) to 1(o) plus Figure 1(s) (no signal peptide). Figure 1 shows a schematic of human P1801 having the alternative CDRs in Figure 1(s).

[0146] As used herein, the terms “P1101”, “Ropeg” and / or “pegylated human interferon α2b” are used interchangeably. P1101 is known in the art, for example, see U.S. Patent Nos. 8,143,214 B2, 8,273,343, 8,617,532 and / or 8,106,160 B2, the contents of which are incorporated herein by reference in their entirety. See also U.S. Patent Application Nos. 20170326206A1, 20220152156A1, 20220362343A1 and / or 20230057788A1, the contents of which are incorporated herein by reference in their entirety. In brief, for example, chemical formulas, methods of manufacture, and uses thereof are disclosed therein. For convenience, its structure is simply shown in Figure 2. More specifically, interferon is a form of P1101 that is functional in human individuals.

[0147] As used herein, the term “mP1101” refers to a structure with the same polyethylene glycolate and linker as P1101, except that the interferon is a mouse form that is functional in rodent individuals.

[0148] ii. Composition and Molecular Profile

[0149] Peptide Sequence

[0150] Figures 1(a) to 1(x) illustrate various amino acid sequence examples of P1801. Table 1 below shows the molecular characteristics of P1801.

[0151] Table 1 Specification 19 / 54 pages 25 CN 122270481 A

[0152]

[0153] In some embodiments, the antibodies of this disclosure comprise heavy chain variable regions from a specific germline heavy chain immunoglobulin gene and / or light chain variable regions from a specific germline light chain immunoglobulin gene.

[0154] In one embodiment, the nucleic acid encoding P1801 and the vector expressing these amino acids contain a nucleic acid molecule encoding a signal peptide (heavy chain SEQ ID NO: 15 and light chain SEQ ID NO: 16), wherein the signal peptide is located at the N-terminus of the heavy chain and / or light chain variable domains. Additionally, in one embodiment, the nucleic acid encoding the light chain signal peptide does not include any introns. These signal peptides can be expressed first in host cells, then lysed by the host cells, and subsequently secreted or released into a solution outside the host cells, thereby generating P1801.

[0155] Nucleic acid molecules encoding antibody P1801

[0156] Another aspect of this disclosure relates to nucleic acid molecules encoding antibodies of this disclosure. Nucleic acids may be present in whole cells, cell lysates, or in partially purified or substantially pure forms. Nucleic acids are “isolated” or “substantially pure” when purified by standard techniques (including alkali / SDS treatment, CsCl bundles, column chromatography, agarose gel electrophoresis, and other techniques well known in the art) and separated from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins). For example, DNA or RNA may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is an eDNA molecule.

[0157] The nucleic acids of this disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as further described below), the cDNA encoding the light and heavy chains of the hybridoma-prepared antibody can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding the antibody can be recovered from the library.

[0158] Once the DNA fragments encoding the VH and VL regions are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding another protein (e.g., antibody constant region or flexible linker). The term "operatively linked" as used in this context means that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain within the frame.

[0159] In one embodiment, isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably ligating the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art, and DNA fragments covering these regions can be obtained by standard PCR amplification. The heavy chain constant regions can be constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, especially the constant regions of IgG1 or IgG4. For Fab fragment heavy chain genes, the VH-encoding DNA is operably ligated to another DNA molecule encoding only the heavy chain CH1 constant region.

[0160] In one embodiment, isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably ligating the VL-encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art, and DNA fragments covering these regions can be obtained by standard PCR amplification. The light chain constant region may be a κ or λ constant region, or a κ constant region.

[0161] In order to generate the scFv gene, the DNA fragments encoding VH and VL are operatively linked to another fragment encoding a flexible adapter (e.g., encoding the amino acid sequence (Gly4-Ser)3), such that the VH and VL sequences can be expressed as adjacent single-stranded proteins, the VL and VH regions of which are joined by a flexible adapter in the specification 20 / 54 pages 26 CN 122270481 A.

[0162] iii. Preparation method

[0163] This disclosure also provides methods for generating, selecting and preparing anti-PD-1 antibodies. The antibodies of this disclosure may be prepared by procedures known in the art. In some embodiments, the antibodies may be prepared in a recombinant manner and expressed using any method known in the art.

[0164] Cloning

[0165] The cloning vector may be constructed according to known techniques or may be selected from a large number of cloning vectors available in the art. While the selected cloning vector may vary depending on the intended host cell, the final product cloning vector is generally self-replicating, has a single target of a specific restriction endonuclease, and / or carries a gene that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors (e.g., pSA3 and pAT28). These and many other commercially available cloning vectors are available from various vendors.

[0166] This disclosure further provides expression vectors. Expression vectors are generally polynucleotides contained according to this disclosure.A replicating polynucleotide construct. This implies that the expression vector, as a free genome or as part of the chromosomal DNA as a whole, must be reproducible in the host cell. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenovirus, adeno-associated virus, retrovirus), granules, and the expression vectors disclosed in PCT Publication WO 87 / 04462. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; and suitable transcriptional control elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational control elements are usually also required, such as ribosome binding sites, translation initiation sites, and stop codons.

[0167] A vector containing the polynucleotide of interest may be introduced into the host cell by any of a variety of suitable methods, including electroporation; transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microblast bombardment; liposome transfection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of vector or polynucleotide is determined by the characteristics of the host cell.

[0168] This disclosure also provides host cells comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding antibodies, peptides, or proteins of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., Escherichia coli or Bacillus subtilis) and yeasts (e.g., Saccharomyces cerevisiae, Saccharomyces pombe, or Kluyveromyces lactis). In one aspect, the level of cDNA expression in the host cell is about 5, 10, or 20 times higher than the expression level of the corresponding endogenous antibody or protein of interest (if present) in the host cell. In one embodiment, P1801 can be prepared using Chinese hamster ovary (CHO) DG44 cells, which are dhfr gene-deficient cell lines (dhfr-) derived from one of the CHO cell lineages.

[0169] Screening for host cells that specifically bind to PD-1 or the PD-1 domain is influenced by immunoassay or fluorescence activated cell sorting (FACS). Cells overexpressing the antibody or protein of interest can be identified.

[0170] Expression vectors can be used to guide the expression of anti-PD-1 antibodies. Those skilled in the art are familiar with the administration of expression vectors used to obtain the expression of exogenous proteins in vivo. See, for example, U.S. Patent Nos. 6,436,908 and 6,413,942.Nos. 6,376,471, all of which are incorporated herein by reference in their entirety. Administration of the expression vector includes local or systemic administration, including injection, oral administration, particle gun or catheter administration, and local administration. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglion, or to the coronary artery, atrium, ventricle, or pericardium. Specification 21 / 54 pages 27 CN 122270481 A

[0171] The vector can be transfected into host cells and stored in a Master Cell Bank. Typically, one or two Master Cell Bank (MCB) vials are removed from a liquid nitrogen container. After thawing at approximately 37°C, the MCB vials are transferred to 125 ml Tri-forest shaking flasks containing pre-warmed Dynamis alkaline medium in a biosafety cabinet. Cells can then be cultured in an INFORS incubator at approximately 37°C with shaking.

[0172] Seed culture passage: After approximately 72 to 96 hours of cell growth, the initial seed culture is transferred to a 500 ml Tri-forest shaking flask containing Dynamis alkaline medium and approximately 30 ml of cells in a biosafety cabinet. This culture passage can be repeated once or multiple times as needed.

[0173] After cell growth, the cell culture medium can be inoculated into a SUB50 containing alkaline medium LC100B. Cell density is determined by sampling the culture and measured using a Beckman Vi-Cell counter. The pH of the batch-fed cell culture is controlled. Dissolved oxygen (DO) is maintained at approximately 45% while stirring. Feed medium and components can be added individually based on a fixed schedule. Antifoaming emulsion is added as needed. The inoculated cell density can be approximately 1.5 × 10⁶ cells / ml. Once the cells reach peak cell density, the cell culture is harvested. After harvesting, the batch-fed culture can be filtered once or multiple times. After filtration, the culture was collected in a 50 L storage bag and stored at approximately 4°C. Methods known in the art for purifying antibodies and other proteins include, for example, those described in U.S. Patent Publications Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is incorporated herein by reference in its entirety.

[0174] General Characteristics

[0175] P1801 blocks the interaction between the immune checkpoint PD-1 and its ligand PD-L1 to disrupt the immunosuppressive mechanism. P1801 may belong to the IgG4 subclass of human antibodies and has been shown to exhibit unique properties. Human IgG4 antibodies can be functionally monovalent in vivo because they may exchange half-molecules (a heavy chain and a light chain) with each other, a process called Fab arm exchange. The Fab domain of P1801 is located within the constant region of the heavy chain and has the amino acid sequence ESKYGPPCPPCP (SEQ ID NO:17) The hinge region of IgG4 is connected to Fc, and this hinge region has an S288P mutation to reduce Fab arm exchange. IgG4 exhibits low affinity for complement component 1q (C1q) and the Fc receptor, causing weak induction of complement toxicity and antibody-dependent cell activation. These properties of IgG4 are preferred in cancer immunotherapy when it is undesirable to recruit host effector functions.

[0176] In another embodiment, P1801 may retain all variable regions and change the IgG type to IgG1, IgG2, IgG3 or other types.

[0177] Hybridoma Screening

[0178] Anti-PD1 antibodies (e.g., P1801) can also be generated by hybridoma screening. For example, murine antibodies are selected as one of the best clones because they show high binding affinity in ELISA. They specifically bind to human PD1 and are able to block the binding of PD1 to PDL1 / PDL2. Additionally, the mouse-type formula of P1801 was humanized and formatted as human immunoglobulin subtype IgG4 because IgG4 has a lower immune response, such as ADCC and CDC, compared to other subtypes IgG1 and IgG3. The DNA sequence of P1801 was codon-optimized for expression. The expression vector for P1801 can be synthesized using DNA 2.0.

[0179] Phage Display

[0180] In some embodiments, antibodies can be prepared and selected using phage display technology. See, for example, U.S. Patent Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150, all of which are incorporated herein by reference in their entirety. Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene lineages from unimmunized donors. According to this technology, the antibody V domain gene frame is cloned into the major or minor coat protein genes of filamentous phages, such as M13 or fd, and displayed as a functional antibody fragment on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on antibody functional characteristics also leads to the selection of genes encoding antibodies that exhibit those characteristics. Therefore, some characteristics are described in the phage-mimicking B cell manual, page 22 / 54, 28 CN 122270481 A. Phage display can be performed in various forms. Several sources of the V gene segment can be used for phage display. Known techniques have reported the isolation of a series of different anti-oxazolinone antibodies from a small randomized library of V genes derived from the spleen of immunized mice. Lineages of V genes from human donors can be constructed, and antibodies against a range of different antigens (including self-antigens) can be isolated essentially using techniques known in the field. In natural immune responses, mutations in antibody genes rapidly aggregate (somatic hypermutation). Some of the introduced changes will confer higher affinity and enhance subsequent antigen stimulation.During this process, B cells displaying high-affinity surface immunoglobulins preferentially replicate and differentiate. This natural process can be simulated using a technique known in the field called "chain shuffling." In this method, the affinity of "primary" human antibodies obtained through phage display is improved by sequentially replacing the heavy and light chain V region genes with naturally occurring variant lineages (lineages) of the V domain genes obtained from never-obtained immunization donors. This technique allows for the production of antibodies and antibody fragments with affinities in the pM-nM range. Strategies for preparing large phage antibody lineages (also known as "the mother-of-all libraries") have been described and are known in the field. Gene shuffling can also be used to derive human antibodies from rodent antibodies, wherein the human antibodies have similar affinity and specificity to the starting rodent antibody. According to this method (also known as "epitope imprinting"), the heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced with a lineage of human V domain genes to produce rodent-human chimeras. Selection of antigens allows for the isolation of human variable regions capable of restoring functional antigen-binding sites, i.e., selection of epitope-controlled (imprinted) conjugates. When this process is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT Publication WO 93 / 06213). Unlike the conventional method of humanizing rodent antibodies by CDR grafting, this technique provides fully human antibodies without rodent-derived framework or CDR residues.

[0181] In some embodiments, antibodies may be prepared using hybridoma technology. It is considered that any mammalian individual, including humans, or antibody-producing cells derived therefrom, can be manipulated to serve as the basis for generating mammalian (including human) hybridoma cell lines. As further described herein, the route and schedule of immunization of the host animal generally conform to techniques known for antibody stimulation and production. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarily, and / or intradermally with an immunogen, including those described herein.

[0182] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using general somatic cell hybridization techniques. Available myeloma cell lines can be used for hybridization. Generally, this technique involves fusing myeloma cells with lymphocytes using a fusion accelerator such as polyethylene glycol or by an electrical method well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parental cells. Any culture medium supplemented with serum or without serum as described herein can be used to culture hybridomas that secrete monoclonal antibodies. As an alternative to cell fusion techniques, EBV immortalized B cells...Cells can be used to generate the PD-1 monoclonal antibody of this disclosure. Hybridomas or other immortalized B cells can be expanded and subcloned as needed, and the antiimmunogen activity of the supernatant can be determined by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

[0183] Hybridomas that can be used as antibody sources encompass all derivatives and progeny cells of parental hybridomas that produce monoclonal antibodies that specifically or preferentially bind to PD-1 or a portion thereof.

[0184] Hybridomas that produce these antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture media or body fluids as needed by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesired activity (if present) can be removed, for example, by passing the formulation through an adsorbent made of an immunogen linked to a solid phase and eluting or releasing the desired antibody from the immunogen. Immunizing a host animal with a PD-1 peptide or a fragment containing the target amino acid sequence can generate an antibody population (e.g., monoclonal antibodies). The PD-1 peptide or fragment containing the target amino acid sequence is used with a bifunctional agent or derivatizing agent, such as maleic benzoyl sulfosuccinimide (via cysteine ​​residue), N-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, SOCl2, or R'N=C=NR (where R and R' are different alkyl groups), to bind to a protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor).

[0185] In one embodiment, the P1801 of interest (monoclonal or polyclonal) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector within the host cell, which can then be amplified and frozen for future use. The generation of recombinant monoclonal antibodies in cell cultures can be performed by cloning antibody genes from B cells using methods known in the art. See, for example, U.S. Patent No. 7,314,622.

[0186] Humanization

[0187] In some embodiments, the polynucleotide sequence can be used for genetic manipulation to “humanize” the antibody or to improve its affinity or other characteristics. The antibody can also be tailored for, for example, dogs, cats, primates, equines, and bovine animals.

[0188] In some embodiments, fully human antibodies can be obtained using commercially available mice engineered to express a specific human immunoglobulin. Mice designed to produce more desirable (e.g., fully human antibodies) or more robust antibodies can also be used.Transgenic animals that respond to an immune response produce humanized or human antibodies. Examples of such technologies include Xenomouse (trademark) from Abgenix, Inc., HuMAb-Mouse (trademark) and TC Mouse (trademark) from Medarex, Inc.

[0189] Antibodies can also be prepared in a recombinant manner as follows: first, antibodies and antibody-producing cells are isolated from a host animal, the gene sequence is obtained, and the antibody is recombinantly expressed in host cells (e.g., CHO cells) using the gene sequence. Another method that can be used is to express the antibody sequence in plants (e.g., tobacco) or transgenic latex. Methods for recombinantly expressing antibodies in plants or latex have been disclosed. Methods for preparing antibody derivatives such as domains, single chains, etc., are also known in the art.

[0190] Antibodies specific to PD-1 can also be isolated using immunoassays and flow cytometry sorting techniques such as fluorescence activated cell sorting (FACS).

[0191] DNA encoding monoclonal antibodies can be easily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies). Hybridoma cells serve as a source of this type of DNA. Once isolated, the DNA can be placed in an expression vector (e.g., the expression vector disclosed in PCT Publication WO 87 / 04462) and subsequently transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not additionally produce immunoglobulins, to synthesize monoclonal antibodies in recombinant host cells. See, for example, PCT Publication WO 87 / 04462. The DNA can also be modified, for example, by replacing homologous murine sequences with coding sequences for constant domains of human heavy and light chains or by covalently attaching all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. In this way, “chimeric” or “hybrid” antibodies having the binding specificity of the PD-1 monoclonal antibodies described herein are prepared.

[0192] Antibody fragments can be generated by the recombinant methods described above or by chemical synthesis, utilizing the protein degradation or other degradation of antibodies. Antibody peptides (especially shorter peptides of up to about 50 amino acids) can be prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, antibodies can be generated by an automated peptide synthesizer employing a solid-phase method. See also U.S. Patent Nos. 5,807,715, 4,816,567, and 6,331,415, page 24 / 54, CN 122270481 A, all of which are incorporated herein by reference in their entirety.

[0193] In some embodiments, the antibody that preferentially binds to PD-1 or its antigen-binding portion may have heavy chain complementarity-determining regions 1 (VHCDR1), 2 (VHCDR2), and / or 3 (VHCDR3) obtained (e.g., derived, identified, or copied) from the heavy chain variable region of the P1801 antibody (e.g., the sequence listed in SEQ ID NO: 5); and light chain complementarity-determining regions 1 (VLCDR1), 2 (VLCDR2), and / or 3 (VLCDR3) obtained from the light chain variable region of the P1801 antibody (e.g., the sequence listed in SEQ ID NO: 6). Those skilled in the art, taking into account the heavy chain variable region sequences or the light chain variable region sequences, will be able to determine all six CDR sequences based on the methods described herein or known in the art.

[0194] In one embodiment, based on the Kabat method, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 shows that P1801 includes: a heavy chain complementarity-determining region 1 (VHCDR1) including the amino acid sequence listed in SEQ ID NO: 9, a heavy chain complementarity-determining region 2 (VHCDR2) including the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) including the amino acid sequence listed in SEQ ID NO: 11; and a light chain complementarity-determining region 1 (VLCDR1) including the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) including the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) including the amino acid sequence listed in SEQ ID NO: 14.

[0195] In another embodiment, based on the Kabat assay, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 18, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0196] In another embodiment, based on the Chothia assay, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 includes: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 9, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 20; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0197] In another embodiment, based on the Chothia method, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 includes: a heavy chain complementarity-determining region 1 (VHCDR1) including the amino acid sequence listed in SEQ ID NO: 18, a heavy chain complementarity-determining region 2 (VHCDR2) including the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) including the amino acid sequence listed in SEQ ID NO: 20; and a light chain complementarity-determining region 1 (VLCDR1) including the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) including the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) including the amino acid sequence listed in SEQ ID NO: 14.

[0198] In one embodiment, based on the Chothia method, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 includes: a heavy chain complementarity-determining region 1 (VHCDR1) including the amino acid sequence listed in SEQ ID NO: 19, a heavy chain complementarity-determining region 2 (VHCDR2) including the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) including the amino acid sequence listed in SEQ ID NO: 20; and a light chain complementarity-determining region 1 (VLCDR1) including the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) including the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR2) including the amino acid sequence listed in SEQ ID NO: 14.Heavy chain complementarity-determining region 3 (VLCDR3).

[0199] In another embodiment, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 based on the Chothia method showed that P1801 includes: heavy chain complementarity-determining region 1 (VHCDR1) including the amino acid sequence listed in SEQ ID NO: 21, heavy chain complementarity-determining region 2 (VHCDR2) including the amino acid sequence listed in SEQ ID NO: 10 and / or heavy chain complementarity-determining region 3 (VHCDR3) including the amino acid sequence listed in SEQ ID NO: 20; and light chain complementarity-determining region 1 (VLCDR1) including the amino acid sequence listed in SEQ ID NO: 12, light chain complementarity-determining region 2 (VLCDR2) including the amino acid sequence listed in SEQ ID NO: 13 and / or light chain complementarity-determining region 3 (VLCDR3) including the amino acid sequence listed in SEQ ID NO: 14.

[0200] In one embodiment, based on the Chothia method, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 includes: a heavy chain complementarity-determining region 1 (VHCDR1) including the amino acid sequence listed in SEQ ID NO: 19, a heavy chain complementarity-determining region 2 (VHCDR2) including the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) including the amino acid sequence listed in SEQ ID NO: 11; and a light chain complementarity-determining region 1 (VLCDR1) including the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) including the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) including the amino acid sequence listed in SEQ ID NO: 14.

[0201] In another embodiment, based on the Chothia method, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 21, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.Chain complementarity-determining region 3 (VLCDR3).

[0202] iv. Characteristics

[0203] At least two known FDA-approved human monoclonal antibodies against PD1 have been approved, namely pembrolizumab (trademark Keytruda) and nivolumab (trademark OPDIVO). Both can treat a variety of diseases, such as certain types of cancer. In addition, the epitopes on hPD1 of pembrolizumab and nivolumab are known in the art.

[0204] In one embodiment, as shown in FIG8, for example, P1801 of this disclosure binds to the same PD1 epitope as commercially available anti-PD1 monoclonal antibodies (e.g., pembrolizumab and / or nivolumab), completely overlapping. In another embodiment, P1801 binds to substantially the same PD1 epitope as commercially available anti-PD1 monoclonal antibodies (e.g., pembrolizumab and / or nivolumab), or has substantially overlapping regions. In another embodiment, P1801 partially binds to the same PD1 epitope region as commercially available anti-PD1 monoclonal antibodies (e.g., pembrolizumab and / or nivolumab). Surprisingly and unexpectedly, although P1801 partially overlaps with the PD1 epitope compared to pembrolizumab and / or nivolumab, suggesting a weaker signature, the kinetic properties of P1801 are substantially the same as, or even better than, those of pembrolizumab and / or nivolumab.

[0205] In yet another embodiment, P1801 only partially covers PD1, but surprisingly, its performance is at least comparable to that of existing commercially available anti-PD1 monoclonal antibodies.

[0206] In one embodiment, this disclosure discloses an anti-human PD1 monoclonal antibody (P1801) whose properties (e.g., molecular characteristics and / or cancer therapeutic efficacy) in various respects are substantially similar to those of commercially available anti-PD1 monoclonal antibodies (e.g., pembrolizumab and / or nivolumab). Exemplary features and properties include, but are not limited to, binding strength against PD1 (as measured by KD), stability / shelf life, and / or the ability to treat cancer. Figure 3 illustrates an example in a mouse model.

[0207] In another embodiment, this disclosure discloses an anti-human PD1 monoclonal antibody (P1801) whose properties (e.g., molecular characteristics and / or cancer therapeutic efficacy) are significantly superior to those of commercially available anti-PD1 monoclonal antibodies. Exemplary features and properties include, but are not limited to, binding strength against PD1 (as measured by KD), stability / shelf life, and / or the ability to treat multiple types of cancer.

[0208] In yet another embodiment, this disclosure discloses a combination of P1101 and an anti-human PD1 monoclonal antibody (P1801), which exhibits significantly superior properties (e.g., molecular properties and / or cancer therapeutic efficacy) compared to commercially available anti-PD1 monoclonal antibodies alone.The antibody is therapeutic and / or has a synergistic effect. Exemplary features and properties include, but are not limited to, binding strength against PD1 (as measured by KD), stability / shelf life, ability to treat cancer and / or shrink tumor size.

[0209] Although the variable regions are altered, those skilled in the art can modify the anti-PD1 antibodies or antigen-binding fragments thereof disclosed herein, such antibodies comprising at least a portion of one or more constant regions that have been deleted or otherwise altered to provide the desired biochemical characteristics (e.g., increased tumor localization or decreased serum half-life compared to antibodies comprising native or unaltered constant regions with substantially the same immunogenicity) (e.g., full-length antibodies or immunoreactive fragments thereof). In some aspects, the constant regions of the modified antibody will comprise human constant regions. Modification of constant regions compatible with the anti-PD1 molecules disclosed herein comprises the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may contain alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or the light chain constant domain (CL). In some aspects, modified constant regions in which one or more of the domains are partially or completely deleted are considered. In some aspects, the modified antibodies will contain a domain-deleted construct or variant (CH2 construct) in which the entire CH2 domain has been removed. In some aspects, the omitted constant region may be replaced by a short amino acid spacer (e.g., residues 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), which provides some of the molecular flexibility normally conferred by the absence of constant regions.

[0210] Anti-PD1 antibodies or their antigen-binding fragments may be further modified to contain additional chemical portions that are not normally protein components. Those derived portions may improve protein solubility, biological half-life, or absorption. The portions may also reduce or eliminate any anticipated side effects of proteins, etc. The portion may include one or more detectable markers or tags (e.g., biotin, reporter enzymes, colorimetric or fluorescent tags, solid carriers, or radiolabels) to facilitate the detection of antibodies or PD-1 in a sample.

[0211] In one embodiment, the complementary site of the anti-PD1 antibody or its antigen-binding fragment of the present disclosure may contain all VH and VL CDRs or only some amino acid residues in some CDRs (e.g., some CDRs may not participate in antigen binding). The complementary site against a specific antigen may be defined, for example, by scanning mutagenesis of amino acid residues within the antibody, particularly CDRs (e.g., scanning mutagenesis of alanine), said amino acid residues considered to be exposed on the surface (e.g., determined by crystallographic modeling) and potentially involved in antigen binding. Subsequent evaluation of the binding of the mutant to the antigen may determine whether the mutated amino acid position participates in antigen binding and thus forms part of the antibody complementary site.

[0212] In another embodiment, the complementary site can be determined by high-resolution cryo-electron microscopy, which can generate an intermolecular density map to determine the binding distance between certain amino acids of one or more CDRs and the antigen.

[0213] In one embodiment, the anti-PD1 antibody or its antigen-binding fragment of the present disclosure includes a heavy chain complementary site, which includes the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 9, 18, 19 or 21 (CDR1), 10 (CDR2) and 11 (CDR3). In another preferred embodiment, the anti-PD1 antibody or its antigen-binding fragment of the present disclosure includes a light chain complementary site, which includes the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 12 (CDR1), 13 (CDR2) and 14 (CDR3). In one embodiment, the anti-PD1 antibody or its antigen-binding fragment of the present disclosure comprises: heavy chain complementary sites comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 9, 10, and 11, and light chain complementary sites comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 12, 13, and 14. In another embodiment, the anti-PD1 antibody or its antigen-binding fragment of the present disclosure comprises: heavy chain complementary sites comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 18, 10, and 11, and light chain complementary sites comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 12, 13, and 14. In another embodiment, the anti-PD1 antibody or antigen-binding fragment thereof disclosed herein comprises: a heavy chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 19, 10, and 11, and a light chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 12, 13, and 14. In yet another embodiment, the anti-PD1 antibody or antigen-binding fragment thereof disclosed herein comprises: a heavy chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 21, 10, and 11, and a light chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 12, 13, and 14.

[0214] Substitution of non-complementary amino acid residues

[0215] Non-complementary amino acids may be substituted by recombination to obtain variable light or heavy chain domains with properties similar to or modified from germline variable domains. "Modified" variable domains also include amino acid deletions and substitutions. For example, N-terminal or C-terminal amino acid residues may be deleted in the modified variable domain.

[0216] Non-complementary amino acid substitutions can be made, for example, to improve stability and / or reduce aggregation tendency. Poor stability can affect the ability of antigen-binding fragments to fold correctly, for example, when expressed in a recombinant manner, resulting in a portion of the expressed fragment being non-functional. Antibodies or their antigen-binding fragments with low stability may also be prone to forming potentially immunogenic aggregates, or have impaired affinity or shelf life. scFv peptides are particularly likely to exhibit problems with stability, solubility, expression, aggregation, degradation products, and overall manufacturability in bacterial and mammalian expression systems. For example, WO 2007 / 109254 discloses framework amino acid substitutions intended to improve the stability of, for example, the VH and / or VL domains in scFv peptides and / or reduce their aggregation tendency. Similarly, substitution of corresponding residues in the VH and VL domains in this case is expected to improve stability and / or reduce aggregation tendency.

[0217] v. Pharmaceutically Acceptable Carriers

[0218] The compositions used in this disclosure, whether P1801 alone, P1101 alone, or any combination thereof, may further comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at certain doses and concentrations and may comprise: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethylbenzyl ammonium chloride; hexahydroxyquaternary ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol); (and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN (trademark), PLURONICS (trademark), or polyethylene glycol (PEG).

[0219] vi. Dosage

[0220] In various embodiments, the concentration of anti-PD1 antibody administered was about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 ...mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg.

[0221] vii. Combination

[0222] Anti-PD-1 antibodies may also be used in combination with other agents to enhance and / or supplement the effectiveness of antibody therapy for cancer, or administered separately, simultaneously, and / or sequentially. One such non-limiting example of an anti-PD1 monoclonal antibody is P1801, wherein the combination is P1801 + P1101. The anti-PD1 monoclonal antibody (e.g., P1801) or P1101 may be referred to as the first or second molecule in the combination. Alternatively or additionally, P1801 may be further optionally combined with another anti-PD1 monoclonal antibody (e.g., pembrolizumab, nivolumab, and / or cimipril).

[0223] In one embodiment, a murine anti-PD-1 antibody (e.g., RMP 1-14) may be used to replace P1801 in a mouse model. Alternatively, a mouse form of mP1101 with interferon may be used in a mouse model.

[0224] viii. Continuous and combined administration

[0225] P1101 may be administered using various regimens and dosages previously reported in the art.

[0226] Continuous administration

[0227] In addition, the length between continuous administration of one or more doses of P1101 or mP1101 to an individual may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks and / or 10 weeks. In another embodiment, P1101 or mP1101 may be continuously administered followed by P1801, wherein the length between one or more doses of P1801 administered to an individual is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, and / or 10 weeks.

[0228] Continuous administration in any of the above sequences (P1101 first or P1801 first) can provide a “synergistic effect” and demonstrate “synergistic effect”, that is, the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by using the compounds separately.

[0229] Combination Application

[0230] Combination application can also provide a “synergistic effect” and demonstrate “synergistic effect”, that is, when active ingredients are used together.The effect achieved is greater than the sum of the effects produced by using the compounds separately. This disclosure also includes a synergistic effect that can be obtained when the active ingredients are: (1) co-formulated and simultaneously administered or delivered in unit dose formulations of the combination; (2) delivered alternately or in parallel as separate formulations; and / or (3) delivered by some other protocol. When delivered in an alternating therapy, the synergistic effect can be obtained when the compounds are administered or delivered sequentially, for example, by different injections made with separate syringes. Generally, during alternating therapy, each active ingredient is administered in an effective dose sequentially (i.e., consecutively), while in combination therapy, two or more active ingredients are administered together in an effective dose.

[0231] In one embodiment, the first molecule and the second molecule can be administered simultaneously to an individual by mixing them and administering them to the individual simultaneously. Alternatively or additionally, the two macromolecules can be administered to the individual immediately in any given order, or one after another closely in any given order. In another embodiment, one macromolecule can be administered first in a given protocol, followed by the second macromolecule in a given protocol. In another embodiment, the frequency and length of the regimen can vary at any length, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days and / or 31 days or longer. The first molecule can be P1101, and the second molecule can be P1801, and vice versa.

[0232] In some embodiments, the first molecule can be administered according to the first regimen during a first treatment period, and the second molecule can be administered according to the second independent regimen during a second independent treatment period, wherein the two regimens and / or treatment periods can be simultaneous, sequential, alternating, or timed in any way. For example, in the case where both molecules are administered once every two weeks, the two regimens can be started on the same day or different days (e.g., the second regimen can be started before or after the first regimen). In another example, where the first molecule is administered once every five days for three months and the second molecule is administered once every two weeks for one year, the two regimens can be started on the same day or different days (e.g., the second regimen can be started before or after the first regimen, on the day the first molecule was administered or a day not during the first molecule treatment period, or after the end of the first molecule treatment period). The dose and / or the time between each administration in the regimen can remain constant or vary. The first molecule can be P1101, and the second molecule can be an anti-PD1 monoclonal antibody, such as P1801. Alternatively, the first molecule can be an anti-PD1 monoclonal antibody, such as P1801, and the second molecule can be P1101.

[0233] In yet another embodiment, the first molecule and the second molecule can be administered alternately in any given order. In one embodiment, the first molecule and the second molecule can be administered in any fixed time interval order. Figure 4(a) illustrates such an example, where a solid arrow indicates the administration of the first molecule to an individual, and a dashed arrow indicates the administration of the second molecule, with each administration 3 days apart. Alternatively, the reverse can also be true, where a solid arrow indicates the administration of the second molecule to an individual, and a dashed arrow indicates the administration of the first molecule, with each administration 3 days apart. In another embodiment, an illustrative example is the administration of P1101 to an individual by a solid arrow, and the administration of an anti-PD1 monoclonal antibody, such as P1801, by a dashed arrow. Alternatively, the reverse can also be true, where a solid arrow indicates the administration of an anti-PD1 monoclonal antibody, such as P1801, to an individual, and a dashed arrow indicates the administration of P1101.

[0234] In a non-limiting embodiment, the two molecules may be administered sequentially, but one molecule may initially be administered in a smaller number of doses (e.g., 3 doses) with a set time period (e.g., 3 days) between each dose, followed by administration of the second molecule in two, three, or four times the number of doses, also with a set time period (e.g., 3 days) between each dose. A non-limiting example can be seen at the top of Figure 4(b). Another illustrative example is that solid arrows indicate administration of P1101 to an individual, and dashed arrows indicate administration of an anti-PD1 monoclonal antibody, such as P1801. The first molecule may be P1101, and the second molecule may be P1801, or vice versa.

[0235] In a non-limiting embodiment, the two molecules may be administered sequentially, but one molecule may initially be administered in the same number of doses (e.g., 6 doses) with a set time period (e.g., 3 days) between each dose, followed by administration of the second molecule in the same number of doses (e.g., 6 doses), also with a set time period (e.g., 3 days) between each dose. Figure 4(b) at the bottom illustrates such a non-limiting example. Simply put, solid arrows indicate administration of P1101 to an individual, and dashed arrows indicate administration of an anti-PD1 monoclonal antibody, such as P1801. Alternatively, solid arrows indicate administration of an anti-PD1 monoclonal antibody, such as P1801, to an individual, and dashed arrows indicate administration of P1101.

[0236] In yet another embodiment, the first and second molecules may be administered alternately, each once, but with an interval of 1 or 2 days between administrations. This regimen may be repeated two, three, four, or five times at fixed time intervals (e.g., every 3, 4, 5, or 6 days). Or, or additionally, either molecule may then be administered once at a single dose at a fixed time interval (e.g., 3, 4, 5, or 6 days). Figure 4(c) at the top illustrates such a non-limiting example. In one embodiment, solid arrows indicate administration of P1101 to an individual,The dashed arrows indicate the administration of an anti-PD1 monoclonal antibody, such as P1801. Alternatively, solid arrows indicate the administration of an anti-PD1 monoclonal antibody, such as P1801, to an individual, and dashed arrows indicate the administration of P1101.

[0237] In one embodiment, the first molecule and the second molecule may be administered alternately once each, but with an interval of 1 or 2 days between the two administrations. This regimen may be repeated two, three, four, or five times at fixed time intervals (e.g., every 3, 4, 5, or 6 days). Such non-limiting examples are illustrated at the bottom of Figure 4(c). In one embodiment, solid arrows indicate the administration of P1101 to an individual, and dashed arrows indicate the administration of an anti-PD1 monoclonal antibody, such as P1801. Alternatively, solid arrows indicate the administration of an anti-PD1 monoclonal antibody, such as P1801, to an individual, and dashed arrows indicate the administration of P1101.

[0238] In a non-limiting embodiment, the two molecules are administered simultaneously or on the same day, but one molecule may initially be administered at a smaller dose than described on page 30 / 54 of the specification (CN 122270481 A), followed by double, triple, or quadruple doses in the second, third, fourth, or fifth administrations, while the second molecule is administered at a constant dose. Alternatively, the reverse may also be true, where the first molecule is administered at a constant dose, while the second molecule may initially be administered at a smaller dose, followed by double, triple, or quadruple doses in the second, third, fourth, or fifth administrations. In one embodiment, the first molecule may be P1101, and the second molecule may be an anti-PD1 monoclonal antibody, such as P1801. Alternatively, the first molecule may be any anti-PD1 monoclonal antibody, such as P1801, and the second molecule may be P1101.

[0239] In another embodiment, the first molecule may be administered daily at a fixed dose for the first 13 days, and the second molecule may be administered starting on day 6, followed by 5 more administrations, all at 3-day intervals. Alternatively, the first 4 administrations of the second molecule may have the same dose, and the dose of the 5th administration may be twice that of the previous 4. In the future, the dose of the 6th administration may be four times that of the previous 4 administrations. A non-limiting example may be illustrated in Figure 5. In one embodiment, the first molecule may be P1101, and the second molecule may be an anti-PD1 monoclonal antibody, such as P1801. Alternatively, the first molecule may be an anti-PD1 monoclonal antibody, such as P1801, and the second molecule may be P1101.

[0240] In another embodiment, the first molecule may be administered ip or sc at intervals of 6, 7, or 8 days (QW × 4 from day 1 to day 22), while the second molecule may be administered ip or sc on days 11, 13, 18, 20, 25, and 27 (BIW × 2, day 11 to day 27). An example is shown in Figure 6. In one embodiment, the first molecule administration may be P1101, and the second molecule...The first molecule may be an anti-PD1 monoclonal antibody, such as P1801. Alternatively, the first molecule may be an anti-PD1 monoclonal antibody, such as P1801, and the second molecule may be P1101.

[0241] In one embodiment, the length between administering a single dose of P1801 or a single dose of P1101 to an individual may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks and / or 10 weeks or longer.

[0242] In another embodiment, the length between administering a single regimen of P1801 and a single regimen of P1101 to an individual may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks and / or 10 weeks or longer.

[0243] ix. Use

[0244] In one embodiment, the use of the anti-PD1 monoclonal antibody of this disclosure enhances the activation of antigen-specific CD4+ or CD8+ T cells, thereby enhancing the individual's immunity to treat, suppress and / or prevent cancer, such as solid tumors. Non-limiting examples of solid tumors include melanoma, hepatocellular carcinoma (HCC), and / or kidney tumors (renal cell carcinoma or RCC).

[0245] In another embodiment, the combination of P1801 and P1101 can further enhance an individual's immunity to treat, suppress, and / or prevent cancers, such as solid tumors, compared to P1801 alone. Non-limiting examples include melanoma, hepatocellular carcinoma (HCC), colorectal cancer (CRC), melanoma, and / or kidney tumors (renal cell carcinoma (RCC)).

[0246] In one embodiment, the P1801 and / or the combination of P1801 and P1101 disclosed herein can be used to treat, suppress, and / or prevent the following diseases:

[0247] Advanced melanoma or adjuvant therapy for melanoma

[0248] The combination of P1801 and / or P1801 with another molecule (e.g., P1101) can be used to treat individuals with unresectable or metastatic melanoma. This treatment may be further optionally combined with pembrolizumab and / or nivolumab.

[0249] Advanced hepatocellular carcinoma (HCC)

[0250] P1801 and / or a combination of P1801 with another molecule (e.g., P1101) may be used to treat patients with hepatocellular carcinoma (HCC). (Instructions for Use, Pages 31 / 54, 37, CN 122270481)Individual A. The individual has previously been treated with sorafenib. This treatment may optionally be further combined with cimiprimab, pembrolizumab, and / or nivolumab.

[0251] Treatment of renal cell carcinoma (RCC) or advanced RCC

[0252] P1801 and / or a combination of P1801 with another molecule (e.g., P1101) may be used as adjuvant therapy for individuals with renal cell carcinoma (RCC) who are at intermediate to high or high risk of recurrence after nephrectomy or after nephrectomy and resection of metastatic lesions. This treatment may optionally be further combined with cimiprimab, pembrolizumab, and / or nivolumab.

[0253] P1801 and / or a combination of P1801 with another molecule (e.g., P1101) may be used as first-line treatment for adult individuals with advanced renal cell carcinoma (RCC). This treatment may be further optionally combined with axitinib and / or cimiprimab, pembrolizumab and / or nivolumab.

[0254] For adult individuals with advanced melanoma

[0255] P1801 or in combination with another molecule (e.g., P1101) may be used to treat a type of skin cancer called melanoma that has spread or is not surgically removable (advanced melanoma). This therapy may be further optionally combined with cimiprimab, nivolumab, pembrolizumab and / or ipilimumab.

[0256] For adult individuals with melanoma whose melanoma and affected lymph nodes have been surgically removed to prevent recurrence

[0257] P1801 or in combination with another molecule (e.g., P1101) may be used to treat adult individuals with melanoma whose melanoma and affected lymph nodes have been surgically removed to prevent recurrence. This therapy may be further optionally combined with cimiprimab, pembrolizumab, and / or nivolumab.

[0258] For certain individuals with advanced renal cell carcinoma

[0259] When the renal cell carcinoma has spread (advanced renal cell carcinoma) and certain individuals with this cancer have not yet received treatment for advanced RCC, P1801 or in combination with another molecule (e.g., P1101) may be used to treat said individuals. This therapy may be further optionally combined with cimiprimab, nivolumab, pembrolizumab, and / or ipilimumab.

[0260] For newly diagnosed individuals with metastatic renal cell carcinoma

[0261] When the renal cell carcinoma has spread (advanced renal cell carcinoma) and the cancer patient has not yet received treatment for advanced RCC, P1801 or in combination with another molecule (e.g., P1101) may be used to treat the individual. This therapy may be further optionally combined with cimiprimab, nivolumab, pembrolizumab, and / or cabozantinib.

[0262] For individuals with previously treated advanced renal cell carcinoma

[0263] When renal cell carcinoma has spread or grown after treatment with other cancer agents, P1801, or in combination with another molecule (e.g., P1101), can be used to treat patients with this type of cancer. This therapy may further optionally be combined with cimiprimab, pembrolizumab, and / or nivolumab.

[0264] For individuals with hepatocellular carcinoma who have previously received sorafenib treatment

[0265] If a patient with hepatocellular carcinoma has previously received sorafenib treatment, then P1801, or in combination with another molecule (e.g., P1101), can be used to treat that individual. This therapy may further optionally be combined with cimiprimab, nivolumab, pembrolizumab, and / or ipilimumab.

[0266] Additionally, P1801, or in combination with another molecule (e.g., P1101), may be used to treat adults and children aged 12 years and older with a type of colon or rectal cancer (colorectal cancer) that has spread to other parts of the body (metastasis), has high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), and who have previously been treated with fluoropyrimidine, oxaliplatin, and irinotecan, and said drugs have not worked or are no longer effective. This therapy may further optionally be combined with cimiprimab, pembrolizumab, and / or nivolumab.

[0267] x. Route and Dosage

[0268] P1801, or in combination with another molecule (e.g., P1101, nivolumab, and / or ipilimumab), may be formulated as an injection for intravenous (IV) administration. In one embodiment, nivolumab (about 10 mg / mL) and ipilimumab (about 5 mg / mL) are injectable formulations for intravenous (IV) administration. In one embodiment, the dosage of P1101 may be between about 50 and about 540 μg. Other non-limiting examples include six consecutive doses of 450 mcg P1101 (Q2W) together with 0.3 mg / kg P1801 (Q2W); six consecutive doses of 450 mcg P1101 (Q2W) together with 0.75 mg / kg P1801 (Q2W); six consecutive doses of 450 mcg P1101 (Q2W) together with 1.5 mg / kg P1801 (Q2W); six consecutive doses of 450 mcg P1101 (Q2W) together with 2.0 mg / kg P1801 (Q2W); or six consecutive doses of 450 mcg P1101 (Q2W) together with 3.0 mg / kg P1801 (Q2W). The treatment period can be during a treatment cycle of 28 days, 35 days, 42 days, 49 days, or 56 days.

[0269] Examples

[0270] Example 1

[0271] Binding Affinity and Inhibition of P1801

[0272] This example aims to measure the binding affinity and inhibitory ability of the P1801 antibody to the antigen. A direct binding ELISA was performed to measure binding specificity and cross-reactivity. Competitive ELISA and cell-based reporter gene assays were performed to measure the inhibitory ability of P1801 PD1 / PD-L1 versus PD1 / PD-L2.

[0273] All incubations should be performed at 25°C, except for antigen-coated culture plates at 4°C. The culture plates should be kept away from airflow and other temperature fluctuations and covered with a culture plate seal. During all washing steps, 300 μL / well of wash buffer was added, and the solution was removed from the culture plates by inverting and wiping them on a paper towel. Each washing step was repeated 3 times.

[0274] The antigen was diluted to the appropriate concentration with 1×DPBS at 50 μL / well overnight and blocked with 1% BSA at 200 μL / well for 2 hours. Sample preparation and incubation: Dilute P1801 samples with 1% BSA, starting with 0.8 μg / mL and performing 2.5-fold serial dilutions for a total of 8 spots. Add two copies of freshly prepared serially diluted sample solution to each well. Seal the plate and incubate at 25°C without shaking for 1 hour. Perform secondary Ab incubation by diluting peroxidase-conjugated goat anti-human IgG Fe fragment-specific Ab to 1:10000 in 1% BSA, then adding 50 μL of freshly prepared diluted secondary Ab solution to each well. Seal the plate and incubate at 25°C without shaking for 40 minutes. Perform color development by adding 50 μL / well of TMB substrate solution, gently tapping the plate to ensure thorough mixing, and allowing it to stand for 2–5 minutes. After incubation, terminate the reaction directly with 50 μL / well of stop solution. Read OD within 10 minutes and protect from light. Ensure all wells are free of air bubbles.

[0275] Inhibition of PD-1 / PD-L1 and PD-1 / PD-L2 was measured by competitive binding ELISA.

[0276] Spreading and blocking were performed, wherein the antigen was diluted to an appropriate concentration at 50 μL / well with 1× DPBS overnight and blocked with 200 μL / well of 1% BSA for 2 hours. Sample preparation and incubation of the competitor P1801: using 1% BSA as dilution buffer, 2-fold serial dilutions were performed starting at 5 μg / mL for a total of 10 spots. Then 50 μL of sample dilution was added to each appropriate well. For the binding agent biotin-hPD-1-mlgG Fe, it was diluted to a working solution of 0.25 μg / mL and then 50 μL was added to each well. The plate was incubated at 25°C with constant shaking (550 rpm) for 2 hours. Anti-streptavidin HRP Ab was diluted to 1:15000 in 1% BSA and then 50 μL was added to each well.Add μL of freshly prepared diluted secondary Ab solution to the wells of the culture plate for secondary Ab incubation. Seal the culture plate and incubate at 25°C with constant shaking (550 rpm) for 40 minutes. Develop color by adding 50 μL / well of TMB substrate solution, gently tapping the culture plate to ensure thorough mixing, and incubating in the dark for 30 minutes. After incubation, terminate the reaction directly with 50 μL / well of stop solution. Read OD within 10 minutes and protect from light. Ensure no air bubbles in all wells. Instructions for use 33 / 54 pages 39 CN 122270481 A

[0277] An analytical method for cell-based reporter gene assays was used to measure the inhibition of PD-1 / PD-L1 by P1801 anti-PD1 antibody. In short, Jurkat cells stably expressing PD-1 (Jurkat / PD1) activate the expression of a luciferase reporter gene driven by the NFAT response element (NFAT-RE) via TCR. However, although PD-1 binds to PD-L1 on Jurkat cells, this interaction blocks TCR-mediated luminescence. When Jurkat cells are activated by a TCR agonist and the PD-1 / PD-L1 interaction is disrupted by anti-PD-1, luciferase is induced (Figure 9). The more anti-PD-1 blocking the interaction, the more luciferase is induced. IC50 can be determined by dose-response curves of the test samples.

[0278] Table 2 below shows the in vitro binding specificity of P1801.

[0279] Table 2

[0280]

[0281] The cross-reactivity of P1801 is reported below in Table 3:

[0282] Table 3

[0283]

[0284] Assay system: direct binding ELISA

[0285]

[0286] Table 4 shows the inhibitory activity of P1801.

[0287] Table 4 Specification 34 / 54 pages 40 CN 122270481 A

[0288]

[0289] In short, P1801 has an affinity of 0.57 nM for human PD-1 (EC50) and 0.44 nM for cynomolgus monkey PD-1 (EC50). No significant difference was observed between the binding affinity of P1801 for human and cynomolgus monkey PD-1. No cross-reactivity was observed between P1801 and mouse PD-1. Competitive human PD-L1 / PD-L2 interaction between P1801 and human PD-1 was observed in both competitive ELISA and cell-based assays. P1801 binds to human PD-1 and cross-reacts with monkey PD-1, and there is no significant difference in binding efficacy (EC50) between human PD-1 and monkey PD-1. P1801 does not cross-react with mouse PD-1. P1801 showed competitive binding in competitive ELISA and cell-based assays.In cell-based reporter gene assays, inhibition of PD-1 binding to PD-L1 and PD-L2 was observed. P1801 reacted with human and cynomolgus monkey PD-1 but not with mouse PD-1.

[0290] Example 2

[0291] Epitope binding of P1801

[0292] The epitope binding sites of P1801 were compared with two existing commercially available anti-PD1 mAbs (i.e., pembrolizumab and nivolumab). Biomolecular interactions were determined using BLI technology. This technology is an optical analysis method that analyzes the interferogram of white light reflected from two surfaces: an internal reference layer and a protein layer deposited on the biosensor tip. Changes in the interferogram are induced by any change in the number of molecules combined with the biosensor tip. In this case, interactions can be measured in real time, thus providing the ability to monitor binding specificity, association-to-dissociation ratio, concentration, fidelity, and accuracy. In addition, only molecules combined with or separated from the biosensor can alter the interferogram and produce a reaction spectrum on the BLI system. However, the absence of bound molecules, changes in the refractive index of the surrounding medium, or changes in flow rate had no effect on the interferogram.

[0293] Here, the blocking effect of the purified anti-PD1 monoclonal antibody P1801 on the PD-1 epitope was tested by Octet BMIA. The purified PD1RB antibody was pre-incubated with PD-1 / His (10 μg / mL antibody; 0.336 μg / mL PD-1) for 1 hour. An anti-human IgG (Fc) sensor was loaded with 3 μg / mL of various baseline antibodies. The association of the pre-formed antibody + antigen complex was measured and compared with the association of the antigen alone. A high binding reaction indicated that the epitopes did not overlap, while a low reaction indicated that the epitopes overlapped. Baseline run for 30 seconds. The antibody loaded on the sensor was run at 4 μg / mL for 700 seconds. Quenching was performed for 480 seconds, and baseline run for 300 seconds. 10 μg / mL of blocking antibody associated with 0.336 μg / mL of PD-1 for 600 seconds. Detailed results can be seen in Figure 8(a) comparing nivolumab and P1801 and Figure 8(b) regarding pembrolizumab. If P1801 binds to the same epitope as either nivolumab or pembrolizumab, the lines would overlap, but they do not. Instead, the two lines marked with arrows do not overlap with any other lines. This indicates non-overlapping binding on the target and represents partial overlap.

[0294] For both benchmark antibodies, P1801 showed a response of 0.0453 against nivolumab and 0.0426 nm against pembrolizumab. Therefore, the results show that P1801 is a partial blocker of PD1 compared to the two commercially available anti-PD1 mAb epitope binding sites, but the other characteristics exhibited by P1801 are comparable, which is surprising and unexpected. Specification 35 / 54 pages 41 CN 122270481 A

[0295] Example 3

[0296] Binding Kinetics

[0297] This example aims to measure the binding affinity of the P1801 antibody to the antigen using a Biacore 8K. First, FcRn and C1q were immobilized onto a CM5 sensor chip. FcRn and C1q were immobilized at 25°C while HBS EP was used as the run buffer. The sensor chip surfaces of flow cells 1 and 2 were activated for 200 seconds (10 μL / min) with a freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Subsequently, FcRn and C1q diluted in 30 mmol / L NaAC (pH 4.5) were injected into flow cell 2 to achieve binding of the appropriate reaction units, while flow cell 1 was set as a blank. After the amine coupling reaction, the remaining active coupling sites on the chip surface were blocked by a 200-second injection of 1 mol / L ethanolamine hydrochloride. To measure the affinity of FcRn and C1q, the assay was performed at 25°C with HBS EP (pH 6.0) as the run buffer. Diluted P1801 antibody was injected onto the surfaces of flow cells 1 and 2 as the associative phase, followed by run buffer as the dissociation phase. Next, the affinity of human PD1 was measured. The assay was performed at 25°C with HBS EP (pH 7.4) as the run buffer. Diluted P1801 antibody (2 μg / ml) was injected onto the surface as the capture phase, human PD1 protein was injected onto the surface as the associative phase, followed by run buffer as the dissociation phase. Finally, P1801 Fc binding was characterized by SPR, where the kinetic data of P1801 antibody for human PD1, FcRn, and C1q were obtained by affinity measurements. All data were processed using Biacore 8K evaluation software version 1.1. Buffer blank injections in flow cell 1 and each cycle were used as a double reference for reaction unit subtraction.

[0298] The data are listed in Table 5 below.

[0299] Table 5

[0300]

[0301] In short, in both drug batches, P1801 bound to human PD-1 with an affinity (KD) of 2.50 and 3.13 nM, and P1801 bound to FcRn and C1q with an affinity (KD) comparable to pembrolizumab.

[0302] Example 4

[0303] Kinetic Measurements for CD64

[0304] This example shows a multi-cycle kinetic analysis of human PD1, cynomolgus monkey PD1, and human CD64 with anti-PD1 Ab. In Example 42 CN 122270481 A on pages 36 / 54 of this specification, the ligand anti-PD1 Ab was captured on the surface of sensor chip protein A. Human PD1, cynomolgus monkey PD1, and human CD64 with anti-PD1 Ab were measured using a Biacore T200.Multi-cycle kinetic analysis of Ab. The anti-PD1 Ab ligand was captured on the surface of sensor chip protein A. The anti-PD1 Ab was first diluted with 1×HBS-EP+ to obtain a final concentration of 3 μg / ml and captured by flow cell 2 of sensor chip protein A. The chip surface contains MabSelect SuRe ligands, allowing for directional and specific binding to the Fc region of the antibody. Assays were performed using the Kinetic / Affinity wizard. The flow path was 2-1 with the ligand injected and captured in flow cell 2, and flow cell 1 used as a reference. A series of analyte concentrations (human PD1, cynomolgus monkey PD1, and human CD64) were sequentially injected into the reference and ligand surfaces as associative phases, with transient dissociation phases obtained by injecting operating buffer. Regeneration solution was then injected as the regeneration phase. All obtained data were fitted to a 1:1 binding model using Biacore T200 evaluation software version 3. Blank injections of flow cell 1 and operating buffer were used as a subtractive dual reference. Results are summarized in Table 6.

[0305] Table 6 Binding affinity of P1801 to CD64 (IgG receptor FcγRI) determined by SPR

[0306]

[0307] Briefly, P1801 binds to human PD-1 with an affinity (KD) of 1.48 nM and to cynomolgus monkey PD1 with an affinity (KD) of 1.62 nM. P1801 has an affinity (KD) of 2.96 × 10⁻¹⁰ M for human CD64 (IgG receptor FcγRI).

[0308] Example 5

[0309] In vivo therapeutic efficacy

[0310] This example will show the in vivo therapeutic efficacy of the test compound P1801 in the treatment of the HuCell MC38-hPDL1 model in female hPD-1 HuGEMM mice, and the effect of P1801 + mP1101. Each mouse was subcutaneously inoculated with 0.1 ml of HuCell MC38 tumor cells (1 × 10⁶) in PBS in the right posterior ventral region to develop a tumor. Treatment was initiated when the tumor reached a mean volume of approximately 65 mm³. Treatment with the test compound P1801 significantly reduced the mean tumor volume compared to the mediator group. Thirty mice participated in the study. All animals were randomly assigned to three different study groups. The mean tumor size at randomization was approximately 65 mm³. The date of tumor randomization was designated as day 0. Morbidity and mortality were assessed daily after tumor cell inoculation. During routine monitoring, tumor growth and any effects of treatment on behavior, such as activity level, food and water consumption, weight gain / loss (measured twice weekly after randomization), and any other abnormalities, were examined. Individual mouse mortality and observed clinical signs were recorded. Tumor volume was measured weekly in two dimensions using calipers.The volume was measured twice, and the volume was expressed in mm3 using the following formula: V = (L×W×W) / 2, where V is the tumor volume, L is the tumor length (longest tumor size) and W is the tumor width (longest tumor size perpendicular to L). Tumor weight was measured at the end of the study.

[0311] On day 23 post-vaccination, the MTV in the isotype control group reached 3489.56 mm3, and the MTVs in the P1801 12mpk treatment group and the nivolumab 12mpk treatment group reached 39.39 mm3 and 236.69 mm3, respectively. Compared with the isotype control group, the test substance P1801 showed significant tumor suppression in the subcutaneous HuCell MC38-hPDL1 model in female hPD-1 HuGEMM mice (TGI: 98.87%) (p<0.001). During follow-up, complete response (CR) was observed in 6 mice in the P1801 treatment group and in 8 mice in the nivolumab treatment group. In terms of survival, the median survival of P1801 treatment exceeded 50 days, significantly longer than that of the isotype control group (p = 0.002). The median survival of the nivolumab 12mpk positive control group also exceeded 50 days, significantly longer than that of the isotype control group (p<0.001). For example, one administration regimen is shown in Figures 6(a) and 6(b).

[0312] Tumor growth inhibition is summarized in Table 7 below:

[0313] Table 7

[0314]

[0315] Data showed that P1801 inhibited MC38-hPD-L1 tumor growth in hPD-1 mice at 12 mg / kg BIW×3 compared with the isotype control group. In addition, P1801 prolonged the survival of tumor-bearing hPD-1 mice compared with the isotype control group. The combination of mP1101 and P1801 also showed efficacy.

[0316] Example 6

[0317] IL2 induction

[0318] This example contains the results of the Whole Blood Cytokine Response Assay for P1801. The assay observed that P1801 enhanced IL-2 production in SEB1-stimulated whole blood in a dose-dependent manner. Heparinized blood from healthy donors was pre-cultured with P1801 and then treated with SEB to induce IL-2 release. IL-2 induces and / or modulates and / or activates T cell responses, thereby inhibiting and / or treating cancer.

[0319] After 2 days of treatment, the supernatant was collected and IL-2 was quantified by ELISA. Whole blood was diluted tenfold with culture medium and...Pre-incubation was carried out for 60 minutes in 96-well plates at 37°C in a 5% CO2 incubator in the absence (no SEB induction and no treatment) or presence of the test substance and NC (0.04, 0.2, 1, 5 and 25 μg / mL). Except for control wells (no SEB induction), whole blood cells were subsequently stimulated with SEB (0.1 μg / mL), and then incubated for 48 hours at 37°C in a 5% CO2 incubator. After incubation, the supernatant was collected by centrifugation at 500 ×g for 5 minutes, aliquoted and stored at -30°C until ELISA analysis. IL-2 was quantified by ELISA following the manufacturer's instructions. Briefly, standards and supernatants were serially diluted with calibration diluent RD5-5. 100 μL of assay diluent RD1W was added to each well, followed by 100 μL of blank standards (31.3 to 2000 pg / mL) and diluted supernatants (2-fold, 4-fold, 8-fold and 16-fold dilution) to designated wells, and the incubation was maintained for two hours. The wells were washed three times with diluted wash buffer, then 200 μL of human IL-2 conjugate was added to each well, and the incubation was maintained for two hours. The washing step was repeated and 200 μL of substrate solution was added to each well, and the incubation was maintained for 10 to 20 minutes. Finally, 50 μL of stop solution was added and the optical density (O.D.) was measured using a microplate reader set at 450 nm and 570 nm. Data were analyzed using Gen5TM software as follows: (A) Average the duplicate readings of blanks, standards and supernatants, then subtract the average O.D. of the blanks to obtain the corrected O.D. (B) Generate a standard curve from corrected O.D. and perform four-parameter logistic (4-PL) curve fitting. The IL-2 concentration of all supernatants read from the standard curve must be multiplied by the dilution factor. (C) Acceptance criteria: a. If the O.D. of the supernatant is lower than the lowest standard (LLOQ3), then the IL-2 value will be represented by the symbol "<LLOQ" and excluded from subsequent calculations. If the RPD4 calculated based on the IL-2 values of the two specified dilutions is higher than 20%, the concentration of the lower dilution will be reported. Otherwise, the average of the concentrations of the two dilutions will be reported.

[0320] The obtained pharmacodynamic results are summarized in Table 8 below:

[0321] Table 8

[0322] SEB-induced IL-2 in human whole blood

[0323]

[0324]

[0325] The results showed that compared with the IgG4 isotype control, P1801 caused a significant difference in SEB-induced IL-2 production in human whole blood at 0.2, 1, 5 and 25 μg / mL. 2. At 0.04, 1, 5 and 25 μg / mL, there was no significant difference in IL-2 production induced by P1801 compared with that induced by nivolumab (P > 0.05).

[0326] For each dose level, N=8.

[0327] IL-2 was not detected in SEB-free whole blood. Statistically significant differences in IL-2 production were observed between P1801 and human IgG4 at concentrations of 0.2, 1, 5, and 25 μg / mL (p values ​​were 0.019, 0.001, 0.001, and 0.015, respectively). No statistically significant differences in IL-2 production were observed between P1801 and nivolumab at concentrations of 0.04, 1, 5, and 25 μg / mL (p value > 0.05). In the P1801 treatment group, the specific fold change relative to IgG4 was at most 1.49-fold, comparable to nivolumab. P1801 enhances IL-2 production in SEB-stimulated whole blood in a dose-dependent manner.

[0328] Example 7

[0329] This example evaluates the pharmacokinetics, anti-drug antibody (ADA), receptor occupancy (RO), and IL-2 release in cynomolgus monkeys following a single intravenous infusion of P1801 at three dose levels. All cynomolgus monkeys assigned to the study were identified as healthy animals by routine blood tests prior to administration. Actual body weight and administration details were measured after the single intravenous injection in the cynomolgus monkeys. Weekly body weight records were taken. During the period from administration to sample collection, slight changes in body weight attributable to blood collection were observed, and no abnormalities were observed. Blood was collected from all surviving animals before administration (days -7 and -1), 24 hours after administration, and 168 hours (day 7), 672 hours (day 28), and 1334 hours (day 56) after administration for IL-2 release analysis. Instructions for Use, pages 39 / 54, CN 122270481 A. IL-2 release from monkey leukocytes (before administration, -7 days) was tested in untreated monkeys treated in vitro with 0.04, 0.2, 1, 5, or 25 μg / mL P1801, nivolumab, or isotype (IgG4). In the SEB stimulation study, P1801 and nivolumab treatments promoted IL-2 release in a dose-dependent manner, while IgG4 treatment only slightly induced IL-2 release, which did not increase with increasing dose.

[0330] IL-2 release from monkey leukocytes was measured in monkeys treated with P1801 and given a single intravenous infusion of 1, 5, or 20 mg / kg P1801. Whole blood was collected from monkeys at five time points in the pharmacokinetic studies (day -1, day 1, day 7, day 28, and day 56). Time-dose-dependent IL-2 release was observed in the 5 and 20 mg / kg groups, but no significant dose-dependent IL-2 release from P1801 was observed. An overview is provided in Tables 9 and 10 below.

[0331] Table 9

[0332]

[0333]

[0334] In short, Table 9 above shows that P1801 promotes significant IL-2 production in monkey PBMCs in a dose-dependent manner.The IgG4 isotype control resulted in a slight increase in IL-2, which did not increase with increasing dose. For each dose level, N=12.

[0335] Table 10

[0336]

[0337] This example shows that P1801 promotes a large amount of IL-2 production in whole blood of monkeys in a time-dependent manner. No dose dependence was observed when P1801 was administered at 1, 5 or 20 mg / kg. For each dose level, N=4 (2 males and 2 females); P value relative to day -1 (ANOVA / Kruskal-Wallis & Wilcoxon rank sum test on group means after Bartlett test).

[0338] Example 8

[0339] In vitro PD-1 receptor occupancy (RO).

[0340] Newly isolated human PBMCs were stimulated for 2 days with anti-CD3 / CD28-coated Dynabeads to induce PD-1 expression, and then cultured with a specified concentration of P1801. P1801-bound PD-1+ T cells were detected using a fluorophore-bound secondary antibody (anti-human IgG4 Fc antibody) and analyzed by flow cytometry. Human PBMCs were isolated from heparinized blood of healthy donors. Mouse anti-human IgG4 Fc antibody containing the fluorophore R-phycoerythrin (PE) and mouse anti-human CD3 antibody containing the fluorophore allophycocyanin (APC) were used. The negative control was a recombinant human IgG4 isotype control antibody purified by Ultra-LEAF™. T cell activators Dynabeads (trademark) human T cell activator CD3 / CD28 were used. Human PBMCs were isolated from heparinized whole blood of healthy donors no more than 6 hours after collection using Ficoll-Paque (trademark) PLUS. Human PBMCs were stimulated for 2 days at 37°C and 5% CO2 incubator using Dynabeads (trademark) human T-cell activators CD3 / CD28 to express PD-1. After two days of activation, PBMCs were suspended and Dynabeads were removed using a magnetic rack (DynaMag) (trademark). Two × 10⁵ PBMCs were incubated in 100 μL staining buffer at room temperature in the dark for 30 minutes in the absence of (untreated) or in the presence of P1801 (0.01, 0.1, and 1 μg / mL), a comparison (0.01, 0.1, and 1 μg / mL), and a negative control (1 μg / mL). PBMCs were washed twice with staining buffer before staining. Anti-hCD3 and anti-hIgG4 Fc antibodies were diluted in staining buffer, and 100 μL of the diluted solution was added to the staining buffer.Antibodies were added to tubes containing PBMCs. The cells were incubated in the dark at room temperature for 20 minutes.

[0341] The PBMCs were washed twice with 1 mL of staining buffer to remove unbound antibodies. The cell pellet was resuspended in 350 μL of staining buffer and analyzed by flow cytometry as soon as possible. Data were analyzed using FCS Express software as follows: (A) CD3-positive regions of the FSC / CD3 dot matrix were circled for further analysis of PD-1 expression, including the percentage of PD-1+ cells (PD-1+%) and the geometric mean fluorescence intensity (Geo MFI) of PD-1 on all CD3+ T cells. (B) PD-1+% was defined as the percentage of CD3+ T cells bound to the anti-PD-1 antibody and calculated by subtracting the untreated group from the treated group using the Overton method. (C) Geo MFI was defined as the geometric mean fluorescence intensity of the PE channel and represented the degree of binding of the anti-PD-1 antibody to PD-1 on the surface of all CD3+ T cells. The data in the summary table are presented as mean ± SE3 of ten donors in eight independent experiments, and P1801 and the comparison were analyzed by one-factor ANOVA4. The results are summarized in Table 11 below.

[0342] Table 11

[0343] P1801 receptor occupancy in human PBMCs

[0344] Specification 41 / 54 pages 47 CN 122270481 A

[0345]

[0346] The results showed that the percentage of P1801 bound to human PBMCs was dose-dependent. At 0.1 and 1 μg / mL, the percentage of PD-1 positive CD3+ T cells of P1801 was not significantly different from that of nivolumab. In addition, the PD-1 binding activity of P1801 showed a dose-dependent effect. At medium and high concentrations of 0.1 and 1 μg / mL, respectively, there was no statistically significant difference in PD-1+% between P1801 and nivolumab (p>0.05). At a high concentration of 1 μg / mL, there was no statistically significant difference in Geo MFI between P1801 and nivolumab (p>0.05).

[0347] Example 9

[0348] In vivo therapeutic efficacy of Renca subcutaneous syngeneic renal cell carcinoma model in female BALB / c mice

[0349] To evaluate the antitumor efficacy of the combination of anti-PD1 antibody and P1101 in a mouse model, two mouse formulations of the molecules were used, namely, the mouse form of mP1101 in which mouse interferon was used instead of human form, and the known mouse anti-PD1 mAb RMP 1-14 as an anti-PD1 mAb. The study design included: randomization to study groups; daily administration, 7 days a week for 3 weeks; post-treatment observation period based on implantation site; subcutaneous or in situ administration. Animals were examined daily, 5 days a week, during the administration and observation period;If subcutaneous implantation is used, body weight is measured twice a week and tumor volume is measured twice a week.

[0350] Table 12 shows a design overview similar to Figures 4(a) to 4(c), illustrating the administration of the combination and the frequency of various molecules. In Table 12 below, Q3D represents administration once every 3 days. ×6 refers to 6 times. SC represents subcutaneous administration. IP represents intraperitoneal injection. ROA represents route of administration.

[0351] Table 12

[0352] Specification 42 / 54 pages 48 CN 122270481 A

[0353]

[0354] Methods

[0355] Renca (CL-00796) cells were maintained in vitro at 37°C and in an air atmosphere of 5% CO2 in DMEM medium supplemented with 10% fetal bovine serum. Cells in the exponential growth phase were collected and quantified by a cell counter, followed by tumor seeding. For tumor inoculation, Renca tumor cells (1 × 10⁶) in 0.1 ml of PBS were subcutaneously inoculated into the right lower ventral region of each mouse to develop tumors. Randomization began when the average tumor size reached approximately 70 mm³. Fifty-six mice were selected for the study. All animals were randomly assigned to seven study groups of eight mice each. Randomization was based on a matted distribution / stratified method (StudyDirector software, version 3.1.399.19). The randomization day was designated as day 0. Treatment began on the same day as randomization (day 1) according to the study design.

[0356] Observations and Data Collection

[0357] Morbidity and mortality were examined daily after tumor cell inoculation. During routine monitoring, any effects of tumor growth and treatment on behavior, such as activity level, food and water consumption, weight gain / loss (measured twice weekly after randomization), eye abnormalities / hair tangles, and any other abnormalities, were examined. The mortality rate and observed clinical signs of individual animals were recorded in detail. Tumor volume was measured twice weekly in two dimensions using calipers after randomization, and the volume was expressed in mm3 using the following formula: "V = (L×W×W) / 2", where V is the tumor volume, L is the tumor length (longest tumor size) and W is the tumor width (longest tumor size perpendicular to L). Drug administration and tumor and body weight measurements were performed in a laminar flow cabinet. Body weight and tumor volume were also measured using StudyDirector software.

[0358] Treatment lasted for 33 weeks. The study ended on day 33. Tumors from euthanized mice were not collected before the end of the study. No samples were taken from any dead mice found.

[0359] Statistical Analysis

[0360] First, statistical analysis was performed using the Bartlett test to test for homogeneity of variance. Second, if Bartlett...If the p-value of the Bartlett test is greater than 0.05, it indicates insufficient evidence to reject the null hypothesis of homogeneity of variance, which is one of the hypotheses of ANOVA. Therefore, ANOVA is performed to test whether any pair of groups has different group means, and a post-hoc Tukey HSD is performed on all paired comparisons, or Dunnett's t-test is performed on each treatment with one control. If the p-value of the Bartlett test is less than or equal to 0.05, it indicates some evidence that the variances may be unequal or that the data do not follow a normal distribution. Next, we perform the Kruskal-Wallis test (page 43 / 54, CN 122270481 A) to test whether any pair of groups has different group medians, and a post-hoc Conover's non-parametric all-pairs comparison test is performed on all paired comparisons, or a Conover non-parametric many-to-one comparison test is performed on each treatment with one control. Both tests use single-step p-value adjustment. In addition, pairwise comparisons were performed without multiple comparison correction, and the nominal / uncorrected p-values ​​were reported directly according to the Welch t-test or the Mann-Whitney U test. The criteria for choosing the Welch t-test or the Mann-Whitney U test were the same as for two-group comparisons.

[0361] All statistical analyses were performed in the R language and environment (version 3.6.0) used for statistical calculations and graphs. Unless otherwise specified, all tests were two-sided, and p-values ​​<0.05 were considered statistically significant. Survival analyses were performed using the Kaplan-Meier model with the log-rank method.

[0362] Results

[0363] First, weight growth curves were measured. Results of weight and weight changes at different time points. Tumor growth curves (mean tumor volume over time) for different groups are shown in Figure 10. Detailed measurements and statistics are shown in Table 13.

[0364] Table 13

[0365]

[0366] In this example, the mean tumor volume of Group 1 reached 3486.36 mm3 on day 22 after the start of treatment. Group 2 was treated with anti-PD1 12 mg / kg, and the TGI value on day 22 was 1.59%. Group 3 was treated with mP1101 0.1 mg / kg, and the TGI value on day 22 was 70.70%. Group 4 was treated with mP1101 0.1 mg / kg Q3D×3 and anti-PD1 12 mg / kg Q3D×6, and the TGI value on day 22 was 64.51%. Group 5 was treated with mP1101 0.1 mg / kgTreatment with Q3D×6 and anti-PD1 12 mg / kg Q3D×6 resulted in a TGI of 79.59% on day 22. Group 6 was treated with mP1101 0.1 mg / kg Q3D×3 followed by anti-PD1 12 mg / kg Q3D×6, resulting in a TGI of 63.67% on day 22. Group 7 was treated with mP1101 0.1 mg / kg Q3D×6 followed by anti-PD1 12 mg / kg Q3D×6, resulting in a TGI of 76.40% on day 22.

[0367] Median survival (MST) was also studied for each group. Mice that reached the humane endpoint were euthanized. Mediator control, anti-PD1 12 mg / kg, mP1101 0.1 mg / kg, mP1101 0.1 mg / kg Q3D×3 and anti-PD1 12 mg / kg Q3D×6, mP1101 0.1 mg / kg Q3D×6 and anti-PD1 12 mg / kg Q3D×6, mP1101 0.1 mg / kg Q3D×3 followed by anti-PD1 12 mg / kg Q3D×6, mP1101 0.1 mg / kg Q3D×6 followed by anti-PD1 12 mg / kg The MSTs for Q3D×6 were 23.00 days, 23.00 days (relative to the carton control group, P>0.05), 31.50 days (relative to the carton control group, P<0.01), 29.00 days (relative to the carton control group, P<0.01), 16.50 days (relative to the carton control group, P>0.05), 30.00 days (relative to the carton control group, P<0.001), and 33.00 days (relative to the carton control group, P<0.01).

[0368] Briefly, in this example, the therapeutic efficacy of the test agent mP1101 was evaluated in a rodent Renca model in female BALB / c mice. Compared with the mediator group, the single agent mP1101 0.1 mg / kg (Group 3) and the combination with anti-PD1 12 mg / kg (Groups 4, 5, 6 and 7) showed significant antitumor efficacy (P<0.001). In addition, the single agent mP1101 0.1 mg / kg (Group 3) and the combination with anti-PD1 12 mg / kg (Groups 4, 6 and 7) prolonged the survival of mice.

[0369] Example 10

[0370] Therapeutic efficacy

[0371] In order to evaluate the antitumor efficacy of anti-PD1 mAb P1801, mammalian individuals were treated with an amount of P1801 that effectively reduces, inhibits and / or treats CRC, RCC and / or melanoma.

[0372] Each individual with RCC or melanoma was treated. Treatment with P1801 resulted in a reduction in mean tumor volume compared to the mediator control group. Thirty individuals were enrolled in the study. All individuals were randomly assigned to three different study groups. The mean tumor size at the time of initial randomization was approximately 65 mm3. The date of tumor randomization was designated as day 0. All individuals were examined daily. During routine monitoring, individuals were examined for tumor growth and any effects of treatment on behavior, such as activity level, food and water consumption, weight gain / loss, and any other abnormalities. Mortality and observed clinical signs were recorded for each individual. Tumor volume was measured twice weekly and expressed in mm3 using the following formula: V = (L×W×W) / 2, where V is the tumor volume, L is the tumor length (longest tumor size), and W is the tumor width (longest tumor size perpendicular to L). Tumor weight was measured at the end of the study.

[0373] The administration protocol is shown in Figure 12. Where appropriate, individuals showed a reduction in the size of melanoma or RCC.

[0374] Example 11

[0375] To evaluate the antitumor efficacy of antiPD1 P1801 mAb alone, P1101 alone with human interferon, and / or the combination of antiPD1 mAb P1801 + P1101, both molecules were used in human form, i.e., humanized forms of P1101 and P1801 with human interferon. The study design included: randomization to the study group; daily administration, 7 days a week for 3 weeks; a post-treatment observation period based on the implantation site; subcutaneous or in situ administration. During the administration and observation period, human individuals were examined daily, 5 days a week; if subcutaneous implantation, body weight was measured twice a week and tumor volume was measured twice a week.

[0376] Table 13 shows an overview of the experimental design, and Figures 4(a) through 4(c) show the administration of the combination and the frequency of the various molecules. In Table 14 below, Q3D represents administration every 3 days. ×6 means 6 times. SC represents subcutaneous administration. IP stands for intraperitoneal injection.

[0377] Table 14 Specification 45 / 54 pages 51 CN 122270481 A

[0378]

[0379] Results showed that in human individuals treated with RCC, CRC and / or melanoma, all of the following reduced tumor size of RCC, CRC and / or melanoma: P1801 treatment alone, P1101 treatment alone, and combination therapy of P1801 and P1101.

[0380] Example 12

[0381] Continuous dose escalation in clinical trials

[0382] To examine the toxicity / tolerance levels of P1801 + P1101 in humans, a phase 1 trial was conducted in humans. The starting dose of P1801 was determined based on preclinical pharmacological and toxicological data. As shown in the example above, in preclinical studies in cynomolgus monkeysA NOAEL of 200 mg / kg was reported in the toxicity study. Taking into account a conversion factor of 3.1 (monkey to human) and a safety margin of 6, the maximum recommended starting dose of P1801 is 10.75 mg / kg (200 mg / kg divided by 3.1, then divided by 6). By making more stringent safety considerations, 0.3 mg / kg was chosen as the starting dose for this example.

[0383] Initially, individuals meeting the criteria (e.g., age >18, having solid tumor RCC, melanoma, hepatocellular carcinoma (HCC) and / or CRC, and some patients may be in advanced stages) will be screened. Individuals will also have a life expectancy of 3 months or more, normal thyroid function, and be negative for HBV, HCV, and HIV. In groups 1 through 5, individuals with advanced solid tumors (particularly including renal cell carcinoma (RCC), melanoma, hepatocellular carcinoma (HCC), and colorectal cancer (CRC)) were treated with six doses of P1101 at 450 mcg every 2 weeks during each 28-day treatment cycle, followed by P1801 every 2 weeks at a predetermined dose level.

[0384] For dose escalation, five different groups were used, as shown in Figure 12. Once the effective dose was determined, group expansion was performed, as shown in Figure 13. PD-L1 expression was measured in all individuals, but only those with positive PD-L1 expression were selected into another group, as shown in Figure 14. Note that in this example, P1101 and P1801 were administered sequentially and are not interchangeable. The results of this example show that the effective doses shown are effective in treating CRC / HCC / RCC / melanoma.

[0385] Example 13

[0386] Complementary Site Determination Specification 46 / 54 pages 52 CN 122270481 A

[0387] The structure and 3D model of P1801 / PD-1 were determined using high-resolution cryo-electron microscopy, thereby obtaining the atomic-resolution structure of P1801 / PD-1, revealing the spatial arrangement of amino acids in the 3D structure and determining the complementary sites.

[0388] To determine the complementary sites, the sample PD1-AcroBio was screened using a grid and the following dataset was obtained. A dataset of sample PD1-AcroBio + PS00066 + PS00067 was collected. The dataset was processed in cryoSPARC 4.4. Micrographs with poorly fitted contrast transfer functions (CTF<4 Å) were removed and 5,306 micrographs were selected for further processing. Particle selection was performed based on a template, and approximately 4 million particles were extracted with a box size of 320 pixels (approximately 265 Å). For initial cleanup, two rounds of 2D classification and selected 2D categories were performed, resulting in approximately 1.5 million particles. The 2D category averages show discrete...The particles are clearly defined, possessing different views and visible secondary structure features. The selected particles are first used in a de novo reconstruction round, followed by four heterogeneous refinements using three decoys, and one non-uniform refinement. The final 128 K particles generate the highest resolution map, which is then selected for further refinement using CTF global refinement, followed by local resolution refinement and reference-based motion correction. The resolution is 2.9 Å based on the gold standard Fourier shell correlation estimate of 0.143 (Figures 16-17). The half-map is further refined using the DeepEMhancer Python package implemented in cryoSPARC. The models for PD1 (α-fold) and the two FABs are docked to the available density using ChimeraX. The variable regions of the FABs are then mutated according to the customer-provided sequences PS00066 and PS00067. The constant regions are not shown. The resulting model undergoes multiple rounds of manual reconstruction (using COOT) and computer-based refinement (Phenix). The glycans were manually constructed at positions ASN 49 and ASN 58. The final model was validated against a half-map and its quality was assessed using MolProbability. The obtained structure provides the location of the protein-protein interface between PD1 and PS00066 / PS00067.

[0389] Sample and mesh preparation for Cryo-EM. PD1-AcroBio + PS00066 + PS00067 was prepared by mixing sample PD1-AcroBio, sample PS00066 and sample PS00067 in a 1:1:1 molar ratio and incubating at room temperature for 30 minutes to obtain a final protein concentration of 0.18 mg / mL for the complex. The 1:1:1 molar ratio of sample PD1-AcroBio, sample PS00066 and sample PS00067 was diluted 17.5-fold in DPBS (provided by the customer) to obtain a final sample concentration of 0.01 mg / mL prior to mesh preparation and imaging. During conventional immersion freezing, an FEI Vitrobot instrument or a manual plunger is used. Simply put, a 3 μl drop of sample suspension is coated onto an EM grid that has been plasma-cleaned using Gatan Solarus (Pleasanton, California). After wiping the sample with filter paper, the grid is immersed in liquid ethane for freezing. The grid is stored in liquid nitrogen until transferred to a transmission electron microscope for imaging.

[0390] Cryo-EM imaging was performed using an FEI Titan Krios microscope. Electron microscopy was performed using an FEI Titan Krios (Hillsboro, Oregon) transmission electron microscope, which operates at 300°C.kV operation, equipped with a Gatan Quantum 967 LS imaging filter, a Volta phase plate, and a Gatan K3 direct detection camera. Glassy ice meshes were clamped into sample holders, transferred to cassettes, and then to a Krios automated feeder, all while maintaining the meshes at a cryogenic temperature (below -170°C). Automated data collection was performed using Leginon software, where high-magnification films were obtained by selecting targets at lower magnifications.

[0391] Preprocessing and 2D classification analysis (cryoSPARC, Relion). Dose-weighted film frame alignment was performed using MotionCor2 or full-frame or patch motion correction in cryoSPARC (Punjani et al., 2017) to account for plateau drift and fasciculation. The contrast transfer function for each micrograph was estimated using CTFfind4, gCTF, or Patch CTF in cryoSPARC (Rohou et al., 2015; Zhang et al., 2016). Individual particles are selected using an automated picking scheme and extracted into a particle stack in Relion or cryoSPARC. The particles can then be 2D aligned and classified without reference in Relion or cryoSPARC.

[0392] CryoSPARC (Cryo-EM Single Particle Analysis Reconstruction and Classification) simplifies cryo-EM data processing, providing capabilities such as particle picking, 2D classification, 3D reconstruction, and refinement. First, the mesh is screened and the data is compiled. Atlases are generated based on the data generated from cryoSPARC 4.4 micrograph data. Figure 16 shows an image generated from the final 3D reconstruction of PD1-AcroBio + PS 00066 + PS00067 using ChimeraX at a nominal resolution of 2.9 Å. The generated atlas with the highest resolution is selected for further refinement using CTF global refinement, followed by local resolution refinement and reference-based motion correction. The resolution of the Fourier shell correlation estimate based on the gold standard of 0.143 is 2.9 Å. The map is further refined using the DeepEMhancer Python package implemented in cryoSPARC. The models for PD1 (α-fold) and the two FABs are docked to the available density using ChimeraX. Figure 17 shows an image of the final 2.9 Å map generated using ChimeraX, colored with local resolution values ​​calculated by cryoSPARC 4.4. The variable regions of the FABs are then made to conform to customer-provided PS00066 and PS00067.The sequence was mutated. The resulting model underwent multiple rounds of manual reconstruction (using COOT) and computer-based refinement (Phenix). The final model was validated against the map and its quality was assessed using MolProbity. The obtained structure provides the location of the protein-protein interface between PD1 and PS00066 / PS00067. The model illustrates the hydrophobic and electrostatic interactions between PD1 and PS00067. The protein-protein contact appears to be primarily driven by hydrophobic interactions, with a cluster of aliphatic and aromatic residues, such as Tyr 32, Pro 33, His 52, Tyr 54 (I:PS00067), Tyr 95 (M:PS00067), and Pro 39, Ala 40, Leu 41, Leu 42, Val 43, Thr 45, Thr 145 (PD1). In addition, Arg 143 and Glu 141 (PD1) can form hydrogen bonds with Glu 99 (I:PS00067) and Tyr 101 (M:PS00067) / His 104 (I:PS00067), respectively.

[0393] Figure 18 is an image of the structure of the P1801 / PD1 complex as resolved by cryo-electron microscopy. Figure 19 is an image of the structure of the P1801 / PL1 / PD1 complex as resolved by cryo-electron microscopy, showing the overlap between P1801 / PD1 and PD-L1 / PD1. Figure 20 shows the epitope location of epitope P1801, illustrating its combination with heavy chain epitopes T59, S60, E61, S62, V64, P83, E84, D85, R86, L128, and A129, and light chain epitopes S87, P130, K131, and A132, where epitopes are shared. Figure 21 shows the epitope location of epitope PD-L1, illustrating its combination with epitopes V64, N66, Y68, S73, N74, Q75, T76, D77, K78, E84, G124, I126, L128, P130, K131, A132, I134, and E136, where epitopes are shared. Figure 22 shows a model of the P1801 CDR at the PD-1 combination interface.

[0394] Figure 23 is a graph showing the binding of epitopes to anti-PD-1 antibodies. Figure 23 shows that all antibodies can bind strongly to PD-1 in the presence of P1801, indicating that their epitopes are different. ISIS-clone19 was selected as the non-competitive Fa. Peresolimab is a humanized immunoglobulin G1 monoclonal antibody that stimulates human PD-1.

[0395] In one embodiment, the heavy chain sequence of the P1801 variable region with defined CDR is

[0396] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATITGGGSYTYYPDSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No: 5).

[0397] The VHCDR1 sequence SSYDMS (SEQ ID NO: 18); the VHCDR2 sequence TITGGGSYTYYPDSVKG (SEQ ID NO: 10); and the VHCDR3 sequence PYLNYFDY (SEQ ID NO: 11) are shown.

[0398] The light chain sequence of the P1801 variable region of CDR was defined as

[0399] EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIYYASQSISGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQSNSWPLTFGQGTKLEIK (SEQ ID No: 6).

[0400] The VLCDR1 sequence RASQSISNNLH (SEQ ID NO: 12); the VLCDR2 sequence YASQSIS (SEQ ID NO: 13); and the VLCDR3 sequence QQSNSWPLT (SEQ ID NO: 14) were shown.

[0401] In another embodiment, the heavy chain sequence of the P1801 variable region of the CDR is defined as

[0402] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATITGGGSYTYYPDSVKGRF Specification 48 / 54 pages 54 CN 122270481 A TISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No: 5).

[0403] The VHCDR 1 sequence GFTFSSYDMS (SEQ ID NO: 19); the VHCDR 2 sequence TITGGGSYTYYPDSVKG (SEQ ID NO: 10); and the VHCDR 3 sequence ASPYLNYFDY (SEQ ID NO: 20) are shown. The light chain sequence of the P1801 variable region of CDR is defined as

[0404] EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIYYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNSWPLTFGQGTKLEIK (SEQ ID No: 6).

[0405] The VLCDR1 sequence RASQSISNNLH (SEQ ID NO: 12); the VLCDR2 sequence YASQSIS (SEQ ID NO: 13); and the VLCDR3 sequence QQSNSWPLT (SEQ ID NO: 14) are shown.

[0406] In one embodiment, the heavy chain sequence of the P1801 variable region showing the CDR with complementary sites is defined as

[0407] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATITGGGSYTYYPDSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No: 5).

[0408] The VHCDR1 sequence GFTFSSYDMS (SEQ ID NO: 19) is shown, and it can also be GlyPheThr PheSer Ser TyrAsp Met Ser, numbered Gly1Phe2Thr3 Phe4Ser5 Ser6 Tyr7Asp8 Met9 Ser10. At all complementary sites, the complementary sites of the VHCDR1 sequence are Phe2Ser5Ser6 and Tyr7. Therefore, Gly1Thr3Phe4Asp8Met9 and Ser10 can individually substitute for any amino acid residue. For example, Gly1 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Thr3 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Phe4 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val; Asp8 can independently replace Ala, Arg, Asn, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Met9 can be used independently.Ser10 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and Ser10 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val.

[0409] The VHCDR2 sequence TITGGGSYTYYPDSVKG (SEQ ID NO: 10) can be Thr IleThr Gly Gly Gly Ser TyrThr Tyr Tyr Pro Asp Ser Val Lys Gly and numbered Thr11 Ile12Thr13 Gly14 Gly15 Gly16 Ser17 Tyr18Thr19 Tyr20 Tyr21 Pro22 Asp23 Ser24 Val25 Lys26 Gly27. At all complementary sites, the VHCDR2 sequence has complementary sites Thr13 Gly14 Gly15 Gly16 Ser17 and Tyr18. Therefore, Thr11, Ile12, Thr19, Tyr20, Tyr21, Pro22, Asp23, Ser24, Val25, Lys26, and Gly27 can individually substitute for any amino acid residue. For example, Thr11 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Ile12 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Thr19 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Tyr20 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; Tyr21 can independently replaceAla, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; Pro22 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Asp23 can independently replace Ala, Arg, Asn, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser24 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; Val25 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Lys26 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr; and Gly27 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0410] The VHCDR3 sequence ASPYLNYFDY (SEQ ID NO: 20) can be AlaSerProTyr Leu AsnTyr PheAsp Tyr and numbered Ala30Ser31Pro32Tyr33 Leu34 Asn35Tyr36 Phe37Asp38 Tyr39. At all complementary sites, the VHCDR3 sequence has four complementary sites: Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39. Therefore, Ala30, Pro32, Tyr36, and Phe37 can individually substitute for any amino acid residue. For example, Ala30 can independently substitute for Arg,Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Pro32 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Tyr36 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu. Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; and Phe37 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val.

[0411] In another embodiment, the heavy chain substitution sequence of the P1801 variable region of the CDR showing the complementary site is defined as

[0412] EVQLVESGGGLVQPGGSLRLSCAASGFTFSYDMSWVRQAPGKGLEWVATITGGGSYTYYPDSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No: 22)

[0413] The VHCDR1 substitution sequence GFTFSYDMS (SEQ ID NO: 21) is shown, which can also be written as GlyPheThr PheSer TyrAsp Met Ser and numbered as Gly1Phe2Thr3 Phe4Ser5 Tyr6Asp7 Met8 Ser9. At all complementary sites, the complementary sites of the VHCDR1 sequence are Phe2, Ser5 and Tyr6. Therefore, Gly1, Thr3, Phe4, Asp8, Met9, and Ser10 can individually substitute any amino acid residue. For example, Gly1 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Thr3 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Trp, Tyr, or Val; Phe4 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val; Asp7 can independently replace Ala, Arg, Asn, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Met8 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr. Trp, Tyr, or Val; and Ser9 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val.

[0414] The VHCDR2 sequence TITGGGSYTYYPDSVKG (SEQ ID NO: 10) can be Thr IleThr Gly Gly Gly Ser TyrThr Tyr Tyr Pro Asp Ser Val Lys Gly and numbered Thr11 Ile12Thr13 Gly14 Gly15 Gly16 Ser17 Tyr18Thr19 Tyr20 Tyr21 Pro22 Asp23 Ser24 Val25 Lys26 Gly27. At all complementary sites, the VHCDR2 sequence has four complementary sites: Thr13, Gly14, Gly15, Gly16, Ser17, and Tyr18. Therefore, Thr11, Ile12, Thr19, Tyr20, Tyr21, Pro22, Asp23, Ser24, Val25, Lys26, and Gly27 can individually substitute for any amino acid residue. For example, Thr11 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Ile12 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, orVal; Thr19 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Tyr20 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; Tyr21 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr. Trp or Val; Pro22 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Asp23 can independently replace Ala, Arg, Asn, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser24 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro. Thr, Trp, Tyr, or Val; Val25 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Lys26 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr; and Gly27 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0415] The VHCDR3 sequence ASPYLNYFDY (SEQ ID NO: 20) can be AlaSerProTyr Leu AsnTyr PheAspThe Tyr sequence is numbered Ala30, Ser31, Pro32, Tyr33, Leu34, Asn35, Tyr36, Phe37, Asp38, and Tyr39. At all complementary sites, the VHCDR3 sequence has Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39. Therefore, Ala30, Pro32, Tyr36, and Phe37 can individually substitute for any amino acid residue. For example, Ala30 can independently replace Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Pro32 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Tyr36 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; and Phe37 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val.

[0416] The VHCDR3 sequence PYLNYFDY (SEQ ID NO: 11) can be ProTyr Leu AsnTyr PheAsp Tyr and numbered Pro32Tyr33 Leu34 Asn35Tyr36 Phe37Asp38 Tyr39. At all complementary positions, the complementary positions of the VHCDR3 sequence are Tyr33, Leu34, Asn35, Asp38, and Tyr39. Therefore, Pro32, Tyr36, and Phe37 can individually substitute any amino acid residue. For example, Tyr36 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; and Phe37 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val.

[0417] VHCDR3 sequence SPYLNYFDY (SEQ ID NO:23) It can be SerProTyr Leu AsnTyr PheAsp Tyr, numbered Ser31Pro32Tyr33 Leu34 Asn35Tyr36 Phe37Asp38 Tyr39. At all complementary sites, the VHCDR3 sequence has Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39. Therefore, Pro32, Tyr36, and Phe37 can individually substitute for any amino acid residue. For example, Pro32 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Tyr36 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; and Phe37 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val.

[0418] In one embodiment, the P1801 variable region light chain sequence that defines the CDR showing the complementary bit is

[0419] EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIYYASQSISGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQSNSWPLTFGQGTKLEIK (SEQ ID No: 6). Specification 51 / 54 pages 57 CN 122270481 A

[0420] The VLCDR1 sequence RASQSISNNLH (SEQ ID NO: 12) is shown, which can also be written as Arg Ala Ser Gln Ser Ile Ser AsnAsnLeu His and numbered Arg1 Ala2 Ser3 Gln4 Ser5 Ile6 Ser7 Asn8Asn9Leu10 His11. In all complementary bits, the complementary bit of the VLCDR1 sequence is Asn9. Therefore, Arg1, Ala2, Ser3, Gln4, Ser5, Ile6, Ser7, Asn8, Leu10, and His11 can individually substitute for any amino acid residue. For example...Arg1 can independently replace Ala, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ala2 can independently replace Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser3 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val. ln4 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser5 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; Ile6 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser7 It can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; Asn8 can independently replace Ala, Arg, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and Leu10 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and His11 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0421] VLCDR2 sequence YASQSIS (SEQ ID NO:13) It can be TyrAla Ser Gln Ser IleSer and numbered Tyr12Ala13 Ser14 Gln15 Ser16 Ile17Ser18. At all complementary sites, the VLCDR2 sequence has Tyr12 and Ser18 as complementary sites. Therefore, Ala13, Ser14, Gln15, Ser16, and Ile17 can individually substitute for any amino acid residue. For example, Ala13 can independently replace Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser14 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; Gln15 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr. Trp, Tyr, or Val; Ser16 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; and Ile17 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0422] The VLCDR3 sequence QQSNSWPLT (SEQ ID NO: 14) can be Gln GlnSer AsnSerTrpPro Leu Thr and numbered Gln20 Gln21Ser22 Asn23Ser24Trp25Pro26 Leu27 Thr28.

[0423] At all complementary sites, the VLCDR3 sequence has Ser22, Asn23, and Trp25. Therefore, Gln20, Gln21, Ser24, Pro26, Leu27, and Thr28 can individually substitute for any amino acid residue. For example, Gln20 can independently substitute for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val; Gln21 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser24 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; Pro26 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met. Phe, Ser, Thr, Trp, Tyr, or Val; Leu27 may independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and Thr28 may independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val.

[0424] Table 15

[0425]

[0426] In the complementary sequences listed in Table 15, the amino acid residues presented in bold and underlined are constant in various proteins. The remaining amino acid residues in the sequence may individually substitute any amino acid residue, such as Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. The condition is that the bolded and underlined residues in the CDR are retained, which may individually substitute the remaining amino acid residues while maintaining function. Therefore, this disclosure contemplates many variations of the CDR sequence, provided that the bolded and underlined residues in the CDR are retained, and one or more of the remaining amino acid residues are substituted. In view of this functional definition, the inventors of this invention have considered many different CDR sequences characterized by the bolded and underlined residues in the CDR, one or more substituted amino acids, and the retention of sequence function.

[0427] It is considered that any embodiment discussed in this specification can be implemented using any method, kit, reagent, or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0428] It should be understood that the specific embodiments described herein are illustrated by way of illustration and are not intended to limit the scope of this disclosure. The main features of this disclosure may be employed in various embodiments without departing from the scope of this disclosure. Those skilled in the art will recognize or be able to determine many equivalents of the specific procedures described herein using only conventional experiments. These equivalents are all considered to be within the scope of this disclosure and are covered by the following claims.

[0429] All publications and patent applications referenced in this specification indicate the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

[0430] When used in conjunction with the term “comprising” in the claims and / or this specification, the use of the word “a / an” may mean “one,” but it also encompasses the meanings of “one or more,” “at least one,” and “one or more.” Although this disclosure supports the definitions of “referring only to alternatives” and “and / or,” the term “or” in the claims is used to mean “and / or” unless explicitly indicated as referring only to alternatives or that the alternatives are mutually exclusive. Throughout this application, the term “about” is used to indicate that a value includes the inherent bias of the apparatus, the method used to determine the value, or the bias that exists between individuals studied.

[0431] As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional unlisted elements or steps.

[0432] As used herein, the term “or a combination thereof” refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if the order is significant in a particular context, it also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing this example, it explicitly includes combinations containing repetitions of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, etc.CBBAAA, CABABB, etc. Those skilled in the art will understand that, unless otherwise apparent from the context, there is generally no limitation on the number of items or terms in any combination.

[0433] All compositions and / or methods disclosed and claimed herein can be prepared and performed according to this disclosure without excessive experimentation. Although the compositions and methods of this disclosure have been described according to preferred embodiments, it will be apparent to those skilled in the art that variations may be made to the compositions and / or methods described herein, as well as the steps or order of steps of the methods, without departing from the concept, spirit, and scope of this disclosure. All such similar substitutions and modifications that will be apparent to those skilled in the art should be considered to be within the spirit, scope, and concept of this disclosure as defined in the appended claims. Instruction Manual, Page 54 / 54, 60 CN 122270481 A, Figure 1(a), Figure 1(b), Figure 1(c), Figure 1(d), Figure 1(f), Figure 1(g); Instruction Manual, Figure 1 / 18, Page 61 CN 122270481 A, Figure 1(h), Figure 1(i), Figure 1(j), Figure 1(k), Figure 1(l), Figure 1(m), Figure 1(n), Figure 1(o); Instruction Manual, Figure 2 / 18, Page 62 CN 122270481 A, Figure 1(p), Figure 1(q), Figure 1(r), Figure 1(s), Figure 1(t), Figure 1(u), Figure 1(v), Figure 1(w), Figure 1(x); Instruction Manual, Figure 3 / 18, Page 63 CN 122270481 A, Figure 2, Figure 3, Figure 4(a); Instruction Manual, Figure 4 / 18, Page 64 CN 122270481 A, Figure 4(b), Figure 4(c) Figure 5 / 18, page 65, CN 122270481 A; Figure 5, Figure 6; Figure 6 / 18, page 66, CN 122270481 A; Figure 7(a), Figure 7(b), Figure 8(a); Figure 7 / 18, page 67, CN 122270481 A; Figure 8(b), Figure 9; Figure 8 / 18, page 68, CN 122270481 A; Figure 10, Figure 11; Figure 9 / 18, page 69, CN 122270481 A; Figure 12; Figure 10 / 18, page 70, CN 122270481 A; Figure 13; Figure 11 / 18, page 71, CN 122270481 A; Figure 14; Figure 12 / 18, page 72, CN 122270481 A; Figure 15, Figure 16; Figure 13 / 18, page 73, CN 122270481 A Figure 17 Figure 18 DescriptionFigure 19 of the instruction manual, page 14 / 18, CN 122270481 A; Figure 20 of the instruction manual, page 15 / 18, CN 122270481 A; Figure 21 of the instruction manual, page 17 / 18, CN 122270481 A; Figure 22 of the instruction manual, CN 122270481 A; Figure 23 of the instruction manual, page 18 / 18, CN 122270481 A.

Claims

1. An isolated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising: Includes the heavy chain complementarity determining region one (VHCDR1) of the amino acid sequences listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21; includes the heavy chain complementarity determining region two (VHCDR2) of the amino acid sequences listed in SEQ ID NO: 10; and / or includes the heavy chain complementarity determining region three (VHCDR3) of the amino acid sequences listed in SEQ ID NO: 11, SEQ ID NO: 20, or SEQ ID NO: 23; and Includes light chain complementarity determining region one (VLCDR1) of the amino acid sequence listed in SEQ ID NO: 12, light chain complementarity determining region two (VLCDR2) of the amino acid sequence listed in SEQ ID NO: 13, and / or light chain complementarity determining region three (VLCDR3) of the amino acid sequence listed in SEQ ID NO:

14.

2. A separated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising one or more paratopes, said one or more paratopes comprising: At least one of the following residues: Phe2, Ser5, Ser6 and Tyr7 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 19; At least one of the following residues: Phe2, Ser5 and Tyr6 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 21; At least one of the following residues: Thr13, Gly14, Gly15, Gly16, Ser17 and Tyr18 of the heavy chain variable sequence (VHCDR2) of SEQ ID NO: 10; At least one of the following residues: Tyr33, Leu34, Asn35, Asp38, and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 11; or At least one of the following residues: Ser31, Tyr33, Leu34, Asn35, Asp38 and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO:

20.

3. A separable antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising one or more complementary sites, said one or more complementary sites comprising: At least one of the following residues: Asn9 of the light chain variable sequence (VLCDR1) of SEQ ID NO: 12; At least one of the following residues: Tyr12 and Ser18 of the heavy chain variable sequence (VLCDR2) of SEQ ID NO: 13; or At least one of the following residues: Ser22, Asn23 and Trp25 of the heavy chain variable sequence (VLCDR3) of SEQ ID NO:

14.

4. A separated antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising one or more complementary sites, said one or more complementary sites comprising: At least one of the following residues: Phe2, Ser5, Ser6 and Tyr7 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 19; At least one of the following residues: Phe2, Ser5 and Tyr6 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 21; At least one of the following residues: Thr13, Gly14, Gly15, Gly16, Ser17 and Tyr18 of the heavy chain variable sequence (VHCDR2) of SEQ ID NO: 10; At least one of the following residues: Tyr33, Leu34, Asn35, Asp38 and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 11; At least one of the following residues: Ser31, Tyr33, Leu34, Asn35, Asp38 and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 20; At least one of the following residues: Asn9 of the light chain variable sequence (VLCDR1) of SEQ ID NO: 12; At least one of the following residues: Tyr12 and Ser18 of the heavy chain variable sequence (VLCDR2) of SEQ ID NO: 13; or At least one of the following residues: Ser22, Asn23 and Trp25 of the heavy chain variable sequence (VLCDR3) of SEQ ID NO:

14.

5. An isolated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising: (i) Contains the heavy chain variable region (VH) of SEQ ID NO: 9 and Contains the light chain variable region (VL) of SEQ ID NO: 6; or (ii) Containing the heavy chain variable region (VH) of SEQ ID NO: 18 and Contains the light chain variable region (VL) of SEQ ID NO: 6; or (iii) The heavy chain variable region (VH) containing SEQ ID NO: 19 and Contains the light chain variable region (VL) of SEQ ID NO: 6; or (iv) The heavy chain variable region (VH) containing SEQ ID NO: 21 and It contains the light chain variable region (VL) of SEQ ID NO:

6.

6. An isolated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising... (i) Containing the heavy chain variable region (VH) of SEQ ID NO: 5 or Contains the heavy chain variable region (VH) of SEQ ID NO: 22; and (ii) Contains the light chain variable region (VL) of SEQ ID NO:

6.

7. An isolated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising: Heavy chain complementarity-determining regions 1 (VHCDR1), 2 (VHCDR2), and / or 3 (VHCDR3) derived from the heavy chain variable region amino acid sequence listed in SEQ ID NO: 5; and Light chain complementarity-determining region 1 (VLCDR1), light chain complementarity-determining region 2 (VLCDR2) and / or light chain complementarity-determining region 3 (VLCDR3) obtained from the amino acid sequence of the light chain variable region listed in SEQ ID NO:

6.

8. The antibody or antigen-binding portion thereof according to any one of claims 1 to 7, further comprising a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7; The HCCR may optionally include one, two, or three conserved amino acid substitutions.

9. The antibody or antigen-binding portion thereof according to any one of claims 1 to 7, further comprising a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8; wherein the LCCR optionally comprises one, two or three conserved modified amino acid substitutions.

10. The antibody or antigen-binding portion thereof according to any one of claims 1 to 7, further comprising: a heavy chain constant region (HCCR) including the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally includes one, two, or three conserved modified amino acid substitutions; and The light chain constant region (LCCR) includes the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally includes one, two or three conserved amino acid substitutions.

11. The antibody or its antigen-binding portion according to claim 10, further comprising... The heavy chain signal peptide (HCSP) at the N-terminus of the VH comprises the amino acids listed in SEQ ID NO:

15.

12. The antibody or antigen-binding portion thereof according to claim 10, further comprising a light chain signal peptide (LCSP) at the N-terminus of the VL, the light chain signal peptide comprising the amino acids listed in SEQ ID NO:

16.

13. The antibody or its antigen-binding portion according to any one of claims 1 to 7, wherein the antibody is a human or humanized antibody.

14. The antibody or its antigen-binding portion according to any one of claims 1 to 7, wherein the antibody is an IgG4 isotype.

15. A pharmaceutical composition for treating, inhibiting, and / or suppressing solid tumors, comprising: (i) the antibody or its antigen-binding portion according to any one of claims 1 to 7; and (ii) Pharmaceutically acceptable carriers.

16. A polynucleotide molecule encoding an antibody or antigen-binding moiety according to any one of claims 1 to 7.

17. An expression vector comprising the polynucleotide molecule of claim 16, wherein the expression vector encoding LCSP does not contain introns.

18. A host cell comprising the expression vector according to claim 17.

19. A method for preventing, treating, and / or suppressing tumors in an individual, comprising the following steps: An effective amount of the antibody or antigen-binding moiety according to claims 1 to 7 is administered to the individual.

20. The method of claim 19, wherein the tumor comprises hepatocellular carcinoma, or a solid tumor comprising melanoma and / or renal cell carcinoma (RCC).

21. A method for regulating the activity of one or more T cells in an individual in vitro, ex vivo, and / or in vivo, the method comprising the following steps: The T cells are contacted with an antibody or its antigen-binding moiety that preferentially binds to human programmed death receptor 1 (hPD-1), wherein the antibody blocks and / or modulates the binding of human PD-L1 and / or human PD-L2 to hPD-1, and wherein the antibody or its antigen-binding moiety comprises: Includes the heavy chain complementarity-determining region 1 (VHCDR1) of the amino acid sequences listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21, includes the heavy chain complementarity-determining region 2 (VHCDR2) of the amino acid sequences listed in SEQ ID NO: 10, and / or includes the heavy chain complementarity-determining region 3 (VHCDR3) of the amino acid sequences listed in SEQ ID NO: 11, SEQ ID NO: 20, or SEQ ID NO: 23; and It includes light chain complementarity-determining region 1 (VLCDR1) of the amino acid sequence listed in SEQ ID NO: 12, light chain complementarity-determining region 2 (VLCDR2) of the amino acid sequence listed in SEQ ID NO: 13, and / or light chain complementarity-determining region 3 (VLCDR3) of the amino acid sequence listed in SEQ ID NO:

14.

22. The method of claim 21, wherein the antibody or its antigen-binding portion further comprises a heavy chain constant region (HCCR), the heavy chain constant region comprising the amino acid sequence listed in SEQ ID NO: 7, and the HCCR optionally comprising one, two or three conserved modified amino acid substitutions.

23. The method of claim 21, wherein the antibody or its antigen-binding portion further comprises a light chain constant region (LCCR), the light chain constant region comprising the amino acid sequence of SEQ ID NO: 8, and wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions.

24. The method of claim 21, wherein the one or more T cells comprise one or more natural killer cells, one or more cytotoxic T cells, one or more helper T cells, one or more monocytes, one or more memory T cells and / or one or more regulatory T cells.

25. The method of claim 21, wherein the antibody or antigen binding portion is at an IC50 concentration of about 1 nM or lower. 50 Blocking the binding of human PD-L1 and human PD-L2 to human PD-1.

26. A method for generating an antibody or antigen-binding partial vector construct, comprising: The host cells according to claim 18 are cultured under conditions that allow expression of the antibody or antigen-binding moiety construct; and The resulting antibody or antigen-binding vector construct is purified and / or recovered from the culture.

27. An antibody or its antigen-binding portion for treating solid tumors, comprising: a. Containing the heavy chain variable region (VH) of SEQ ID NO: 5 or the heavy chain variable region (VH) of SEQ ID NO: 22; b. Contains the light chain variable region (VL) of SEQ ID NO: 6; and Optionally, another therapeutic agent may be included.

28. The antibody or antigen-binding portion thereof according to claim 27, further comprising: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO:7, wherein the HCCR optionally comprises one, two, or three conserved amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO:8, wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions; and Optionally, another therapeutic agent may be included.

29. The antibody or its antigen-binding portion according to claim 27, wherein the other therapeutic agent comprises P1101, P1801 and / or another anti-human PD1 antibody.

30. The antibody or its antigen-binding portion according to claim 28, wherein the other therapeutic agent comprises P1101, P1801 and / or another anti-human PD1 monoclonal antibody.

31. The antibody or its antigen-binding portion according to any one of claims 28 to 30, wherein the solid tumor comprises hepatocellular carcinoma, renal cell carcinoma, and / or melanoma.

32. A combination for treating, inhibiting, preventing, and / or suppressing a solid tumor in an individual, wherein the combination comprises... Antibody or its antigen-binding portion; and Another treatment, The antibody or its antigen-binding portion comprises: a. Containing the heavy chain variable region (VH) of SEQ ID NO: 5 or the heavy chain variable region (VH) of SEQ ID NO: 22; and b. Contains the light chain variable region (VL) of SEQ ID NO:

6.

33. The combination of claim 32, wherein the antibody or its antigen-binding portion further comprises: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally comprises one, two or three conserved amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two or three conserved amino acid substitutions.

34. The combination according to any one of claims 32 to 33, wherein the therapeutic agent comprises P1101, P1801 and / or another anti-human PD1 monoclonal antibody.

35. The combination of claim 34, wherein the P1101, P1801 and / or another anti-human PD1 monoclonal antibody are administered sequentially and / or approximately simultaneously.

36. The combination of claim 35, wherein P1101 and / or P1801 is administered prior to another anti-human PD1 monoclonal antibody.

37. The combination according to claim 32 or 33, wherein the antibody or its antigen-binding portion and the other therapeutic agent are administered as a single composition or as separate compositions.

38. The combination of any one of claims 32 to 37, wherein the solid tumor comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

39. A recombinant protein comprising... (i) the antibody or antigen-binding moiety according to claim 1; and (ii) An optional tag sequence for expressing detection and / or purification.

40. The isolated antibody or its antigen-binding portion according to any one of claims 1 to 2, 5 to 7 or 27, in a method of treating cancer, the method comprising administering the isolated antibody or its antigen-binding portion to an individual.

41. The isolated antibody or its antigen-binding portion according to claim 40, wherein the cancer comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

42. Use of an isolated antibody or its antigen-binding portion according to any one of claims 1 to 2, 5 to 7 or 27 for the manufacture of a medicament for therapeutic and / or preventive treatment of cancer.

43. Use of the isolated antibody or its antigen-binding portion according to claim 42, wherein the cancer comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

Images