Anti-pd-1 / ctla-4 / vegf antibodies and use thereof

Abstract

Provided are anti-PD-1 antibodies and tri-specific antibodies, or antigen-binding fragments thereof, that specifically bind to PD-1, CTLA-4, and VEGF, polynucleotides encoding the antibodies, or antigen-binding fragments thereof, and methods of making and using the same.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480036734.7 (22) Application Date 2024.04.26 (66) Domestic Priority Data PCT / CN2023 / 107464 2023.07.14 CN (85) PCT International Application Entering National Phase Date 2025.12.01 (86) PCT International Application Application Data PCT / CN2024 / 089982 2024.04.26 (87) PCT International Application Publication Data WO2025 / 016011 EN 2025.01.23 (71) Applicant Jiahe Biopharmaceutical Co., Ltd. Address: Room 401, Building 6, No. 690 Bibo Road, China (Shanghai) Pilot Free Trade Zone, Pudong New Area, Shanghai 201203, China Room 17 (72) Inventors: Lv Yaxuan, Peng Fei, Xu Juehua, Qian Zhen, Cao Huanhuan, Yang Xueyan, Ding Qian, Tan Yongcong, Han Shuhua, Du Qinglin (74) Patent Agency: China Patent Agency (Hong Kong) Limited 72001 Patent Attorney: Li Zhiqiang, Huang Nian (51) Int.Cl. C07K 16 / 28 (2006.01) A61K 39 / 395 (2006.01) (54) Invention Title: Anti-PD-1 / CTLA-4 / VEGF Antibody and Its Use (57) Abstract: Provided are an anti-PD-1 antibody and a trispecific antibody or antigen-binding fragment thereof that specifically binds to PD-1, CTLA-4 and VEGF, a polynucleotide encoding the antibody or antigen-binding fragment thereof, and methods for preparing and using the above. Claims (3 pages), Description (38 pages), Sequence Listing (electronic publication), Figures (11 pages), CN 121335922 A 2026.01.13 CN 1 21 33 59 22 A 1. An antibody or antigen-binding fragment thereof that binds to PD-1 (programmed cell death protein 1), comprising: a heavy chain antibody variable domain (VHH), said VHH comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein VHH CDR1 comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 1, VHH CDR2 comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and VHH CDR3 comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 3. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein said VHH comprises CDRs 1, 2, and 3, each having an amino acid sequence as shown in SEQ ID NO: 1, 2, and 3. 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the VHH comprises the same as SEQ ID NO:4. An amino acid sequence having at least 80% identity. 4. The antibody or antigen-binding fragment of claim 3, wherein the VHH comprises, or is composed of, the amino acid sequence of SEQ ID NO: 4. 5. The antibody or antigen-binding fragment of any one of claims 1-4, wherein the antibody or antigen-binding fragment specifically binds to PD-1. 6. The antibody or antigen-binding fragment of any one of claims 1-5, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment of the same. 7. An antibody or antigen-binding fragment of the same, comprising VHH CDRs 1, 2, and 3 of the antibody or antigen-binding fragment of any one of claims 1-6. 8. The antibody or antigen-binding fragment of any one of claims 1-7, wherein the antibody or antigen-binding fragment comprises two or more heavy-chain antibody variable domains. 9. An antibody or antigen-binding fragment of the same, which cross-competes with the antibody or antigen-binding fragment of any one of claims 1-8. 10. A multispecific antibody or antigen-binding fragment of the same, comprising a first domain specifically binding to PD-1 and a second domain specifically binding to CTLA-4. 11. The multispecific antibody or antigen-binding fragment thereof according to claim 10, wherein a) the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1), the VH1 comprising complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 1, 2, and 3, respectively; and / or b) the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2), the VH2 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 7, 8, and 9, respectively. 12. The multispecific antibody or antigen-binding fragment thereof according to claim 10 or 11, wherein a) the first domain specifically binding to PD-1 comprises or is composed of the heavy chain variable region (VH1) of SEQ ID NO: 4; and / or b) the second domain specifically binding to CTLA-4 comprises or is composed of the heavy chain variable region (VH2) of SEQ ID NO: 10. 13. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 10-12, wherein the antibody further comprises one or more additional domains that specifically bind to antigens other than PD-1 and CTLA-4.14. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 10-13, further comprising a domain specifically binding to VEGF. 15. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 10-14, which is an anti-PD-1 / CTLA-4 / VEGF trispecific antibody or antigen-binding fragment thereof, comprising a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to VEGF. 16. The multispecific antibody or antigen-binding fragment thereof according to claim 15, wherein a) the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1), the VH1 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 1, 2, and 3, respectively; b) the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2), the VH2 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 7, 8, and 9, respectively; and / or c) the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) and a light chain variable region (VL3), the VH3 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 13, 14, and 15, respectively, and the VL ... 19, 20, and 21 light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3). 17. The multispecific antibody or antigen-binding fragment thereof according to claim 16, wherein the VH1 comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 4, and / or the VH2 comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 10. 18. The multispecific antibody or antigen-binding fragment thereof according to claim 16, wherein a) the first domain specifically binding to PD-1 comprises, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 4; b) the second domain specifically binding to CTLA-4 comprises, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 10; and / or c) the third domain specifically binding to VEGF comprises, and is composed of, the heavy chain variable region (VH) of SEQ ID NO: 16.19. The antibody or antigen-binding fragment thereof according to any one of claims 1-18, wherein the antibody further comprises the Fc region of IgG. 20. The antibody or antigen-binding fragment thereof according to any one of claims 10-19, wherein the first domain, second domain, third domain and / or Fc region are directly interconnected via one or more linkers. 21. The antibody or antigen-binding fragment thereof according to any one of claims 20, wherein the linkers are the same or different, and / or wherein the linkers are flexible linkers, and / or wherein the linkers are peptide linkers. 22. The antibody or antigen-binding fragment thereof according to any one of claims 15-21, wherein the antibody comprises the heavy chain of SEQ ID NO: 27 and the light chain of SEQ ID NO: 23, or comprises them. 23. The antibody or antigen-binding fragment thereof according to any one of claims 1-22, wherein the antibody is an isotype of IgG1, IgG2, IgG3 or IgG4, optionally comprising one, two, three, four, five, six, seven, eight, nine or ten substitutions in the Fc region. 24. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-23 and a pharmaceutically acceptable carrier. 25. A polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-23. 26. A carrier comprising the polynucleotide according to claim 25. 27. An isolated host cell comprising the carrier according to claim 26. 28. A method of generating an antibody or antigen-binding fragment thereof, the method comprising culturing a host cell according to claim 27 under conditions expressing the antibody or antigen-binding fragment thereof, and recovering and purifying the antibody or antigen-binding fragment generated by the host cell. 29. A method of treating a subject with cancer, the method comprising administering to a subject in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-23 or a pharmaceutical composition according to claim 24, for a duration sufficient to treat the cancer. 30. The method according to claim 29, wherein the cancer is a solid tumor or a hematologic malignancy. 31. The method of claim 30, wherein the solid tumor is melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, stomach cancer, ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal cancer, etc.32. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-23 or the pharmaceutical composition of claim 24 in the preparation of a medicament for treating a subject with cancer of need. 33. The use according to claim 32, wherein the cancer is a solid tumor or a hematologic malignancy. 34. The use according to claim 33, wherein the solid tumor is melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, stomach cancer, ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary cancer, endometriosis, cervical cancer, or a metastatic lesion of cancer. 35. The antibody or antigen-binding fragment thereof according to any one of claims 1-23, or the pharmaceutical composition according to claim 24, for treating cancer in a subject in need. 36. The antibody or antigen-binding fragment thereof or pharmaceutical composition according to claim 35, wherein the cancer is a solid tumor or a hematologic malignancy. 37. The antibody or antigen-binding fragment thereof or pharmaceutical composition according to claim 36, wherein the solid tumor is melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, stomach cancer, ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary cancer, endometriosis, cervical cancer, or a metastatic lesion of cancer. Claims 3 / 3 Page 4 CN 121335922 A Anti-PD-1 / CTLA-4 / VEGF Antibody and Its Use Technical Field

[0001] This invention relates to anti-PD-1 antibodies, trispecific antibodies that specifically bind to PD-1, CTLA-4 and VEGF or antigen-binding fragments thereof, polynucleotides encoding the antibody or antigen-binding fragments thereof, and methods for preparing and using the above. Background Art

[0002] PD-1 (programmed cell death protein 1) is a receptor mainly expressed on the surface of T cells. PD-1 regulates T cell function by inhibiting T cell activation and proliferation and promoting T cell exhaustion (Liu et al., 2021; Sharpe and Pauken,PD-1 is activated by its ligands PD-L1 and PD-L2, which are expressed on certain tumor cells and other antigen-presenting cells. In preclinical and clinical studies, PD-1 inhibition has been shown to enhance T-cell activity and promote anti-tumor immune responses (Chen et al., 2021; B. Zhao, Zhao and Zhao, 2020). PD-1 inhibitors, such as nivolumab (Opidivo®) and pembrolizumab (Keytruda®), have been approved by the FDA for the treatment of several cancer types, including lung cancer, melanoma, and bladder cancer. Keytruda is used to treat several types of cancer, including melanoma, non-small cell lung cancer, and head and neck cancer. Nivolumab is used to treat cancers including melanoma, non-small cell lung cancer, and renal cell carcinoma.

[0003] VEGF (vascular endothelial growth factor) is a protein that plays a crucial role in angiogenesis (the process of forming new blood vessels). Because angiogenesis is essential for the growth and metastasis of solid tumors, VEGF is an important target for cancer therapy. VEGF is secreted by cells (including tumor cells) in the tumor microenvironment (TME). The presence of VEGF promotes the proliferation and migration of cells (such as tumor cells) (Melincovici et al., 2018; Simons, Gordon, and Claesson-Welsh, 2016). In preclinical and clinical studies, VEGF inhibition has been shown to interfere with tumor angiogenesis and reduce tumor growth (Baraniskin et al., 2019; Garcia et al., 2020; Y. Zhao et al., 2022). VEGF inhibitors, such as bevacizumab (Avastin®), which block the binding of VEGF to VEGFR1 and VEGFR2, thereby inhibiting downstream signaling and angiogenesis, have been approved by the FDA for the treatment of several cancer types, including colorectal cancer, non-small cell lung cancer, and hepatocellular carcinoma.

[0004] Cytotoxic lymphocyte-associated molecule-4 (CTLA-4, CD152) is another immune checkpoint protein that regulates T cell function. As the first clinically targeted immune checkpoint receptor expressed on T cells, CTLA-4 shares the same ligands as CD28, namely CD80 / B7-1 and CD86 / B7-2. CTLA-4 is constitutively expressed on Foxp3+ regulatory T cells (Tregs) and upregulated on other activated T cells (Jago, Yates, Câmara, Lechler, and Lombardi, 2004; Ś ledzińska, Menger,Bergerhoff, Peggs, and Quezada, 2015). The mechanism of CTLA-4 is thought to be by inhibiting T cell activation by outcompeting CD28 in binding to CD80 / B7-1 and CD86 / B7-2 (Chikuma, 2017). CTLA-4 inhibitors, such as ipilimumab, have been approved by the FDA for the treatment of several cancer types, including melanoma and prostate cancer.

[0005] Ipilimumab, the most well-known anti-CTLA4 antibody, was approved in 2011 for the treatment of advanced melanoma. Ipilimumab has demonstrated potent cancer immunotherapy effects in clinical practice as a monotherapy (Hodi et al., 2010) and as part of a combination therapy with nivolumab (Larkin et al., 2015). However, CTLA-4 therapy has shown serious immunotherapy-related adverse events (irAEs) (Calabrese, Calabrese, and Cappelli, 2018), especially when combined with nivolumab therapy, which reduces patient tolerance to the antibody due to systemic activation of T cells by blocking the B7-CTLA-4 pathway (Bertrand, Kostine, Barnetche, Truchetet, and Schaeverbeke, 2015; Hodi, 2010). Nevertheless, CTLA-4 remains an important immunotherapy target because it can induce durable immunity in cancer patients (Maio et al., 2015; Schadendorf et al., 2015). The main challenge in producing CTLA-4 antibodies is to improve their safety and efficacy.

[0006] In summary, PD-1, CTLA-4, and VEGF are important targets for cancer therapy, and their inhibition has shown promising results in both preclinical and clinical studies. PD-1 and CTLA-4 inhibitors enhance T cell activity and promote anti-tumor immune responses, while VEGF inhibitors interfere with tumor angiogenesis and reduce tumor growth. However, drugs like ipilimumab have various side effects. Therefore, further research is needed to optimize the use of these inhibitors and explore their potential in combination with other cancer therapies.

[0007] This invention provides a separated antibody or antigen-binding fragment thereof that binds to PD-1 (programmed cell death protein 1), comprising: a heavy chain antibody variable domain (VHH), said VHH comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 1, and the VHH CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 1.2. An amino acid sequence having at least 80% identity, and the VHHCDR3 region comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 3.

[0008] In one embodiment, VHH comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively. In one embodiment, VHH comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 4. In one embodiment, VHH comprises the amino acid sequence of SEQ ID NO: 4, or is composed of the amino acid sequence of SEQ ID NO: 4.

[0009] In one embodiment, the antibody or antigen-binding fragment specifically binds to PD-1.

[0010] In one embodiment, the antibody or antigen-binding fragment is a humanized antibody or an antigen-binding fragment thereof.

[0011] The present invention also provides an isolated antibody or an antigen-binding fragment thereof comprising the VHH CDRs 1, 2, and 3 of the antibody or antigen-binding fragment described above.

[0012] In one embodiment, the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

[0013] The present invention also provides an isolated antibody or antigen-binding fragment thereof, which cross-competes with the above-described antibody or antigen-binding fragment thereof.

[0014] The present invention also provides an isolated multispecific antibody or antigen-binding fragment thereof, comprising a first domain specifically binding to PD-1 and a second domain specifically binding to CTLA-4. In one embodiment, the antibody or antigen-binding fragment thereof further comprises one or more additional domains specifically binding to antigens other than PD-1 and CTLA-4. In one embodiment, the antibody or antigen-binding fragment thereof further comprises a domain specifically binding to VEGF.

[0015] In one embodiment, an anti-PD-1 / CTLA-4 / VEGF trispecific antibody or antigen-binding fragment thereof is provided, comprising a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to VEGF.

[0016] In one embodiment, the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1), said VH1 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 1, 2, and 3, respectively. Specification 2 / 38 pages 6 CN 121335922 A

[0017] In one embodiment, the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1), said VH1 comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 4. In one embodiment, specifically...The first domain that binds to PD-1 comprises, or is composed of, the heavy chain variable region (VH1) of SEQ ID NO: 4.

[0018] In one embodiment, the second domain that specifically binds to CTLA-4 comprises a heavy chain variable region (VH2), wherein the VH2 comprises heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 7, 8, and 9, respectively.

[0019] In one embodiment, the second domain that specifically binds to CTLA-4 comprises a heavy chain variable region (VH2), wherein the VH2 comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 10. In one embodiment, the second domain that specifically binds to CTLA-4 comprises, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 10.

[0020] In one embodiment, the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) and a light chain variable region (VL), wherein the VH3 comprises heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 13, 14, and 15, respectively; and the VL comprises light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) of SEQ ID NO: 19, 20, and 21, respectively.

[0021] In one embodiment, the third domain specifically binding to VEGF comprises, or is composed of, the heavy chain variable region (VH3) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO: 22.

[0022] In one embodiment, the third domain specifically binding to VEGF comprises, or is composed of, the heavy chain of SEQ ID NO: 17 and the light chain of SEQ ID NO: 23. In one embodiment, the third domain that specifically binds to VEGF comprises a full-length antibody comprising two heavy chains and two light chains interconnected by disulfide bonds, or an antigen-binding fragment thereof, or composed thereof. In one embodiment, the antigen-binding fragment of the full-length antibody is a Fab fragment or an scFv fragment.

[0023] In one embodiment, the isolated antibody or its antigen-binding fragment further comprises the Fc region of IgG. In one embodiment, the Fc region belongs to IgG1. In one embodiment, the Fc region belongs to IgG1 LALA (IgG1 with the Leu234Ala / Leu235Ala mutation).

[0024] In one embodiment, the antibody or its antigen-binding fragment according to the invention belongs to IgG1, IgG2, IgG3, orThe IgG4 isotype optionally includes one, two, three, four, five, six, seven, eight, nine, or ten substitutions in the Fc region.

[0025] In one embodiment, a first domain specifically binding to PD-1 is located at the N-terminus of the Fc region.

[0026] In one embodiment, a second domain specifically binding to CTLA-4 is located at the N-terminus of the Fc region.

[0027] In one embodiment, a third domain specifically binding to VEGF is located at the N-terminus of the Fc region.

[0028] In one embodiment, a first domain specifically binding to PD-1 is located at the C-terminus of the Fc region.

[0029] In one embodiment, a second domain specifically binding to CTLA-4 is located at the C-terminus of the Fc region.

[0030] In one embodiment, a third domain specifically binding to VEGF is located at the C-terminus of the Fc region.

[0031] In one embodiment, the first domain specifically binding to PD-1, the second domain specifically binding to CTLA-4, the third domain specifically binding to VEGF, and the Fc region are directly interconnected or interconnected via one or more adapters. In one embodiment, the adapters may be the same or different. In one embodiment, the adapter is a flexible adapter. In one embodiment, the adapter is a peptide adapter. In one embodiment, the adapter is GGGGSGGGGS.

[0032] In one embodiment, the third domain specifically binding to VEGF is a Fab fragment or an scFv fragment, and the specification page 3 / 38 7 CN 121335922 A and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention is represented from the N-terminus to the C-terminus by the following formula: Third domain – hinge region – Fc – adapter – second domain – adapter – first domain.

[0033] In one embodiment, the third domain specifically binding to VEGF is a full-length antibody. In one embodiment, a first domain specifically binding to PD-1 is optionally linked via a linker to the C-terminus of any one or both heavy chains of the full-length antibody, and a second domain specifically binding to CTLA-4 is optionally linked via a linker to the C-terminus of the first domain specifically binding to PD-1. In another embodiment, a second domain specifically binding to CTLA-4 is optionally linked via a linker to the C-terminus of any one or both heavy chains of the full-length antibody, and a first domain specifically binding to PD-1 is optionally linked via a linker to the C-terminus of the second domain specifically binding to CTLA-4. In another embodiment, the first domain specifically binding to PD-1 and the second domain specifically binding to CTLA-4 are linked to the C-terminus of two heavy chains of the full-length antibody in the same or different order. In another embodiment, the two domains specifically binding to PD-1 are linked to the C-terminus of the first and second heavy chains of the full-length antibody.Each of the second CTLA-4 binding domains is connected to the C-terminus of the heavy chain of the full-length antibody via a linker, and each of the two first domains specifically binding to PD-1 is connected to the C-terminus of the two second domains specifically binding to CTLA-4 via a linker.

[0034] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention comprises, or is composed of, the heavy chain of SEQ ID NO: 27 and the light chain of SEQ ID NO: 23.

[0035] In one embodiment, the antibody according to the invention comprises, or is composed of, one or two heavy chains and one or two light chains. In one embodiment, the antibody according to the invention comprises, or is composed of, two heavy chains and two light chains. In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention comprises, a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to VEGF. In one embodiment, a first domain specifically binding to PD-1 includes a heavy chain variable region (VH1), which contains heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 1, 2, and 3, respectively; a second domain specifically binding to CTLA-4 includes a heavy chain variable region (VH2), which contains heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 7, 8, and 9, respectively; and a third domain specifically binding to VEGF includes a heavy chain variable region (VH3) and a light chain variable region (VL), where VH3 contains heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 13, 14, and 15, respectively, and VL contains light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (HCDR3) of SEQ ID NOs: 19, 20, and 21, respectively. (LCDR3). In one embodiment, the first domain specifically binding to PD-1 comprises, or is composed of, the heavy chain variable region (VH1) of SEQ ID NO: 4; the second domain specifically binding to CTLA-4 comprises, or is composed of, the heavy chain variable region (VH2) of SEQ ID NO: 10; and the third domain specifically binding to VEGF comprises, or is composed of, the heavy chain variable region (VH3) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO: 22. In one embodiment, the heavy chain comprises, SEQ ID NO: 4.The amino acid sequence of NO: 27 and the light chain of SEQ ID NO: 23, or composed thereof.

[0036] The present invention also provides a pharmaceutical composition comprising the antibody of the present invention or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0037] The present invention also provides a polynucleotide encoding a heavy chain variable region (VH) or a heavy chain encoding the antibody of the present invention or an antigen-binding fragment thereof.

[0038] The present invention also provides a polynucleotide encoding the antibody of the present invention or an antigen-binding fragment thereof.

[0039] The present invention also provides a carrier comprising the polynucleotide of the present invention.

[0040] The present invention also provides a host cell comprising the carrier of the present invention. Specification 4 / 38 pages 8 CN 121335922 A

[0041] The present invention also provides a method for producing the antibody of the present invention, the method comprising culturing the host cell of the present invention under conditions expressing the antibody or an antigen-binding fragment thereof, and recovering the antibody produced by the host cell.

[0042] The present invention also provides a method for treating a subject with cancer, the method comprising administering to a subject in need a therapeutically effective amount of an isolated antibody or an antigen-binding fragment thereof for a duration sufficient to treat the cancer.

[0043] The present invention also provides the use of the isolated antibody or antigen-binding fragment thereof of the present invention in the preparation of a medicament for treating cancer in subjects in need.

[0044] The present invention also provides the isolated antibody or antigen-binding fragment thereof of the present invention for treating cancer in subjects in need. Brief Description of the Drawings

[0045] Figure 1 shows a schematic structural diagram of the trispecific antibody GBD214-33-03 (anti-PD1 / CTLA4 / VEGF trispecific antibody) according to the present invention.

[0046] Figure 2 shows the binding specificity of different antibodies to human PD1 protein. The results show the binding of GBD214-33-03 to PD1 in HEK293-PD1 cells. GBD214-33-03 showed strong binding to HEK293-PD1 cells with a median fluorescence intensity (MFI) similar to that of its PD1 parent GBD002-hS019-WS (anti-PD1 antibody) and pembrolizumab.

[0047] Figure 3 shows the performance of different antibodies in PD1 reporter gene assays. PD1 reporter gene assays showed that GBD214-33-03 had a similar maximum luminescent signal to pembrolizumab and AK104 (an anti-PD1 / CTLA4 bispecific antibody from Akesobio, Inc.). This result indicates that GBD214-33-03 can block the interaction between PD1 and its ligand PD-L1.

[0048] Figure 4 shows the performance of different antibodies in a mixed lymphocyte reaction (MLR) assay. The results show that GBD214-33-03 had the highest maximum IFN-γ in the MLR assay.The results show that GBD214-33-03 exhibits similar immunogenicity to its parent, GBD002-hS019-WS, and pembrolizumab. This indicates that GBD214-33-03 possesses similar potent immunogenicity to pembrolizumab.

[0049] Figure 5 shows the VEGF binding of different antibodies. The results show that GBD214-33-03 exhibits VEGF binding similar to that of bevacizumab.

[0050] Figure 6 shows the VEGF blocking of different antibodies. The results show that GBD214-33-03 inhibits the binding of VEGFR2 to VEGF165, similar to bevacizumab.

[0051] Figure 7 shows the performance of different antibodies in VEGF reporter gene assays. The results show that GBD214-33-03 inhibits VEGF165-induced downstream NFAT signaling by blocking the binding of VEGFR2 to VEGF165, similar to bevacizumab.

[0052] Figures 8a and 8b show the binding specificity of different antibodies to human CTLA4 protein. The results show that in CHOK1-CTLA4 cells, GBD214-33-03 has a lower MFI compared to ipilimumab, suggesting that GBD214-33-03 binds to the CTLA4 portion in CHOK1-CTLA4 cells. The CTLA4 parent GBD008-hS005-3-2 (anti-CTLA4 antibody) has a weaker CTLA4 binding ability compared to ipilimumab. In CHOK1-PD1-CTLA4 cells, GBD214-33-03 shows a stronger binding than its PD1 parent GBD002-hS019-WS.

[0053] Figures 9a to 9d show the blocking effect of different antibodies on the binding of CD80 and CD86 to CTLA4. The results showed that GBD214-33-03 partially inhibited the binding of CD80 / CD86 to CTLA4 in CHOK1-CTLA4 and CHOK1-PD1-CTLA4 cells. GBD214-33-03 showed weaker blocking ability compared to ipilimumab in both CHOK1-CTLA4 and CHOK1-PD1-CTLA4 cells.

[0054] Figure 10 shows the downregulation of the PD1 receptor in CHOK1-PD1-CTLA4 cells by different antibodies. The results showed that the downregulation of PD1 in the cell datasheet, page 5 / 38, CN 121335922 A, was due to CTLA4 internalization. The percentage of PD1 downregulation was measured. GBD214-33-03 showed similar PD1 internalization compared to AK104.

[0055] Figures 11a and 11b show the in vivo antitumor efficacy of different antibodies in the A375 PBMC mouse model. The results showed that in the A375 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg) was effective.The AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg) combination (1 mg / kg, 5 mg / kg, and 20 mg / kg), AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), pembrolizumab, bevacizumab, and ipilimumab-LALA (ipilimumab with Leu234Ala / Leu235Ala mutations) showed better efficacy compared to the combination of pembrolizumab, bevacizumab, and ipilimumab-LALA (ipilimumab with Leu234Ala / Leu235Ala mutations) (15 mg / kg + 15 mg / kg + 15 mg / kg). For all treatment groups, the mean body weight of mice decreased from day 7, with a decrease of no more than 15%.

[0056] Figures 12a and 12b show the in vivo antitumor efficacy of different antibodies in the HT29 PBMC mouse model. The results showed that in the HT29 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) had better potency than the combination of AK112 (1 mg / kg, 5 mg / kg, and 20 mg / kg), AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), pembrolizumab, bevacizumab, and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg). For all treatment groups, the mean body weight of mice remained relatively stable, with a decrease of no more than 15%. Detailed Description

[0057] All publications referenced in this specification, including but not limited to patents and patent applications, are incorporated herein by reference as if fully illustrated.

[0058] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0059] Although any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, exemplary materials and methods are described herein. The following terms will be used in describing and claiming the invention.

[0060] As used in this specification and the appended claims, unless the context clearly specifies otherwise, the singular forms “an,” “a,” and “the / described” include plural references. Thus, for example, a reference to “a cell” includes a combination of two or more cells, and so on.

[0061] “Specific binding” or “specifically binding” or “binding” means that an antibody binds to an antigen or an epitope within an antigen with a higher affinity than to other antigens. Typically, an antibody binds to an antigen or an epitope within an antigen with an affinity of about 1 × 10⁻⁸ M or less, for example…The antibody binds to an equilibrium dissociation constant (KD) of approximately 1 × 10⁻⁹ M or less, approximately 1 × 10⁻¹⁰ M or less, approximately 1 × 10⁻¹¹ M or less, or approximately 1 × 10⁻¹² M or less, where the KD is typically at most 1 / 100 of the KD of its binding to nonspecific antigens (e.g., BSA, casein). The dissociation constant can be determined using standard procedures. However, antibodies that specifically bind to antigens or epitopes within antigens may exhibit cross-reactivity with other related antigens, such as with the same antigens (homologs) from other species, such as humans or monkeys, for example, cynomolgus cyno, chimpanzee chimp, or common marmoset. When a monospecific antibody binds specifically to one antigen or one epitope, a bispecific antibody binds specifically to two different antigens or two different epitopes.

[0062] "Antibody" means, in a broad sense, and includes immunoglobulin molecules, including monoclonal antibodies (including mouse, human, humanized, and chimeric monoclonal antibodies), antigen-binding fragments, bispecific or multispecific antibodies, dimer antibodies, tetramer antibodies or multimer antibodies, single-chain antibodies, domain antibodies, and any other modified configuration of an immunoglobulin molecule containing the antigen-binding site of the desired specificity specification 6 / 38 page 10 CN 121335922 A. "Full-length antibody" comprises two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and their multimers (e.g., IgM). Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (which contains domain CH1, hinge CH2, and CH3). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), which alternate with framework regions (FRs). Each VH and VL contains three CDRs and four FR segments, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0063] The “complementarity-determining region (CDR)” is the “antigen binding site” in the antibody. CDRs can be defined using various terms: (i) complementarity-determining regions (CDRs) (three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3)) based on sequence variability (Wu and Kabat, (1970) J Exp Med 132:211-50; Kabat et al., Sequencesof Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) “Hypervariable region”, “HVR”, or “HV” (three in VH (H1, H2, H3) and three in VL (L1, L2, L3)) refers to a region of highly variable structure within the variable domain of an antibody, as defined by Chothia and Lesk (Chothia and Lesk, (1987) Mol Biol 196:901-17). The International Immunogenetics (IMGT) database (http: / / www_imgt_org) provides standardized numbering and definitions of antigen-binding sites. The correspondence between CDR, HV, and IMGT classifications is described in Lefranc et al., (2003) Dev Comparat Immunol 27:55-77. Unless otherwise expressly stated in the specification, the terms “CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2,” and “LCDR3” as used herein include CDR as defined by any of the foregoing methods, Kabat, Chothia, or IMGT.

[0064] Immunoglobulins can be classified into five classes based on the amino acid sequence of their heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into the following isotypes: IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be classified into two distinct types based on the amino acid sequence of its constant domain, namely kappa (κ) and lambda (λ).

[0065] “Isolated antibody” refers to an antibody or antibody fragment that is substantially free of other antibodies with different antigen specificities (e.g., isolated antibodies that specifically bind to PD-1 are substantially free of antibodies that specifically bind to antigens other than PD-1). In the case of the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention, the trispecific antibody specifically binds to PD-1, CTLA-4 and VEGF, and is substantially free of antibodies that specifically bind to antigens other than PD-1, CTLA-4 and VEGF. "Isolated antibody" encompasses antibodies isolated to a high degree of purity, such as antibodies of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0066] “Recombinant” refers to antibodies and other proteins prepared, expressed, generated, or isolated through recombinant methods.

[0067] “Epitope” refers to the antigenic moiety that an antibody specifically binds to. Epitopes are typically composed of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups of moieties such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural features and specific charge features. Epitopes can be composed of continuous and / or discontinuous amino acids that form a conformational spatial unit. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are close to each other in three-dimensional space through the folding of the protein molecule. The antibody “epitope” depends on the method used to identify the epitope.

[0068] “Multispecific” refers to an antibody that specifically binds to at least two different antigens or two different epitopes within an antigen (e.g., three, four, or five different antigens or epitopes). “Trispecific” antibody refers to an antibody that specifically binds to three different antigens or three different epitopes within the same antigen. Trispecific antibodies may have cross-reactivity with other related antigens, such as cross-reactivity with the same antigen (homologous) from other species, such as humans or monkeys, for example, cynomolgus (Macaca fascicularis, cynomolgus, cyno), chimpanzee (Pan troglodytes, specification 7 / 38 page 11 CN 121335922 A chimpanzee, chimp), or common marmoset (Callithrix jacchus, common marmoset, marmoset), or may bind to epitopes shared between two or more different antigens.

[0069] A “vector” is a polynucleotide capable of replicating within a biological system or moving between such systems. Vector polynucleotides typically contain elements such as origin of replication, polyadenylation signals, or selection markers that function to facilitate the replication or maintenance of these polynucleotides within the biological system. Examples of such biological systems may include cells, viruses, animals, plants, and reconstructed biological systems utilizing biological components capable of replicating vectors. The polynucleotide containing the vector can be a DNA or RNA molecule or a hybrid thereof.

[0070] "Expression vector" means a vector that can be used in a biological system or reconstructed biological system to guide the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0071] "Polynucleotide" means a synthetic molecule containing nucleotide chains covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

[0072] "Overexpression," "overexpressing," and "overexpressing" are used interchangeably.And refers to samples such as cancer cells, malignant cells, or cancerous tissues that have measurably higher levels of PD-1, CTLA-4, or VEGF or their ligands compared to a reference sample. Overexpression can be caused by gene amplification or increased transcription or translation. Protein expression and overexpression in a sample can be measured in live or lysed cells using known assays such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassay. Polynucleotide expression and overexpression in a sample can be measured, for example, using fluorescence in situ hybridization, Southern blotting, or PCR techniques. A protein or polynucleotide is overexpressed when the level of a protein or polynucleotide in a sample is at least 1.5-fold or statistically significant compared to a reference sample. The selection of a reference sample is known.

[0073] “Sample” means a similar fluid, cell, or tissue isolated from a subject, and a collection of similar fluids, cells, or tissues present in the subject. Exemplary samples are biological fluids, such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, teardrops, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites (such as ascites associated with non-solid tumors), fluids from the pleural cavity, pericardial cavity, peritoneal cavity, abdominal cavity and other body cavities, fluids collected by bronchial lavage, liquid solutions that have come into contact with the subject or biological sources (e.g., cell and organ culture media, including cell or organ conditioned media, lavage fluid, etc.), tissue biopsies, tumor tissue removed by fine needle aspiration or surgery.

[0074] “Cancer cells” or “tumor cells” refer to cancer cells, precancerous cells or transformed cells in vivo, in vitro or in tissue culture that have spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. Although transformation can result from infection with a transforming virus and the incorporation of new genomic nucleic acids, the uptake of exogenous nucleic acids or their spontaneous occurrence or occurrence after exposure to carcinogens, thereby mutating endogenous genes. Transformation / cancer is exemplified by: morphological changes in vitro, in vivo, and ex vivo; immortalization of cells; abnormal growth control; lesion formation; proliferation; malignancy; adjustment of tumor-specific marker levels; invasiveness; and tumor growth in suitable animal hosts (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd edition 1994)).

[0075] “about” means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless otherwise expressly stated elsewhere in the embodiments or specification, in the context of a particular analysis, result, or embodiment, “about” means within a standard deviation according to practice in the art, or within a range not exceeding 5%, whichever is greater.

[0076] "Anti-PD-1 / CTLA-4 / VEGF trispecific antibody", "PD-1 / CTLA-4 / VEGF trispecific antibody", "PD-1 / CTLA-4 / VEGF antibody", or "trispecific anti-PD-1 / CTLA-4 / VEGF antibody" refers to a molecule containing at least one binding domain specifically binding to PD-1, at least one binding domain specifically binding to CTLA-4, and at least one binding domain specifically binding to VEGF. The binding domains specifically binding to PD-1, CTLA-4, and VEGF are typically VH / VL pairs or VH only. Trispecific anti-PD-1 / CTLA-4 / VEGF antibodies can be monovalent or bivalent in binding to PD-1, CTLA-4, or VEGF.

[0077] "Valence" refers to the presence of a specific number of binding sites in the molecule that are specific to the antigen. Therefore, the terms “monovalent,” “divalent,” “tetravalent,” and “hexavalent” refer to the presence of one, two, four, and six antigen-specific binding sites in the molecule, respectively.

[0078] The term “single-chain Fv” or “scFv” antibody refers to an antibody fragment containing the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired antigen-binding structure. For a review of scFv, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315. See also International Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203. In one embodiment, the scFv comprises a VH region, a peptide linker, and a VL region (VH-VL form) from the N-terminus to the C-terminus. In another embodiment, the scFv comprises a VL region, a peptide linker, and a VH region (VL-VH form) from the N-terminus to the C-terminus.

[0079] As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, which comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the two domains are forced to pair with a complementary domain of another chain, resulting in two antigen-binding sites. A more sufficiency description of diabodies is found, for example, EP 404,097, WO93 / 11161 and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA90: 6444-6448. For reviews of engineered antibody variants, see Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.

[0080] “Fab” comprises the VH and CH1 regions of the heavy chain and the VL and CL regions of the light chain, which are generally linked together by disulfide bonds and have a single antigen-binding site. According to this disclosure, the VH, CH1, VL, and CL regions in Fab can be arranged in various ways to confer antigen-binding capability. For example, the VH and CH1 regions can be on one polypeptide chain, while the VL and CL regions can be on separate polypeptide chains. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide chain, optionally arranged in different orders.

[0081] "Antigen-specific CD4+ or CD8+ T cells" refers to CD4+ or CD8+ T cells activated by a specific antigen or its immunostimulatory epitope.

[0082] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably.

[0083] "Treatment" or "treatment" refers to a therapeutic treatment aimed at slowing (reducing) undesirable physiological changes or diseases (such as the development or spread of tumors or tumor cells) or providing beneficial or desired clinical outcomes during treatment. Beneficial or desired clinical outcomes include symptom relief, reduction of disease severity, stabilization (i.e., no worsening) of the disease state, delay or slowing of disease progression, absence of metastasis, improvement or relief of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also refer to an extended survival compared to the expected survival of a subject without treatment. Subjects requiring treatment include those who already have undesirable physiological changes or diseases, and those who are prone to having physiological changes or diseases.

[0084] "Therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome at the necessary dose and time. The therapeutic effective amount of the antibody of the present invention may vary depending on factors such as disease state, individual age, sex and weight, and the ability of the antibody of the present invention to elicit the desired response in the individual. Exemplary indications of an effective treatment or treatment combination include, for example, improvement in the patient's health, reduction in tumor burden, cessation or slowing of tumor growth, and / or failure of cancer cells to metastasize to other parts of the body.

[0085] Unless otherwise expressly stated, the amino acid residues in the constant region of the antibody throughout this specification are numbered according to the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0086] This document uses conventional single-letter and three-letter amino acid codes, as shown in Table 1.

[0087] Table 1: Single-letter and three-letter amino acid codes Amino acid three-letter code Single-letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine ​​Cys C Glutamic acid Gln E Glutamine Glu Q Glycine Gly G Histidine His H Isoleucine Ile I Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Composition of substances The present invention provides anti-PD-1 antibodies, anti-PD-1 / CTLA-4 antibodies, and in particular trispecific antibodies or antigen-binding fragments thereof that specifically bind to PD-1, CTLA-4 and VEGF. The present invention provides polynucleotides or complementary nucleic acids thereof encoding the antibodies of the present invention or antigen-binding fragments thereof, vectors, host cells, and methods for preparing and using them.

[0088] In the context of this disclosure, the antibodies of the present invention include, where applicable, their antigen-binding fragments. For example, isolated anti-PD-1 antibodies or their antigen-binding fragments can be simply referred to as "isolated anti-PD-1 antibodies," and so on.

[0089] Antibody This invention provides an isolated antibody or antigen-binding fragment thereof that binds to PD-1 (programmed cell death protein 1), comprising: a heavy chain antibody variable domain (VHH), said VHH comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1, and the VHH CDR2 region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, and 93% identity with SEQ ID NO: 2 (page 10 / 38 of specification, CN 121335922 A).The VHH contains amino acid sequences with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 98%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3.

[0090] In one embodiment, the VHH contains CDRs 1, 2, and 3, which have the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively.

[0091] In one embodiment, the VHH contains amino acid sequences with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. In one embodiment, VHH comprises, or is composed of, the amino acid sequence of SEQ ID NO: 4.

[0092] In one embodiment, the antibody or antigen-binding fragment binding to PD-1 comprises a heavy chain having an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. In one embodiment, the antibody or antigen-binding fragment binding to PD-1 comprises a heavy chain comprising, or is composed of, the amino acid sequence of SEQ ID NO: 5.

[0093] In one embodiment, the antibody or antigen-binding fragment specifically binds to PD-1. In one embodiment, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0094] The present invention also provides isolated antibodies or antigen-binding fragments thereof comprising VHH CDRs 1, 2, and 3 of the antibodies or antigen-binding fragments thereof described above.

[0095] In one embodiment, the antibody or antigen-binding fragment thereof comprises VHH CDRs 1, 2, and 3, each comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1, 2, and 3, respectively. In one embodiment, the antibody or antigen-binding fragment thereof comprises VHH CDRs 1, 2, and 3 shown in SEQ ID NO: 1, 2, and 3, respectively.

[0096] In one embodiment, the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains. In one embodiment, the antibody or antigen-binding fragment comprises two, three, four, five, six, seven, eight, nine, or ten VHHs.

[0097] The present invention also provides isolated antibodies or antigen-binding fragments thereof that cross-compete with the antibodies or antigen-binding fragments described above. In one embodiment, the antibody or antigen-binding fragment thereof cross-compete with antibodies or antigen-binding fragments comprising VHH CDRs 1, 2, and 3, each of VHH CDRs 1, 2, and 3 comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1, 2, and 3, respectively. In one embodiment, the antibody or its antigen-binding fragment cross-competes with antibodies or their antigen-binding fragments comprising VHH CDR 1, 2 and 3 as shown in SEQ ID NO 1, 2 and 3, respectively. In one embodiment, the antibody or its antigen-binding fragment cross-competes with an antibody or its antigen-binding fragment comprising VHH comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 4. In one embodiment, the antibody or its antigen-binding fragment cross-competes with an antibody or its antigen-binding fragment comprising VHH comprising, or consisting of, the amino acid sequence of SEQ ID NO: 4.

[0098] The present invention also provides isolated multispecific antibodies or their antigen-binding fragments comprising a first domain specifically binding to PD-1 and a second domain specifically binding to CTLA-4. In one embodiment, the antibody further describes (page 11 / 38, CN 121335922 A) as containing one or more additional domains that specifically bind to antigens other than PD-1 and CTLA-4. In one embodiment, the additional domains that specifically bind to antigens other than PD-1 and CTLA-4 include a third domain that specifically binds to VEGF.

[0099] In one embodiment, an isolated anti-PD-1 / CTLA-4 / VEGF trispecific antibody or an antigen-binding fragment thereof is provided, comprising a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and a third domain that specifically binds to VEGF.

[0100] In some embodiments, the antibody or antigen-binding fragment of the present invention enhances the activation of antigen-specific CD4+ or CD8+ T cells.

[0101] In some embodiments, the antibody or antigen-binding fragment of the present invention inhibits the binding of PD-1 to PD-L1 and PD-L2.

[0102] In some embodiments, the antibody or antigen-binding fragment of the present invention inhibits the binding of CTLA-4 to CD80 and CD86.

[0103] In some embodiments, the antibody or antigen-binding fragment of the present invention inhibits the binding of VEGF to VEGFR1 and / or VEGFR2.

[0104] In some embodiments, the antibody or antigen-binding fragment of the present invention induces PD-1 internalization on the cell surface.

[0105] In one embodiment, the first domain that specifically binds to PD-1 comprises or is composed of a heavy chain variable region (VH1), said VH1 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), wherein HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1, 2, and 3, respectively.

[0106] In one embodiment, the first domain that specifically binds to PD-1 comprises or is composed of a heavy chain variable region (VH1), said VH1 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 1, 2, and 3, respectively.

[0107] In one embodiment, the second domain that specifically binds to CTLA-4 comprises or is composed of a heavy chain variable region (VH2), said VH2 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), wherein HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7, 8, and 9, respectively.

[0108] In one embodiment, the second domain that specifically binds to CTLA-4 comprises or is composed of a heavy chain variable region (VH2), said VH2 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 7, 8, and 9, respectively.

[0109] In one embodiment, the third domain that specifically binds to VEGF comprises or is composed of a heavy chain variable region (VH3) and a light chain variable region (VL), wherein the VH3 comprises heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), wherein each of HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13, 14, and 15, respectively; and the VL comprises light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3), wherein each of LCDR1, LCDR2, and LCDR3 comprises at least 80%, 81%, 82%, and 83% identity with SEQ ID NO: 19, 20, and 21, respectively. Amino acid sequences with 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0110] In one embodiment, the third domain that specifically binds to VEGF comprises or consists of a heavy chain variable region (VH3) and a light chain variable region (VL) as specified on page 12 / 38 of the specification, CN 121335922 A, said VH3 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 13, 14, and 15, respectively, and said VL comprising light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) of SEQ ID NO: 19, 20, and 21, respectively.

[0111] In one embodiment, the first domain specifically binding to PD-1 comprises or is composed of the heavy chain variable region (VH1) of SEQ ID NO: 4, wherein VH1 optionally has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen conserved amino acid substitutions. Optionally, any substitution is not within the CDR.

[0112] In one embodiment, the first domain comprises or is composed of the heavy chain variable region (VH1), wherein the VH1 has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. Optionally, any difference from the sequence of SEQ ID NO: 4 is not within the CDR.

[0113] In one embodiment, the second domain specifically binding to CTLA-4 comprises or is composed of the heavy chain variable region (VH2) of SEQ ID NO: 10, wherein VH2 optionally has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen conserved amino acid substitutions. Optionally, any substitution is not within the CDR.

[0114] In one embodiment, the second domain comprises or is composed of the heavy chain variable region (VH2), wherein the VH2 has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 10. Optionally, any difference from the sequence of SEQ ID NO: 10 is not within the CDR.

[0115] In one embodiment, the third domain that specifically binds to VEGF comprises, or is composed of, the heavy chain variable region (VH3) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO: 22, wherein VH3 and VL optionally each have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen conserved amino acid substitutions. Optionally, any substitution is not within the CDR.

[0116] In one embodiment, the third domain comprises, or is composed of, a heavy chain variable region (VH3) and a light chain variable region (VL), wherein the VH3 has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16, and the VL has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22. Optionally, any differences from the sequences of SEQ ID NO: 16 and / or SEQ ID NO: 22 are not within the CDR.

[0117] In one embodiment, the first, second, and third domains are independently chimeric antibodies, humanized antibodies, human antibodies, single-chain antibodies, Fv, Fab, F(ab′)2, Fd, single-chain Fv molecules (scFv), biantibodies, or single-domain antibodies (dAb).

[0118] In one embodiment, the first, second, and third domains are independently humanized. In one embodiment...In the embodiment, the first and second domains are independently humanized VHH.

[0119] In one embodiment, the antibody according to the invention comprises only one heavy chain. In one embodiment, the antibody according to the invention comprises one heavy chain and one light chain. In one embodiment, the antibody according to the invention comprises two heavy chains. In one embodiment, the antibody according to the invention comprises two heavy chains and two light chains.

[0120] In one embodiment, the antibody according to the invention consists of one heavy chain. In one embodiment, the antibody according to the invention consists of one heavy chain and one light chain. In one embodiment, the antibody according to the invention consists of two heavy chains. In one embodiment, the antibody according to the invention consists of two heavy chains and two light chains.

[0121] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody includes a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and / or a third domain that specifically binds to VEGF. In one embodiment, a first domain specifically binding to PD-1 includes a heavy chain variable region (VH1), wherein the VH1 includes heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 1, 2, and 3, respectively; a second domain specifically binding to CTLA-4 includes a heavy chain variable region (VH2), wherein the VH2 includes heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 7, 8, and 9, respectively; and / or a third domain specifically binding to VEGF includes a heavy chain variable region (VH3) and a light chain variable region (VL), wherein the VH3 includes heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 13, 14, and 15, respectively, and the VL includes light chain complementarity-determining regions 1 (LCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 19, 20, and 21, respectively. (LCDR2) and 3 (LCDR3).

[0122] In one embodiment, the first domain specifically binding to PD-1 comprises, or is composed of, the heavy chain variable region (VH1) of SEQ ID NO: 4; the second domain specifically binding to CTLA-4 comprises, or is composed of, the heavy chain variable region (VH2) of SEQ ID NO: 10; and / or the third domain specifically binding to VEGF comprises, and is composed of, the heavy chain variable region (VH3) of SEQ ID NO: 16 and SEQ ID NO: 16.22 light chain variable region (VL), or composed of them.

[0123] In one embodiment, the third domain that specifically binds to VEGF comprises, or is composed of, the heavy chain of SEQ ID NO: 17 and the light chain of SEQ ID NO: 23. In one embodiment, the third domain that specifically binds to VEGF comprises, or is composed of, a full-length antibody or antigen-binding fragment thereof comprising two heavy chains and two light chains interconnected by disulfide bonds. In one embodiment, the antigen-binding fragment of the full-length antibody is a Fab fragment or an scFv fragment.

[0124] In one embodiment, the antibody or antigen-binding fragment thereof further comprises the Fc region of IgG. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG1, IgG2, IgG3, or IgG4 isotype. In one embodiment, the antibody or antigen-binding fragment thereof is an IgG1 LALA isotype. As is known in the art, the term "LALA" refers to the introduction of an amino acid substitution L234A / L235A (Kabat nomenclature).

[0125] In one embodiment, a first domain specifically binding to PD-1 is located at the N-terminus of the Fc region. In one embodiment, a second domain specifically binding to CTLA-4 is located at the N-terminus of the Fc region. In one embodiment, a third domain specifically binding to VEGF is located at the N-terminus of the Fc region.

[0126] In one embodiment, a first domain specifically binding to PD-1 is located at the C-terminus of the Fc region. In one embodiment, a second domain specifically binding to CTLA-4 is located at the C-terminus of the Fc region. In one embodiment, a third domain specifically binding to VEGF is located at the C-terminus of the Fc region.

[0127] In one embodiment, the first domain specifically binding to PD-1, the second domain specifically binding to CTLA-4, the third domain specifically binding to VEGF, and the Fc region are directly interconnected or interconnected via one or more linkers (such as flexible linkers). In one embodiment, the linkers may be the same or different. In one embodiment, the linker is a peptide linker, and most preferably, the linker is a peptide linker lacking a proteolytic cleavage site. In some embodiments, the amino acid residues of the linker are selected from G, A, S, P, E, T, D, and K. In some embodiments, the linker is GGGGSGGGGS.

[0128] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention is represented from the N-terminus to the C-terminus by the following formula: Third domain – hinge region – Fc – linker – second domain – linker – first domain.

[0129] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention is represented from the N-terminus to the C-terminus by the following formula: Specification 14 / 38 pages 18 CN 121335922 A Third domain – Hinge region – Fc – Connector – First domain – Connector – Second domain.

[0130] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention is represented from the N-terminus to the C-terminus by the following formula: Second domain – Hinge region – Fc – Connector – Third domain – Connector – First domain.

[0131] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention is represented from the N-terminus to the C-terminus by the following formula: First domain – hinge region – Fc – adapter – third domain – adapter – second domain.

[0132] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention is represented from the N-terminus to the C-terminus by the following formula: Second domain – hinge region – Fc – adapter – first domain – adapter – third domain.

[0133] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention is represented from the N-terminus to the C-terminus by the following formula: First domain – hinge region – Fc – adapter – second domain – adapter – third domain.

[0134] In one embodiment, the third domain specifically binding to VEGF is a full-length antibody. In one embodiment, the first domain specifically binding to PD-1 is optionally linked to the C-terminus of any one or both heavy chains of the full-length antibody via a linker, and the second domain specifically binding to CTLA-4 is optionally linked to the C-terminus of the first domain specifically binding to PD-1 via a linker. In one embodiment, the second domain specifically binding to CTLA-4 is optionally linked to the C-terminus of any one or both heavy chains of the full-length antibody via a linker, and the first domain specifically binding to PD-1 is optionally linked to the C-terminus of the second domain specifically binding to CTLA-4 via a linker. In one embodiment, the first domain specifically binding to PD-1 and the second domain specifically binding to CTLA-4 are linked to the C-terminus of two heavy chains of the full-length antibody in the same or different order. In one embodiment, the two domains specifically binding to PD-1 and CTLA-4 are linked to the C-terminus of two heavy chains of the full-length antibody.Each of the second CTLA-4 binding domains is connected to the C-terminus of the heavy chain of the full-length antibody via a linker, and each of the two first domains specifically binding to PD-1 is connected to the C-terminus of the two second domains specifically binding to CTLA-4 via a linker (see Figure 1).

[0135] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention comprises one heavy chain and one light chain, or is composed of the latter, wherein the heavy chain contains the amino acid sequence of SEQ ID NO: 27, and the light chain contains the amino acid sequence of SEQ ID NO: 23.

[0136] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention comprises two heavy chains and two light chains, or is composed of the latter, wherein the heavy chains contain the amino acid sequence of SEQ ID NO: 27, and the light chains contain the amino acid sequence of SEQ ID NO: 23.

[0137] In one embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 27. In one embodiment, the light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 23. In one embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 27, and the light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 23.

[0138] The term "identity" or "homology" between two amino acid or nucleotide sequences is determined by sequence alignment. (Page 15 / 38 of specification, CN 121335922 A) If the two sequences to be compared are of different lengths, sequence alignment preferably involves determining the percentage of amino acids or nucleotide residues in the shorter sequence that are identical to those in the longer sequence. Sequence alignment can be performed using a computer.The procedure is routinely determined. Differences that arise in comparisons between a given sequence and the sequences described above in this disclosure may be caused by, for example, addition, deletion, substitution, insertion, or recombination.

[0139] In some embodiments, the CDR sequence of the antibody of the present invention may contain any conserved modification.

[0140] "Conserved modification" refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody containing an amino acid sequence. Conserved modifications include amino acid substitution, addition, and deletion. Conserved substitution is that amino acids are replaced by amino acid residues having similar side chains. The family of amino acid residues with similar side chains is well-defined and includes amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), amino acids with aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), amino acids with aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amino acids with amides (e.g., asparagine, glutamine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with sulfur-containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide can be substituted with alanine, as previously described for scanning mutagenesis targeting alanine (MacLennan et al., Acta Physiol. Scand. Suppl. 643:55-67, 1998; Sasaki et al., Adv. Biophys. 35:1-24, 1998). Amino acid substitutions of the antibodies of the present invention can be performed by known methods, such as by PCR mutagenesis (US Patent No. 4,683,195). Alternatively, variant libraries can be generated using known methods, such as using random codons (NNK) or non-random codons, such as DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants can be tested for their properties using the assays described herein.

[0141] Generation of the monospecific antibodies of the present invention In some embodiments, the antibodies of the present invention are human.

[0142] In some embodiments, the antibodies of the present invention are humanized.

[0143] The monospecific antibodies of the present invention described herein (e.g., those specifically binding to PD-1, CTLA-4, or VEGF)Antibodies can be produced using a variety of techniques. For example, monoclonal antibodies can be produced using the hybridoma method described in Kohler and Milstein, Nature 256:495, 1975. In the hybridoma method, mice or other host animals (such as hamsters, rats, alpacas, or monkeys) are immunized with human or cynomolgus monkey PD-1, CTLA-4, or VEGF, or fragments of PD-1, CTLA-4, or VEGF (such as the extracellular domains of PD-1, CTLA-4, or VEGF). Then, spleen cells from the immunized animal are fused with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Clones generated from a single immortalized hybridoma cell are screened to produce antibodies with desired properties, such as binding specificity, cross-reactivity or lack thereof, and affinity for the antigen.

[0144] A variety of host animals can be used to produce the antibodies of the present invention. For example, Balb / c mice can be used to generate mouse anti-human PD-1, CTLA-4, or VEGF antibodies. Alpacas can be used to generate anti-human PD-1 or CTLA-4 VHH. Antibodies prepared in Balb / c mice and other non-human animals can be humanized using various techniques to produce more human-like sequences.

[0145] Exemplary humanization techniques, including the selection of human receptor frameworks, are known and include CDR transplantation (US Patent No. 5,225,539), SDR transplantation (US Patent No. 6,818,749), surface remodeling (Padlan, (1991) Mol Immunol 28:489-499), surface remodeling of specificity-determining residues (US Patent Publication No. 2010 / 0261620), human framework adaptation (US Patent No. 8,748,356), or hyperhumanization (US Patent No. 7,709,226). In these methods, the CDR of the parent antibody is transferred to a human frame, which can be selected based on its overall homology with the parent frame, similarity based on CDR length, or canonical structural identity, or a combination thereof.

[0146] Humanized antibodies can be further optimized to improve their selectivity or affinity for the desired antigen by incorporating altered frame-supporting residues via techniques such as those described in International Patent Publications WO1090 / 007861 and WO1992 / 22653 to maintain binding affinity (reverse mutation), or by introducing variations at any CDR to improve antibody affinity.

[0147] Transgenic animals, such as mice or rats carrying a human immunoglobulin (Ig) locus in their genome, can be used to produce human antibodies against target proteins, and are described in, for example, U.S. Patent No. 6,150,584, International Patent Publication No. WO99 / 45962, International Patent Publication No. WO2002 / 066630, WO2002 / 43478, WO2002 / 043478 and WO1990 / 04036, Lonberg et al. (1994) Nature 368:856-9, Green et al. (1994) Nature Genet. 7:13-21, Green and Jakobovits (1998) Exp. Med. 188:483-95, Lonberg and Huszar (1995) Int Rev Immunol 13:65-93, Bruggemann et al. (1991) Eur J Immunol 21:1323- 1326, Fishwild et al., (1996) Nat Biotechnol 14:845-851; Mendez et al., (1997) Nat Genet 15:146-156; Green (1999) J Immunol Methods 231:11-23; Yang et al., (1999) Cancer Res 59:1236-1243; Brüggemann and Taussig (1997) Curr Opin Biotechnol 8:455-458. In these animals, endogenous immunoglobulin loci can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the animal's genome using homologous or non-homologous recombination, transchromosome, or minigene. Companies such as Regeneron (http: / / www.regeneron.com), Harbour Antibodies (http: / / www.harbourantibodies.com), Open Monoclonal Technology, Inc. (OMT) (http: / / www.omtinc.net), KyMab (http: / / www.kymab.com), Trianni (http: / / www.trianni.com), and Ablexis (http: / / www.ablexis.com) can be engaged to provide human antibodies against selected antigens using the technologies described above.

[0148] Human antibodies can be selected from phage display libraries in which phages are engineered to express human immunoglobulins or portions thereof, such as Fab, single-chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., (2000) J Mol Biol 296:57-86; Krebs et al., (2001) J Immunol Meth 254:67-84; Vaughan et al., (1996) Nature Biotechnology 14:309-314; Sheets et al., (1998) PITAS (USA) 95:6157-6162; Hoogenboom and Winter (1991) J Mol Biol 227:381; Marks et al., (1991) J Mol Biol 222:581). The antibodies of the present invention can be isolated, for example, from a phage display library expressing the variable regions of the heavy and light chains of an antibody as a fusion protein with the pIX coat protein of a phage, as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Patent Publication No. WO09 / 085462. The library can be screened for phage binding to human and / or cynomolgus monkey PD-1, CTLA-4, or VEGF, and the obtained positive clones can be further characterized, Fab can be isolated from the clonal lysate, and expressed as full-length IgG. Such phage display methods for isolating human antibodies are described, for example, in: U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, 5,427,908, 5,580,717, 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081.

[0149] The preparation of immunogenic antigens and the generation of monoclonal antibodies can be carried out using any suitable technique, such as recombinant protein generation. Immunogenic antigens can be administered to animals in the form of purified proteins or in the form of protein mixtures comprising whole-cell, cell, or tissue extracts, or the antigen can be de novo formed in an animal from nucleic acids encoding the antigen or a portion thereof.

[0150] The anti-PD-1 antibody GBD002-hS019-WS obtained from alpacas in this disclosure has the amino acid and nucleotide sequences shown in Table 2.

[0151] Table 2. Amino acid and nucleotide sequences of the GBD002-hS019-WS antibody

[0151] Page 18 / 38, CN 121335922 AThe anti-CTLA-4 antibody GBD008-hS005-3-2 obtained from alpacas in this disclosure has the amino acid and nucleotide sequences shown in Table 3.

[0152] Table 3. Amino acid and nucleotide sequences of the GBD008-hS005-3-2 antibody Specification 19 / 38 pages 23 CN 121335922 A The anti-VEGF antibody in this disclosure, as a variant of bevacizumab, has the amino acid and nucleotide sequences shown in Table 4.

[0153] Table 4. Amino acid and nucleotide sequences of the anti-VEGF antibody Specification 20 / 38 pages 24 CN 121335922 A Specification 21 / 38 pages 25 CN 121335922 A Generation of the multispecific antibody of the present invention The bispecific PD-1 / CTLA-4 antibody of the present invention can be generated by combining the PD-1 binding VH (or VH / VL) domain isolated and characterized herein with the CTLA-4 binding VH (or VH / VL) domain. Alternatively, bispecific PD-1 / CTLA-4 antibodies may be engineered using the VH (or VH / VL) domain of a publicly available monospecific anti-PD-1 or anti-CTLA antibody, and / or by mixing the PD-1 or CTLA-4 VEGF-binding VH (or VH / VL) domain identified herein with a publicly available PD-1 or CTLA-4-binding VH (or VH / VL) domain. Trispecific, tetraspecific, and other similar antibodies may be generated in a similar manner.

[0154] For example, the trispecific PD-1 / CTLA-4 / VEGF antibody of the present invention may be generated by combining the PD-1-binding VH (or VH / VL) domain, the CTLA-4-binding VH (or VH / VL) domain, and the VEGF-binding VH (or VH / VL) domain isolated and characterized herein. Alternatively, the trispecific PD-1 / CTLA-4 / VEGF antibody may be engineered using the VH (or VH / VL) domain of a publicly available monospecific anti-PD-1, anti-CTLA, or anti-VEGF antibody, and / or by mixing the PD-1, CTLA-4, or VEGF-binding VH (or VH / VL) domain identified herein with a publicly available PD-1, CTLA-4, or VEGF-binding VH (or VH / VL) domain.

[0155] Exemplary anti-PD-1 antibodies that can be used to engineer trispecific PD-1 / CTLA-4 / VEGF antibodies include, for example, pembrolizumab and nivolumab. Exemplary anti-CTLA-4 antibodies that can be used to engineer trispecific PD-1 / CTLA-4 / VEGF antibodies include, for example, ipilimumab. Exemplary anti-CTLA-4 antibodies that can be used to engineer trispecific PD-1 / CTLA-4 / VEGF antibodies include, for example, ipilimumab.Anti-VEGF antibodies include, for example, bevacizumab. Exemplary monospecific domains may also be derived from bispecific antibodies, such as the anti-PD-1×CTLA-4 bispecific antibody catonilimab and MEDI5752, the anti-PD-1 / CTLA-4 bispecific antibody AK104, and the anti-PD-1 / VEGF bispecific antibody AK112.

[0156] The resulting trispecific PD-1 / CTLA-4 / VEGF antibodies can be tested for binding to PD-1, CTLA-4, and VEGF using the methods described herein, as well as their desired functional properties, such as enhanced activation of antigen-specific CD4+ and CD8+ T cells.

[0157] Typically, standard methods are used to mutate molecules (such as constant domains of antibodies) at the DNA level.

[0158] The trispecific PD-1 / CTLA-4 / VEGF antibody GBD214-33-03 constructed in this disclosure has the amino acid and nucleotide sequences shown in Table 5, page 22 / 38, CN 121335922 A.

[0159] Table 5. Amino acid and nucleotide sequences of the GBD214-33-03 antibody. Page 23 / 38, CN 121335922 A. Page 24 / 38, CN 121335922 A. Polynucleotides, vectors, and host cells. This invention also provides antibodies or antigen-binding fragments thereof, wherein antibody VH and / or VL are encoded by polynucleotides. The polynucleotides may be complementary deoxyribonucleic acid (cDNA) and may be codon-optimized for expression in a suitable host. Codon optimization is a well-known technique.

[0160] The present invention also provides isolated polynucleotides encoding the VH, VL, heavy chain, and / or light chain of the antibody of the present invention.

[0161] The present invention also provides isolated polynucleotides encoding the VH, VL, or VH and VL of the antibody of the present invention.

[0162] The present invention also provides isolated polynucleotides encoding the VH of SEQ ID NO: 4, 10, and / or 16.

[0163] The present invention also provides isolated polynucleotides encoding the heavy chain and / or light chain of the antibody of the present invention.

[0164] In one embodiment, the present invention provides isolated polynucleotides encoding the heavy chain of SEQ ID NO: 5 or 11. In one embodiment, the present invention provides isolated polynucleotides encoding the heavy chain of SEQ ID NO: 17 or 27 and / or the light chain of SEQ ID NO: 23.

[0165] In one embodiment, the present invention provides isolated polynucleotides comprising the same components as SEQ ID NO: 6 or 12.The nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment, the present invention provides isolated polynucleotides comprising the nucleotide sequence of SEQ ID NO: 6 or 12.

[0166] In one embodiment, the present invention provides a separate polynucleotide comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence of SEQ ID NO: 18 or 28, and / or a separate polynucleotide comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence of SEQ ID NO: 24. In one embodiment, the present invention provides a separate polynucleotide comprising the nucleotide sequence of SEQ ID NO: 18 or 28 and / or the nucleotide sequence of SEQ ID NO: 24.

[0167] The VH or VL or antigen-binding fragment thereof encoding the antibody of the present invention, or the polynucleotide sequence encoding the heavy or light chain of the antibody of the present invention, can be operatively linked to one or more regulatory elements (such as promoters or enhancers) to enable the nucleotide sequence to be expressed in the intended host cell. The polynucleotide can be cDNA.

[0168] The present invention also provides vectors comprising the polynucleotides of the present invention. Such vectors can be plasmid vectors, viral vectors, baculovirus expression vectors, transposon-based vectors, or any other vector suitable for introducing the synthetic polynucleotides of the present invention into a given organism or genetic background in any way. For example, a polynucleotide encoding the variable region (which is optionally linked to a constant region) of the light chain and / or heavy chain of the antibody of the present invention is inserted into an expression vector. The light chain and / or heavy chain can be cloned into the same or different expression vectors. DNA segments encoding immunoglobulin chains can be operatively linked to a control sequence in the expression vector, the control sequence ensuring the expression of the immunoglobulin polypeptide. Such control sequences include signal sequences, promoters (e.g., naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected for compatibility with the host cells chosen for antibody expression. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the protein encoded by the incorporated polynucleotide.

[0169] Suitable expression vectors typically replicate in a host organism either as a free organism or as part of the host's chromosomal DNA. Typically, expression vectors contain selection markers, such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance, to enable the detection of cells transformed with the desired DNA sequence.

[0170] Suitable promoter and enhancer elements are known in the art. Exemplary promoters for expression in eukaryotic cells include: light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus immediate early promoters; herpes simplex virus thymidine kinase promoters; early and late SV40 promoters; promoters present in long terminal repeat sequences from retroviruses; mouse metallothionein-I promoters; and various known tissue-specific promoters. The selection of suitable vectors and promoters is entirely within the capabilities of those skilled in the art.

[0171] Exemplary vectors that can be used are bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, California, USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden); and eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.4 (Lonza) and pEE12.4 (Lonza).

[0172] The present invention also provides host cells comprising one or more vectors of the present invention. “Host cell” means a cell to which a vector has been introduced. It should be understood that the term "host cell" refers not only to the specific target cell, but also to its progeny and stable cell lines derived from the specific target cell. Because certain changes may occur in the offspring due to mutations or environmental influences, such progeny may differ from the parent cells, but are still included within the scope of the term "host cell" as used herein. Such host cells can be eukaryotic, prokaryotic, plant, or archaea cells. Examples include *Escherichia coli*, bacilli (such as *Bacillus subtilis*), and other Enterobacteriaceae (such as *Salmonella*, *Serratia*, and various *Pseudomonas* species).The species (e.g., *Saccharomyces cerevisiae*) is an example of a prokaryotic host cell. Other microorganisms (such as yeast) can also be used for expression. Yeasts (e.g., *Saccharomyces cerevisiae*) and *Pichia* are examples of suitable yeast host cells. Exemplary eukaryotic cells may be of mammalian, insect, bird, or other animal origin. Mammalian eukaryotic cells include immortalized cell lines, such as hybridoma cell lines or myeloma cell lines, such as SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NSO (European Center for Cell Culture Collection (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human bone marrow tumor cell line is U266 (ATCC CRL-TIB-196), as described on pages 26 / 38 of the manual, CN 121335922 A. Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHOK1SV (LonzaBiologics, Walkersville, MD), Potelligent® CHOK2SV (Lonza), CHO-K1 (ATCC CRL-61), or DG44.

[0173] The present invention also provides a method for producing the antibodies of the present invention, the method comprising culturing the host cells of the present invention under conditions expressing the antibodies, and recovering the antibodies produced by the host cells. Methods for preparing and purifying antibodies are well known in the art. Once synthesized (whether chemically synthesized or recombinantly synthesized), the whole antibody, its dimer, single light chain and / or heavy chain, or other antibody fragments (such as VH and / or VL) can be purified according to standard procedures including ammonium sulfate precipitation, affinity column, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (generally see Scopes, Protein Purification (Springer-Verlag, NY, (1982)). The target antibody can be substantially pure, for example at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99% or more pure, for example free from contaminants other than the target antibody, such as cell debris, macromolecules, etc.

[0174] The polynucleotide sequence of the present invention can be incorporated into a vector using standard molecular biology methods. Host cell transformation, culture, antibody expression, and purification are all performed using known methods.

[0175] Another embodiment of the present invention is a method for generating the antibody of the present invention or an antigen-binding fragment thereof, the method comprising:The VH, VL, heavy chain, and / or light chain polynucleotides encoding the antibody or antigen-binding fragment of the present invention are incorporated into an expression vector; host cells are transformed with the expression vector; host cells are cultured in a culture medium under conditions in which the VH, VL, heavy chain, and / or light chain are expressed and form the antibody or antigen-binding fragment; and the antibody or antigen-binding fragment is recovered from the host cells or culture medium.

[0176] Pharmaceutical Composition / Administration The present invention provides pharmaceutical compositions comprising the antibody or antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier. For therapeutic use, the antibody or antigen-binding fragment of the present invention may be prepared as a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment of the present invention as an active ingredient in a pharmaceutically acceptable carrier. “Carrier” refers to a diluent, adjuvant, excipient, or solvent used for administering the antibody of the present invention. Such solvents may be liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They can be sterilized using conventional, known sterilization techniques (e.g., filtration). The compositions may contain desired pharmaceutically acceptable adjuvants to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants. The concentration of the antibody or its antigen-binding fragment in such pharmaceutical formulations may range from less than about 0.5% by weight (typically) to at least about 1% by weight to at most 15% by weight or 20% by weight, and may be selected based primarily on the desired dose, liquid volume, viscosity, and other factors, depending on the specific administration method chosen. Suitable solvents and formulations, including other human proteins (e.g., human serum albumin), are described, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, edited by Troy, D.B., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691–1092, with particular reference to pp. 958–989.

[0177] The therapeutic use of the antibody or its antigen-binding fragment of the present invention can be administered via any suitable route for delivering the antibody or its antigen-binding fragment to the host, such as parenteral administration, for example intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, or transmucosal (oral, intranasal, vaginal, rectal) administration, in tablets, capsules, solutions, powders, gels, or granules. (Instructions for use 27 / 38 pages, 31 CN 121335922 A)The agent; and contained in a syringe, implantation device, osmotic pump, cartridge, micropump; or other means as known in the art and understood by those skilled in the art. Site-specific administration can be achieved, for example, intratumoral, intrabronchial, intraperitoneal, intracystic, intracartilaginous, intracavitary, intrabody cavity, intracerebellum, intravenous, colonic, cervical, gastric, intrahepatic, intracardiac, intraosseous, intrapelvic, pericardial, intraperitoneal, intrapleural, prostate, lung, rectum, kidney, retina, spinal cord, synovium, pleural cavity, uterus, intravascular, intrabladder, lesion, vagina, rectum, sublingual, sublingual, intranasal, or percutaneous delivery.

[0178] The antibody or antigen-binding fragment of the present invention can be administered to the subject via any suitable route, for example, via intravenous (iv) infusion or bolus injection parenteral administration, intramuscular or subcutaneous or intraperitoneal administration. Intravenous infusion can be administered over, for example, 15, 30, 60, 90, 120, 180, or 240 minutes, or over, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.

[0179] The dose given to the subject is sufficient to alleviate or at least partially suppress the disease being treated (“therapeutic effective dose”), and may sometimes be 0.005 mg to about 100 mg / kg, for example about 0.05 mg to about 30 mg / kg or about 5 mg to about 25 mg / kg, or about 4 mg / kg, about 8 mg / kg, about 16 mg / kg or about 24 mg / kg, or for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg, but may even be higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg / kg.

[0180] Fixed unit doses, such as 50, 100, 200, 500, or 1000 mg, may also be administered, or the dose may be determined based on the patient's body surface area, such as 500, 400, 300, 250, 200, or 100 mg / m². Patients are typically treated with 1 to 8 doses (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 doses), but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses may also be administered.

[0181] Administration of the antibody or its antigen-binding fragment of the present invention may be repeated after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. Repeated treatment courses are also possible, as is long-term administration. Repeated administration can be at the same dose or at different doses. For example, the antibody or its antigen-binding fragment of the present invention can be administered at 8 mg / kg or 16 mg / kg.Administer 8 mg / kg once a week for 8 weeks, then 8 mg / kg or 16 mg / kg every two weeks for another 16 weeks, then 8 mg / kg or 16 mg / kg every four weeks via intravenous infusion.

[0182] For example, the antibody or antigen-binding fragment of the present invention can be used as a daily dose of about 0.1-100 mg / kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, daily after the start of treatment, on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, etc. The antibody or antigen-binding fragment thereof of the present invention may be administered prophylactically for at least one day of any of the following periods: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or alternatively, for at least one week of any of the following periods: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks, or any combination thereof, using a single or fractionated dose or any combination thereof every 24, 12, 8, 6, 4, or 2 hours.

[0183] The antibody or antigen-binding fragment thereof of the present invention may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of cancer progression events, and / or reduce the risk of relapse when cancer is in remission.

[0184] The antibody or antigen-binding fragment thereof of the present invention may be lyophilized for preservation and reconstituted in a suitable carrier prior to use. This technique has proven effective for conventional protein formulations and can be employed using known lyophilization and reconstitution techniques.

[0185] Methods and Uses The antibodies or antigen-binding fragments thereof of the present invention have diagnostic utility in vitro and in vivo, as well as therapeutic and preventive utility. For example, the antibodies or antigen-binding fragments thereof of the present invention can be administered in vitro or ex vivo to cultured cells, or to subjects seeking treatment, prevention, and / or diagnosis of various conditions, such as cancer and infectious diseases. Specification 28 / 38 pages 32 CN 121335922 A

[0186] The present invention provides a method for altering the immune response of a subject, the method comprising administering to the subject the antibody or antigen-binding fragment of the present invention for a duration sufficient to alter the immune response.

[0187] In some embodiments, the immune response is enhanced, stimulated, or upregulated.

[0188] In some embodiments described herein, the subject is a human patient.

[0189] In some embodiments described herein, the subject is a human patient who requires an enhanced immune response.

[0190] In some embodiments, the subject is immunocompromised.

[0191] In some embodiments, the subject is at risk of immunocompromise. Immunocompromised subjects may be undergoing or have undergone chemotherapy or radiotherapy.

[0192] In some embodiments, the subject is immunocompromised due to infection or at risk of immunocompromise due to infection.

[0193] The antibodies or antigen-binding fragments thereof of the present invention are suitable for treating subjects with a condition that can be treated by enhancing a T-cell-mediated immune response.

[0194] In some embodiments, the antibodies or antigen-binding fragments thereof used in the methods of the present invention are those defined in this disclosure.

[0195] In some embodiments, the trispecific PD-1 / CTLA-4 / VEGF antibody used in the methods of the present invention is GBD214-33-03. The amino acid and nucleotide sequences of this antibody are shown in Table 5.

[0196] The present invention also provides a method for inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of the present invention for a duration sufficient to inhibit tumor cell growth.

[0197] The present invention also provides a method for treating cancer by administering a therapeutically effective amount of an antibody of the present invention or an antigen-binding fragment thereof to a subject in need (for a duration sufficient to treat cancer).

[0198] Cancer can be a hyperproliferative condition or symptom, a solid tumor, a hematologic malignancy, a soft tissue tumor, or a metastatic lesion.

[0199] “Cancer” is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. Examples of cancer include solid tumors, hematologic malignancies, soft tissue tumors, and metastatic lesions. Exemplary solid tumors include malignancies of various organ systems, such as sarcomas and carcinomas (including adenocarcinomas and squamous cell carcinomas), such as those affecting the liver, lungs, breast, lymph nodes, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., kidneys, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers, small bowel cancers, and esophageal cancers. Squamous cell carcinoma includes malignant tumors such as those in the lungs, esophagus, skin, head and neck, oral cavity, anus, and cervix.

[0200] In some embodiments, the cancer is melanoma.

[0201] Metastatic lesions of the above-described cancers can also be treated or prevented using the methods and antibodies of the present invention described herein.

[0202] Exemplary cancers whose growth can be inhibited or reduced using the antibodies of the present invention or their antigen-binding fragments include cancers that are responsive to immunotherapy. Examples of such cancers include melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, etc.Cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, lung cancer, metastatic malignant melanoma, clear cell carcinoma, hormone-refractory prostate adenocarcinoma, non-small cell lung cancer, or head and neck cancer. The antibodies of the present invention described herein or their antigen-binding fragments can be used to treat refractory or recurrent malignant tumors.

[0203] Other exemplary cancers that can be treated with the antibodies of the present invention or their antigen-binding fragments include: anal cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer and CNS cancer, fallopian tube cancer, vaginal cancer, vulvar cancer, skin or intraocular malignant melanoma, astroesophageal cancer, testicular cancer, ovarian cancer, pancreatic cancer, rectal cancer, uterine cancer, etc. Primary CNS lymphoma; tumors of the central nervous system (CNS), cervical cancer, choriocarcinoma, rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, eye cancer; intraepithelial neoplasia, renal cancer, laryngeal cancer, liver cancer; small cell lung cancer, neuroblastoma, oral cancer (e.g., lip, tongue, mouth and pharynx), nasopharyngeal carcinoma, retinoblastoma, rhabdomyosarcoma, respiratory system cancer, sarcoma, thyroid cancer, urinary system cancer, liver cancer, anal region cancer, fallopian tube cancer, vaginal cancer, vulvar cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, Merkel cell carcinoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including asbestos-induced cancers) and other cancers and sarcomas, as well as combinations of the above cancers.

[0204] Exemplary hematologic malignancies that can be treated with the antibodies or antigen-binding fragments of the present invention include leukemia, lymphoma, and myeloma, such as precursor B-cell lymphoblastic leukemia / lymphoma and B-cell non-Hodgkin lymphoma, acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL) (including low-grade, intermediate-grade, and high-grade FL), cutaneous follicular center lymphoma, marginal zone B-cell lymphoma (MALT type, lymph node type, and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma, etc. Large cell lymphoma (DLBCL), Burkitt lymphoma (BL), plasmacytoma, multiple myeloma (MM), plasma cell leukemia, post-transplant lymphoproliferative disorder, Waldenström macroglobulinemia, plasma cell disease, anaplastic large cell lymphoma (ALCL), T-cell acute lymphoblastic leukemia, primary systemic amyloidosis (e.g., light chain amyloidosis), prolymphocytic / granulocytic lymphomaCellular leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), large granular lymphocytic (LGL) leukemia, NK cell leukemia, and Hodgkin's lymphoma.

[0205] "Plasma cell disease" refers to a disease characterized by clonal plasma cells and includes multiple myeloma, light chain amyloidosis, and Waldenström macroglobulinemia. Light chain amyloidosis and Waldenström macroglobulinemia can occur independently of multiple myeloma. They can also coexist with multiple myeloma and develop before or after the development of multiple myeloma.

[0206] Exemplary B-cell non-Hodgkin's lymphomas include lymphomatoid granuloma, primary exudative lymphoma, intravascular large B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain diseases (including γ, μ, and α diseases), lymphomas induced by treatment with immunosuppressants (such as cyclosporine-induced lymphoma), and methotrexate-induced lymphoma.

[0207] In some embodiments, the subject has a tumor expressing PD-L1.

[0208] In some embodiments, the subject has a tumor expressing CTLA-4.

[0209] In some embodiments, the subject has a tumor expressing VEGF.

[0210] In some embodiments, the subject has been treated with an anti-PD-1 antibody.

[0211] In some embodiments, the subject is refractory to treatment with an anti-PD-1 antibody.

[0212] In some embodiments, the subject has a tumor that has relapsed after treatment with an anti-PD-1 antibody.

[0213] In some embodiments, the subject has been treated with an anti-PD-1 antibody (e.g., KEYTRUDA® (pembrolizumab)).

[0214] In some embodiments, the subject has been treated with an anti-PD-1 antibody (e.g., OPDIVO® (nivolumab)).

[0215] In some embodiments, the subject is refractory to treatment with an anti-PD-1 antibody (e.g., KEYTRUDA® (pembrolizumab)).

[0216] In some embodiments, the subject is refractory to treatment with an anti-PD-1 antibody (e.g., OPDIVO® (nivolumab)).

[0217] In some embodiments, the subject has a tumor that has relapsed after treatment with an anti-PD-1 antibody (e.g., KEYTRUDA® (pembrolizumab)).

[0218] In some embodiments, the subject has a tumor that has relapsed after treatment with an anti-PD-1 antibody (e.g., OPDIVO® (nivolumab)).

[0219] In some embodiments, the subject has been treated with an anti-PD-L1 antibody (e.g., MEDI-4736, MDX-1105, averulin).

[0220] In some embodiments, the subject is refractory to treatment with anti-PD-L1 antibodies (e.g., MEDI-4736, MDX-1105, avelumab, or atezolizumab).

[0221] In some embodiments, the subject has a tumor that has relapsed after treatment with anti-PD-L1 antibodies (e.g., MEDI-4736, MDX-1105, avelumab, or atezolizumab).

[0222] In some embodiments, the subject has been treated with or is being treated with anti-PD-L2 antibodies.

[0223] In some embodiments described herein, the subject is refractory to treatment with anti-PD-L2 antibodies.

[0224] In some embodiments, the subject has a tumor that has relapsed after treatment with anti-PD-L2 antibodies.

[0225] Various qualitative and / or quantitative methods can be used to determine the relapse or refractory nature of a disease. Symptoms that may be associated with relapse or resistance include, for example, a decline or stagnation in a patient's health, or the recurrence or worsening of various symptoms associated with solid tumors, and / or the spread of cancer cells from one site to other organs, tissues, or cells in the body.

[0226] The present invention also provides a method of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of the antibody of the present invention, wherein the subject is being treated with or has been treated with an anti-PD-1 antibody.

[0227] The present invention also provides a method of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of the antibody of the present invention, wherein the subject is being treated with or has been treated with an anti-PD-L1 antibody.

[0228] The present invention also provides a method of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of the antibody of the present invention, wherein the subject is being treated with or has been treated with an anti-PD-L2 antibody.

[0229] Combination therapy for cancer treatment The antibody of the present invention may be administered in combination with a second therapeutic agent.

[0230] The antibody or antigen-binding fragment of the present invention may be administered in combination with one, two, three, four, five, or six other therapeutic agents.

[0231] "In combination with" means administering the antibody of the present invention simultaneously with at least one second therapeutic agent as a single agent, or as a single agent in any order. Generally, each agent will be administered at a dose and / or schedule determined according to that agent.

[0232] In some embodiments, the second therapeutic agent modulates the activity of molecules involved in the cancer immune cycle, such molecules being involved in stimulating or inhibiting pathways in the release of cancer cell antigens, cancer antigen presentation, T cell initiation, etc.The second therapeutic agent plays a role in priming and activation, T cell migration to the tumor, T cell infiltration into the tumor, T cell recognition of cancer cells, and killing of cancer cells. The cancer immune cycle is described in Chen and Mellman (2013) Immunity 39:1-10. In some embodiments, the second therapeutic agent modulates the activity of molecules involved in regulating the activity of regulatory T cells (Tregs), co-stimulatory or co-inhibitory ligands expressed on the tumor, activation or inhibition receptors on natural killer (NK) cells, or immunosuppressive factors in the tumor microenvironment. Combination cancer immunotherapy is described in Manoney et al., (2015) Nature Reviews 14:561-584.

[0233] As is known, second therapeutic agents generally enhance the activity of stimulatory molecules and suppress the activity of inhibitory molecules. Therefore, "modulation" refers to the enhancement of the immune response by the second therapeutic agent, whether the agent itself is an agonist or antagonist of a particular molecule. Specification 31 / 38 pages 35 CN 121335922 A

[0234] The efficacy of the combinations described herein can be tested in animal models known in the art. Examples

[0235] The present invention will now be described with reference to the following specific, non-limiting examples.

[0236] Example 1: Generation of anti-PD-1 antibody, anti-CTLA-4 antibody and anti-VEGF antibody Anti-human PD-1 antibody and anti-human CTLA-4 antibody were generated by immunizing alpacas with recombinant human PD-1 and human CTLA-4 extracellular domain (ECD) proteins, respectively. Total RNA was extracted from PBMCs and cDNA was synthesized and amplified. The framework region of the alpaca VH gene was replaced with the human framework region by CDR transplantation technology and cloned into an expression vector to generate the corresponding humanized antibody clone. The resulting anti-PD-1 antibody GBD002-hS019-WS has the heavy chain variable region of SEQ ID NO: 4. The resulting anti-CTLA-4 antibody has the heavy chain variable region (GBD008-hS005-3-2) of SEQ ID NO: 10.

[0237] The amino acids of the heavy and light chain variable regions of the anti-VEGF antibody can be referenced from the commercial antibody bevacizumab (Avastin), and are shown in SEQ ID NO: 16 and SEQ ID NO: 22, respectively. Biointron was commissioned to synthesize an anti-VEGF antibody as a variant of bevacizumab, the sequence of which is shown in Table 4. This anti-VEGF antibody contains two heavy chains having the amino acid sequence of SEQ ID NO: 17 and two light chains having the amino acid sequence of SEQ ID NO: 23. In this anti-VEGF antibody, the Fc fragment is different from the Fc fragment of bevacizumab.

[0238] Example 2: Production of GBD214-33-03 antibodyConstruction of the trispecific antibody GBD214-33-03: The DNA sequences encoding the heavy chain containing the N-terminus to C-terminus of the anti-VEGF antibody (SEQ ID NO: 17), the variable region of the heavy chain of the anti-CTLA-4 antibody (SEQ ID NO: 10), and the variable region of the heavy chain of the anti-PD-1 antibody (SEQ ID NO: 4), and the DNA sequence encoding the light chain of the anti-VEGF antibody (SEQ ID NO: 23) were cloned into the pcDNA3.3 expression vector to obtain the trispecific antibody. Adapter 1 and / or adapter 2 were added as flexible links to the heavy chain of GBD214-33-03. The obtained trispecific antibody was named "GBD214-33-03". A schematic diagram of the structure of GBD214-33-03 is shown in Figure 1.

[0239] Example 3. FACS analysis of the specificity of binding of anti-PD-1 antibody to human PD1 protein on cell surface. HEK293-PD1 cells (1×10⁵ / well) were washed twice in FACS buffer (PBS + 2% BSA) and resuspended in 100 μl of FACS buffer containing serially diluted (1:5) anti-PD1 mAb and incubated at 4°C for 1 h. The cells were then washed twice in FACS buffer and the binding antibody was detected by incubation at 4°C for 1 h with APC anti-human IgG Fc (Biolegend, catalog number 410712). Subsequently, the cells were washed twice in FACS buffer and then collected and analyzed using a Fortessa flow cytometer (BD Bioscience). The results are shown in Figure 2. The results show that GBD214-33-03 binds to PD1 in HEK293-PD1 cells. GBD214-33-03 showed strong binding to HEK293-PD1 cells, with a median fluorescence intensity (MFI) similar to its PD1 parent GBD002-hS019-WS and pembrolizumab.

[0240] Example 4. PD1 reporter gene assay in Jurkat-NFAT-Luc2-PD1 cells (ELISA) CHOK1-PD-L1 cells (4×104 / well) were harvested and suspended in PRMI 1640 medium (Gibco, catalog 31800022) containing 10% FBS (Gibco, catalog 10099-141) and grown overnight in 96-well plates at 37°C. On the second day, the supernatant was discarded, and Jurkat-NFAT-Luc2-PD1 cells (5×104 / well) and serially diluted antibody were added to each well. The cells were incubated at 37°C for 5 hours. Subsequently, Bio-Glo luciferase assay buffer (Promega, catalog) was prepared.Cells were added (G7940), incubated at room temperature for up to 10 minutes, and then read using an EnVision 2105 multimode microplate reader (PerkinElmer). The results are shown in Figure 3. PD1 reporter gene assay showed that GBD214-33-03 had a similar maximum luminescent signal to pembrolizumab and AK104. The results indicate that GBD214-33-03 can block the interaction between PD1 and its ligand PD-L1.

[0241] Example 5. Mixed lymphocyte reaction (MLR) assay of antibody CD14+ cells were isolated from PBMCs using an isolation kit (Stem Cell, catalog number 17858) and according to the manufacturer's instructions. Subsequently, CD14+ cells were seeded at 1×10⁶ cells / ml, 3–5 ml, in 6-well plates (3–5×10⁶ / well) of medium supplemented with 50 ng / ml IL-4 and 50 ng / ml GMCSF. Cytokines were replaced every 2–3 days. On day 6, to induce mature DCs, 1 μg / ml LPS (Sigma-Aldrich, catalog number L6529) was added to the medium, and the cells were cultured at 37°C for 24 hours. Mature DCs were then collected on day 7 for MLR assay. For the MLR assay, fresh T cells were isolated according to the manufacturer's instructions (Stem Cell, catalog number 17951), and seeded at 2×10⁵ cells / well into the assay plate. Serially diluted antibody was added to the assay plate, and then mature DCs were seeded at 1×10⁴ cells / well. The assay plate was incubated at 37°C for 5 days, and the supernatant was collected for IFN-γ detection using the HTRF kit (Cisbio, catalog number 62HIFNGPEH) according to the manufacturer's instructions. The results are shown in Figure 4. The results showed that the maximum IFN-γ production of GBD214-33-03 in the mixed lymphocyte reaction (MLR) assay was similar to that of its parent GBD002-hS019-WS and pembrolizumab. The results indicate that GBD214-33-03 has a potent immunogenicity similar to that of pembrolizumab.

[0242] Example 6. VEGF binding assay (ELISA) VEGF165 protein (1 µl / ml) (Kactus, catalog number VEG-HM065) was coated onto a 96-well flat-bottom microplate at 37°C for 2 hours. The plate was washed 6 times with PBST and blocked at 37°C for 2 hours by adding 300 µl of blocking buffer. Aspirate the supernatant and wash the plate three times with PBST. Add serially diluted antibody to each well. Incubate the plate at 37°C for 1 day.The plate was incubated for 1 hour and then washed 6 times with PBST. Anti-human Fc (Jackson ImmunoResearch Inc., catalog number 109-035-008) and SA-HRP (Jackson ImmunoResearch Inc., catalog number 016-030-084) were added to the plate and the plate was incubated at 37°C for 1 hour. Subsequently, 100 µl of TMB solution was added to each well and the plate was incubated at room temperature for 5 minutes, followed by the addition of 50 µl of TMB solution. The optical density (OD450) of each well was measured using an EnVision 2105 multimode microplate reader (PerkinElmer). The results are shown in Figure 5. The results show that GBD214-33-03 has similar VEGF binding to bevacizumab as that of bevacizumab.

[0243] Example 7. VEGF Blocking Assay (ELISA) VEGF165 protein (1 µl / ml) (Kactus, catalog number: VEG-HM065) was coated onto a 96-well flat-bottom microplate at 37°C for 2 hours. The plate was washed 6 times with PBST and blocked at 37°C for 2 hours by adding 300 µl of blocking buffer. The supernatant was aspirated, and the plate was washed 3 times with PBST. Serially diluted antibody and VEGFR2-mFc-biotin (Kactus, catalog number: VGF-HM3R2B) were added to each well. The plate was incubated at 37°C for 1 hour and then washed 6 times with PBST. Anti-human Fc (Jackson ImmunoResearch Inc., catalog number 109-035-008) and SA-HRP (Jackson ImmunoResearch Inc., catalog number 016-030-084) were added to the plate, and the plate was incubated at 37°C for 1 hour. Subsequently, 100 µl of TMB solution was added to each well, and the plate was incubated at room temperature for 5 minutes, followed by the addition of 50 µl of TMB solution. The optical density (OD450) of each well was measured using an EnVision 2105 multimode microplate reader (PerkinElmer). The results are shown in Figure 6. The results show that GBD214-33-03 inhibits the binding of VEGFR2 to VEGF165, similar to bevacizumab.

[0244] Example 8. VEGF reporter gene assay of antibody (ELISA method) HEK293-NFAT-KDR-Luc cells (4 × 10⁴ cells / well) were harvested and resuspended in PRMI 1640 medium (Gibco, catalog number 31800022) containing 10% FBS (Gibco, catalog number 10099-141), and the cells were seeded at 4 × 10⁴ cells / well and grown overnight at 37°C in 96-well white plates. On the second day, the supernatant was discarded, and the cells were transferred to each well.Add VEGF165 (15 ng / ml) (Kactus, catalog number VEG-HM065) and serially diluted antibody to each well. Incubate cells at 37°C for 5 hours. Next, add Bio-Glo luciferase assay buffer (Promega, catalog number 33 / 38, page 37, CN 121335922 A G7940) to each well. Incubate the plate at room temperature for up to 10 minutes, then read the results using an EnVision 2105 multimode microplate reader (PerkinElmer). The results are shown in Figure 7. The results show that GBD214-33-03 can inhibit VEGF165-induced downstream NFAT signaling by blocking the binding of VEGFR2 to VEGF165, similar to bevacizumab.

[0245] Example 9. FACS analysis of the binding specificity of anti-CTLA4 antibody to human CTLA4 protein on cell surface. CHOK1-CTLA4 cells (1×10⁵ / well) and CHOK1-PD1-CTLA4 cells (1×10⁵ / well) were harvested and collected and resuspended in 100 μl FACS buffer (PBS + 2% BSA). Serially diluted anti-CTLA4 antibody was added to the plate and the cells were incubated at 4°C for 1 h. The cells were then washed twice with FACS buffer and the binding antibody was detected by incubation at 4°C for 1 h with APC anti-human IgG Fc (Biolegend, catalog number 410712). The cells were washed twice with FACS buffer and then collected. The cells were analyzed using a Fortessa flow cytometer (BD Bioscience). The results are shown in Figures 8a and 8b. The results showed that GBD214-33-03 had a lower MFI compared to ipilimumab in CHOK1-CTLA4 cells, suggesting that GBD214-33-03 partially binds to CTLA4 in CHOK1-CTLA4 cells. The CTLA4 parent GBD008-hS005-3-2 of GBD214-33-03 had a weaker binding affinity to CTLA4 compared to ipilimumab. In CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed a stronger binding affinity to its PD1 parent GBD002-hS019-WS.

[0246] Example 10. FACS analysis of the blocking of CD80 and CD86 binding to CTLA4 by anti-CTLA4 antibody. CHOK1-CTLA4 cells or CHOK1-PD1-CTLA4 cells (1×105 / well) were harvested and collected, and the cells were resuspended in 100 μl of serially diluted anti-CTLA4 antibody and CD80-biotin (SinoBiological, catalog number).Cells were incubated in FACS buffer containing either CD80 / CD86-B (10698-H49H-B) or CD86-Biotin (SinoBiological, catalog number 10699-H03H-B). Cells were incubated at 4°C for 30 min and then washed twice with FACS buffer. Antibody binding was detected by incubation at 4°C for 30 min with APC anti-human IgG Fc (Biolegend, catalog number 410712). Cells were then collected after washing twice with FACS buffer and analyzed using a Fortessa flow cytometer (BD Bioscience). The results are shown in Figures 9a to 9d. The results show that GBD214-33-03 partially inhibits the binding of CD80 / CD86 to CTLA4 in CHOK1-CTLA4 and CHOK1-PD1-CTLA4 cells. In both CHOK1-CTLA4 and CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed weaker blocking ability compared to ipilimumab.

[0247] Example 11. Affinity of Antibodies with PD1, CTLA4, and VEGF (Octet Method) The binding kinetics of antibodies with recombinant human PD1, CTLA-4, or VEGF were qualitatively and quantitatively analyzed using the Octet method (Octet RH-16, Sartorius) for protein interactions. The binding rate (Kon) and dissociation rate (Koff) were calculated using a simple one-to-one binding model (Octet evaluation software version 12.2). The equilibrium dissociation constant (kD) was calculated as the ratio of Koff / Kon. The results are shown in Tables 6, 7, and 8. The results in Table 6 show that all antibodies bound to the CTLA-4 antigen, the results in Table 7 show that all antibodies bound to the PD1 antigen, and the results in Table 8 show that all antibodies bound to the VEGF antigen.

[0248] Table 6. Affinity results of antibody with PD1 Table 7. Affinity results of antibody with CTLA4 Specification 34 / 38 pages 38 CN 121335922 A Table 8. Affinity results of antibody with VEGF Example 12. Determination of PD-1 receptor downregulation in CHOK1-PD1-CTLA4 cells by flow cytometry.

[0249] CHOK1-PD1-CTLA4 cells were harvested and the cell density was adjusted to 5×106 / ml. Cells were added at 50 µl / well to F12K+10% FBS medium in 96-well U-plates. Serially diluted antibody was added to the cells. Cells were incubated at 4°C for 30 min and washed twice with FACS buffer at 4°C. Thereafter, all procedures were performed at 4°C, including pipetting and centrifugation. Subsequently, a working concentration of 10 µg / ml of non-competitive PD-1-AF647 antibody was added to the plate and the cells were incubated at 4°C.The cells were incubated for another 30 minutes. The cells were washed twice with FACS buffer at 4°C and then fixed with 4% paraformaldehyde (100 µL / well) at room temperature for 10 minutes. After fixation, the cells were washed once with FACS buffer and analyzed using a Fortessa flow cytometer (BD Bioscience). The results are shown in Figure 10. The results showed that PD1 in the cells was downregulated due to CTLA4 internalization. The percentage of PD1 downregulated was measured. GBD214-33-03 cells showed similar PD1 internalization as AK104.

[0250] Example 13. In vivo antitumor efficacy of GBD214-33-03 antibody in A375 PBMC mouse model.

[0251] NSG mice (n=6, 4 weeks old) were subcutaneously inoculated with A375 human melanoma cancer cells (5×106 cells) premixed with hPBMC (6×105 cells). When the tumor was established (approximately 220 mm3), pembrolizumab (15 mg / kg), bevacizumab (15 mg / kg), ipilimumab-LALA (15 mg / kg), three doses of AK112 (1 mg / kg, 5 mg / kg, and 20 mg / kg), three doses of AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), three doses of GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg), and a combination of pembrolizumab, bevacizumab, and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg) were administered intraperitoneally. Treatment was given twice weekly for a total of five doses. Tumor growth and mouse body weight were monitored every three days and reported as mean tumor volume and mean body weight. Results are shown in Figures 11a and 11b. The results showed that in the A375 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg and 20 mg / kg) had better potency than the combination of AK112 (1 mg / kg, 5 mg / kg and 20 mg / kg), AK104 (1 mg / kg, 5 mg / kg and 20 mg / kg), pembrolizumab, bevacizumab and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg). For all treatment groups, the mean body weight of mice decreased from day 7, with a decrease of no more than 15%.

[0252] Example 14. In vivo antitumor potency of GBD214-33-03 antibody in HT29 PBMC mouse model. Specification 35 / 38 pages 39 CN 121335922 A

[0253] Used with hPBMCPremixed HT29 human colon cancer cells (6 × 10⁵ cells) and 5 × 10⁶ cells were subcutaneously inoculated into NSG mice (n=6 per group, 4 weeks old). When tumors were established (approximately 250 mm³), pembrolizumab (15 mg / kg), bevacizumab (15 mg / kg), ipilimumab-LALA (15 mg / kg), three doses of AK112 (1 mg / kg, 5 mg / kg, and 20 mg / kg), three doses of AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), three doses of GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg), and a combination of pembrolizumab, bevacizumab, and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg) were administered intraperitoneally. Treatment was given twice weekly for a total of five doses. Tumor growth and mouse body weight were monitored every three days and reported as mean tumor volume and mean body weight. The results are shown in Figures 12a and 12b. The results showed that in the HT29 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg and 20 mg / kg) had better potency than the combination of AK112 (1 mg / kg, 5 mg / kg and 20 mg / kg), AK104 (1 mg / kg, 5 mg / kg and 20 mg / kg), pembrolizumab, bevacizumab and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg). For all treatment groups, the mean body weight of mice remained relatively stable, with a decrease of no more than 15%.

[0254] In summary, the present invention has developed a trispecific antibody that specifically binds to PD-1, CTLA-4 and VEGF to block checkpoints expressed on T cells. The TsAb exhibits potent receptor-binding and ligand-blocking activity against PD-1 and VEGF, while only partially blocking the interaction between CTLA-4 and its ligand. The designed partial blocking activity against CTLA-4 aims to reduce peripheral irAE toxicity. In mouse models, the trispecific antibody of this invention showed potent antitumor activity.

[0255] References: Baraniskin, A., Buchberger, B., Pox, C., Graeven, U., Holch, J. W., Schmiegel, W., and Heinemann, V. (2019). Efficacy of bevacizumab in first-line treatment of metastatic colorectal cancer: A systematicreview and meta‑analysis. Eur J Cancer, 106, 37‑44. doi:10.1016 / j.ejca.2018.10.009 Bertrand, A., Kostine, M., Barnetche, T., Truchetet, M. E. and Schaeverbeke, T. (2015). Immune related adverse events associated with anti‑CTLA‑4 antibodies: systematic review and meta‑analysis. BMC Med, 13, 211. doi: 10.1186 / s12916‑015‑0455‑8 Calabrese, L. H., Calabrese, C. and Cappelli, L. C. (2018). Rheumatic immune‑related adverse events from cancer immunotherapy. Nat Rev Rheumatol, 14 (10), 569‑579. doi:10.1038 / s41584‑018‑0074‑9 Chen, S., Zhang, Z., Zheng, X., Tao, H., Zhang, S., Ma, J., . . . Hu, Y. (2021). Response Efficacy of PD‑1 and PD‑L1 Inhibitors in Clinical Trials: A Systematic Review and Meta‑Analysis. Front Oncol, 11, 562315. doi:10.3389 / fonc.2021.562315 Chikuma, S. (2017). CTLA‑4, an Essential Immune‑Checkpoint for T‑Cell Activation. Curr Top Microbiol Immunol, 410, 99‑126. doi:10.1007 / 82_2017_61 Garcia, J., Hurwitz, H. I., Sandler, A. B., Miles, D., Coleman, R. L .,Deurloo, R. and Chinot, O. L. (2020). Bevacizumab(Avastin®) in cancer treatment: A review of 15 years of clinical experience and future outlook. Cancer Treat Rev, 86, 102017. doi:10.1016 / j.ctrv.2020.102017 Hodi, F. S. (2010). Overcoming immunological tolerance to melanoma: Targeting CTLA‑4. Asia Pac J Clin Oncol, 6 Suppl 1, S16‑23. doi:10 .1111 / Description Page 36 of 38 40 CN 121335922 A j.1743‑7563.2010.01271.x Hodi, F. S., O'Day, S. J., McDermott, D. F., Weber, R. W., Sosman, J. A., Haanen, J.B., . . . Urba, W. J. (2010). Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med, 363(8), 711‑723. doi: 10.1056 / NEJMoa1003466 Jago, C. B., Yates, J., Câmara, N. O., Lechler, R. I. and Lombardi, G. (2004). Differential expression of CTLA‑4 among T cell subsets. Clin Exp Immunol, 136(3), 463‑471. doi:10.1111 / j.1365‑2249.2004.02478.x Larkin, J., Chiarion‑Sileni, V., Gonzalez, R., Grob, J. J., Cowey, C. L., Lao, C. D., .. . Wolchok, J. D. (2015). CombinedNivolumab combined with Ipilimumab versus monotherapy for previously untreated melanoma. N Engl J Med, 373(1), 23-34. doi: 10.1056 / NEJMoa1504030 Liu, J., Chen, Z., Li, Y., Zhao, W., Wu, J. and Zhang, Z. (2021). PD-1 / PD-L1 Checkpoint Inhibitors in Tumor Immunotherapy. Front Pharmacol, 12, 731798. doi:10.3389 / fphar.2021.731798 Maio, M., Grob, J. J., Aamdal, S., Bondarenko, I., Robert, C., Thomas, L., . . . Wolchok, J. D. (2015). Five-year survival rates for treatment-naive patients with advanced melanoma receiving ipilimumab combined with dacarbazine in a phase III trial. J Clin Oncol, 33(10), 1191-1196. doi:10.1200 / jco.2014.56.6018 Melincovici, C. S., Boşca, A. B., Şuşman, S., Mărginean, M., Mihu, C., Istrate, M., . . . Mihu, C. M. (2018). Vascular endothelial growth factor (VEGF) — a key factor in normal and pathological angiogenesis. Rom J Morphol Embryol, 59(2), 455-467. Schadendorf, D., Hodi, F. S., Robert, C., Weber, J. S., Margolin, K., Hamid, O., . . . Wolchok, J. D. (2015). PooledAnalysis of Long‑Term Survival Data From Phase II and Phase III Trials of Ipilimumab in Unresectable or Metastatic Melanoma .J Clin Oncol , 33 (17) , 1889‑1894 . doi:10 .1200 / jco.2014.56.2736 Sharpe, A. H. and Pauken, K. E. (2018) .The diverse functions of the PD1 inhibitory pathway.Nat Rev Immunol, 18(3) , 153‑167. doi:10.1038 / nri.2017.108 Simons , M ., Gordon , E . and Claesson‑Welsh , L .(2016) . Mechanisms and regulation of endothelial VEGF receptor signalling.Nat Rev Mol Cell Biol, 17 (10) , 611‑625. doi:10.1038 / nrm.2016.87 Ś ledzińska, A., Menger, L., Bergerhoff, K., Peggs, K. S. and Quezada,S. A. (2015) . Negative immune checkpoints on T lymphocytes and their relevance to cancer immunotherapy .Mol Oncol , 9 (10) , 1936‑1965 . doi:10 .1016 / j.molonc.2015.10.008 Zhao, B., Zhao, H. and Zhao, J . (2020) .Efficacy of PD‑1 / PD‑L1 blockade Description Page 37 / 38 41 CN 121335922 A monotherapy in clinical trials.Ther Adv Med Oncol, 12 , 1758835920937612. doi: 10.1177 / 1758835920937612 Zhao, Y., Guo, S., Deng, J.,Shen, J., Du, F., Wu, doi:10.7150 / ijbs.70958. Instruction Manual Page 38 / 38, 42 CN 121335922 A, Figure 1, Figure 2; Instruction Manual Appendix 1 / 11, Page 43 CN 121335922 A, Figure 3, Figure 4; Instruction Manual Appendix 2 / 11, Page 44 CN 121335922 A, Figure 5, Figure 6; Instruction Manual Appendix 3 / 11, Page 45 CN 121335922 A, Figure 7, Figure 8a; Instruction Manual Appendix 4 / 11, Page 46 CN 121335922 A, Figure 8b, Figure 9a; Instruction Manual Appendix 5 / 11, Page 47 CN 121335922 A, Figure 9b, Figure 9c; Instruction Manual Appendix 6 / 11, Page 48 CN 121335922 A, Figure 9d, Figure 10; Instruction Manual Appendix 7 / 11, Page 49 CN 121335922 A, Figure 11a; Instruction Manual Appendix 8 / 11, Page 50 CN 121335922 A, Figure 11b. Figure 12a, page 9 / 11, CN 121335922 A; Figure 12b, page 10 / 11, CN 121335922 A; Figure 12b, page 11 / 11, CN 121335922 A

Claims

1. An antibody or antigen-binding fragment thereof that binds to PD-1 (programmed cell death protein 1) comprises: a heavy chain antibody variable domain (VHH), said VHH comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 1, the VHH CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and the VHH CDR3 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

3.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VHH comprises CDRs 1, 2 and 3, which have amino acid sequences as shown in SEQ ID NO: 1, 2 and 3, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the VHH comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

4.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VHH comprises or is composed of the amino acid sequence of SEQ ID NO:

4.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment specifically binds to PD-1.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

7. An antibody or an antigen-binding fragment thereof comprising VHH CDR 1, 2 and 3 of the antibody or antigen-binding fragment thereof according to any one of claims 1-6.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

9. An antibody or an antigen-binding fragment thereof, which cross-competes with an antibody or an antigen-binding fragment thereof according to any one of claims 1-8.

10. A multispecific antibody or an antigen-binding fragment thereof, comprising a first domain specifically binding to PD-1 and a second domain specifically binding to CTLA-4.

11. The multispecific antibody or its antigen-binding fragment according to claim 10, wherein... a) The first domain that specifically binds to PD-1 includes a heavy chain variable region (VH1), wherein the VH1 includes complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 1, 2, and 3, respectively; and / or b) The second domain that specifically binds to CTLA-4 includes a heavy chain variable region (VH2), which includes heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 7, 8, and 9, respectively.

12. The multispecific antibody or its antigen-binding fragment according to claim 10 or 11, wherein... a) The first domain that specifically binds to PD-1 comprises, or is composed of, the heavy chain variable region (VH1) of SEQ ID NO: 4; and / or b) The second domain that specifically binds to CTLA-4 includes, or is composed of, the heavy chain variable region (VH2) of SEQ ID NO:

10.

13. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 10-12, wherein the antibody further comprises one or more additional domains that specifically bind to antigens other than PD-1 and CTLA-4.

14. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 10-13, further comprising a domain specifically binding to VEGF.

15. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 10-14, wherein it is an anti-PD-1 / CTLA-4 / VEGF trispecific antibody or antigen-binding fragment thereof, comprising a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to VEGF.

16. The multispecific antibody or its antigen-binding fragment according to claim 15, wherein... a) The first domain that specifically binds to PD-1 includes a heavy chain variable region (VH1), which includes heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 1, 2, and 3, respectively. b) The second domain that specifically binds to CTLA-4 includes a heavy chain variable region (VH2), which contains heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 7, 8, and 9, respectively; and / or c) The third domain that specifically binds to VEGF comprises a heavy chain variable region (VH3) and a light chain variable region (VL3), wherein the VH3 comprises heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 13, 14, and 15, respectively, and the VL3 comprises light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) of SEQ ID NO: 19, 20, and 21, respectively.

17. The multispecific antibody or antigen-binding fragment thereof according to claim 16, wherein the VH1 comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 4, and / or the VH2 comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

10.

18. The multispecific antibody or its antigen-binding fragment according to claim 16, wherein... a) The first domain that specifically binds to PD-1 includes, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 4; b) The second domain that specifically binds to CTLA-4 comprises, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 10; and / or c) The third domain that specifically binds to VEGF comprises, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO:

22.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1-18, wherein the antibody further comprises the Fc region of IgG.

20. The antibody or antigen-binding fragment thereof according to any one of claims 10-19, wherein the first domain, the second domain, the third domain and / or the Fc region are directly interconnected via one or more adapters.

21. The antibody or antigen-binding fragment thereof according to any one of claims 20, wherein the adapter is the same or different, and / or wherein the adapter is a flexible adapter, and / or wherein the adapter is a peptide adapter.

22. The antibody or antigen-binding fragment thereof according to any one of claims 15-21, wherein the antibody comprises, or is composed of, the heavy chain of SEQ ID NO: 27 and the light chain of SEQ ID NO:

23.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1-22, wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 isotype, optionally comprising one, two, three, four, five, six, seven, eight, nine or ten substitutions in the Fc region.

24. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-23 and a pharmaceutically acceptable carrier.

25. A polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-23.

26. A vector comprising the polynucleotide according to claim 25.

27. An isolated host cell comprising the vector according to claim 26.

28. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell according to claim 27 under conditions expressing the antibody or an antigen-binding fragment thereof, and recovering and purifying the antibody or antigen-binding fragment produced by the host cell.

29. A method of treating a subject with cancer, the method comprising administering to a subject in need a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1-23 or a pharmaceutical composition according to claim 24, for a duration sufficient to treat the cancer.

30. The method of claim 29, wherein the cancer is a solid tumor or a hematologic malignancy.

31. The method of claim 30, wherein the solid tumor is melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, stomach cancer, ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary cancer, endometriosis, cervical cancer, or metastatic lesions of cancer.

32. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-23 or the pharmaceutical composition according to claim 24 in the preparation of a medicament for treating cancer in a subject of need.

33. The use according to claim 32, wherein the cancer is a solid tumor or a hematologic malignancy.

34. The use according to claim 33, wherein the solid tumor is melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, stomach cancer, ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary cancer, endometriosis, cervical cancer, or metastatic lesions of cancer.

35. The antibody or antigen-binding fragment thereof according to any one of claims 1-23, or the pharmaceutical composition according to claim 24, for treating cancer in a subject in need.

36. The antibody or antigen-binding fragment thereof or pharmaceutical composition according to claim 35, wherein the cancer is a solid tumor or a hematologic malignancy.

37. The antibody or antigen-binding fragment thereof or pharmaceutical composition according to claim 36, wherein the solid tumor is melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, stomach cancer, ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary cancer, endometriosis, cervical cancer, or metastatic lesions of cancer.

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