PD-1 binding antibodies and uses thereof

Abstract

The present application relates to an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human PD-1. Also provided are nucleic acid molecules encoding the antibody or antigen-binding portion thereof, expression vectors, host cells, and methods for expressing the antibody or antigen-binding portion thereof. Also provided are bispecific molecules and pharmaceutical compositions comprising the antibody or antigen-binding portion thereof, and methods of treatment using the PD-1 antibody or antigen-binding portion thereof.

Description

[0001] Related applications and citations are included.

[0002] This application claims priority to U.S. Patent Application No. 63 / 072,421, filed on August 31, 2020.

[0003] All documents cited or referenced herein (including, but not limited to, all literature, patents, and published patent applications cited herein) (“Documents Referenced herein”), all documents cited or referenced in the Documents Referenced herein, and any manufacturer’s manuals, instructions, product specifications, and product pages mentioned herein or in any of the documents incorporated herein by reference, are incorporated herein by reference and may be used in practicing this invention. More specifically, all referenced documents are incorporated herein by reference as if they were specifically and individually incorporated. Any Genbank sequences mentioned in this disclosure are incorporated by reference to sequences with the earliest valid filing date of this disclosure. Invention Field

[0004] This application generally relates to isolated monoclonal antibodies, particularly mouse-derived, chimeric, or humanized monoclonal antibodies, or their antigen-binding moieties, which bind to PD-1 with high affinity and functionality. It also provides nucleic acid molecules encoding the antibody or its antigen-binding moieties, expression vectors, host cells, and methods for expressing the antibody or its antigen-binding moieties. This application also provides bispecific molecules, immunoconjugates, chimeric antigen receptors, and pharmaceutical compositions that may contain the antibody or its antigen-binding moieties, as well as treatment methods using the PD-1 antibody or its antigen-binding moieties of this application. Background Technology

[0005] The immune system damages harmful invaders while simultaneously protecting healthy cells. This balance between immune defense and self-tolerance is crucial for normal physiological function and is achieved through multiple checks and balances in the immune response. For example, effector immune cells can only function fully after passing through immune checkpoints. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD-1) are the two earliest discovered inhibitory immune checkpoint receptors that act as brakes on the immune response and provide immunosuppressive signals. CTLA4 is induced early in the T-cell activation process and competes with CD28 for binding to CD80 / CD86. PD-1 is subsequently expressed and binds to programmed cell death 1 ligand 1 (PD-L1) and / or PD-L2 to counteract the positive signaling induced by TCR / CD28 (Sharpe AH, Pauken KE. (2018) Nat Rev Immunol. 18(3): 153-167).

[0006] PD-1 is a transmembrane protein containing a proximal immunoreceptor tyrosine repressor motif (ITIM) and a distal tyrosine switching motif (ITSM) (Thomas, ML (1995) J Exp Med 181: 1953-6; Vivier, E and Daeron, M (1997) Immunol Today 18: 286-91). Upon binding to PD-L1, PD-1 activation leads to ITSM phosphorylation, thereby recruiting phosphatases 1 / 2 and SLAM-related proteins containing the Src homology 2 domain, dephosphorylating proximal TCR and CD28 signaling molecules such as ZAP70, and inactivating downstream signaling pathways such as PI3K / AKT and Ras-MEK-ERK pathways (Sharpe AH, Pauken KE. (2018) ibid.; Yokosuka T et al., (2012) J Exp Med. 209(6): 1201-17). Depending on the circumstances, the PD-1-PD-L1 signaling pathway can induce apoptosis, dysfunction, and exhaustion of effector T cells, proliferation and function of intrinsic lymphocytes, and / or proliferation of Treg cells (Qin W et al., (2019) FrontImmunol.10:2298).

[0007] Expression of PD-L1 and PD-L2 in hematopoietic and non-hematopoietic tissues can prevent tissue inflammation and help maintain homeostasis, while expression of PD-L1 and / or PD-L2 in cancer cells can help cancer cells evade immune surveillance. Specifically, the binding of PD-L1 on tumor cells to PD-1 on cytotoxic T cells leads to CD8... + T cell dysfunction and apoptosis, and PD-L1 makes tumor cells resistant to CD8. + T-cell lysis (Azuma T et al., (2008) Blood 111(7): 3635-3643). Several PD-1 or PD-L1 inhibitors have been approved by the FDA for cancer treatment, including two PD-1 antibodies, nivolumab and pembrolizumab, and three PD-L1 antibodies, avelumab, atezolizumab, and durvalumab. Nivolumab ( Bristol Myers Squibb has been approved for the treatment of non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer (BC), colorectal cancer (CRC) with microsatellite instability or mismatch repair deficiency (MSIH / dMMR), hepatocellular carcinoma (HCC), classical Hodgkin lymphoma (cHL), melanoma, and head and neck squamous cell carcinoma (HNSCC). Pembrolizumab ( Merck has been approved for the treatment of melanoma, HNSCC, cervical cancer, cHL, NSCLC, BC, gastric and gastroesophageal cancer, and all advanced solid tumors with MSIH / dMMR. A meta-analysis of 19 randomized clinical trials involving 11,379 patients showed that PD-1 therapy had significantly better survival outcomes than PD-L1 therapy (Duan J et al., (2020) JAMA Oncol. 6(3): 375-384).

[0008] In human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infections, CD8... + High expression of PD-1 was also found on T cells. PD-1 / PD-L1 blockade can restore CD8+ expression. + T cells and CD4 + The functions of T cells, eliminating the inhibitory effect of liver-resident NK cells on T cells, promoting cytokine production, and reducing viral load (Barber DL et al., (2006) Nature. 439: 682-687; Qin W et al., (2019) ibid.). PD-1 and / or PD-L1 therapy has shown beneficial effects on sepsis, and nivolumab and BMS-936559 are in clinical trials for the treatment of severe sepsis (Riva A, Chokshi S. (2018) Hepatol Iht. 12(3): 223-36).

[0009] In clinical applications, some patients do not respond to currently approved PD-1 antibodies. Therefore, there is a need for other monoclonal antibodies that can enhance drug properties.

[0010] References to or designations of any documents in this application are not an admission that such documents are prior art. Summary of the Invention

[0011] This application provides isolated monoclonal antibodies, such as mouse-derived, chimeric, or humanized monoclonal antibodies, or their antigen-binding sites, which bind to PD-1 (e.g., human PD-1) and, compared with prior art PD-1 antibodies such as nivolumab and pembrolizumab, have i) comparable (if not higher) binding affinity / capacity to human, monkey, and mouse PD-1, ii) comparable (if not higher) blocking activity against PD-1-PD-L1 interaction, and / or iii) comparable (if not higher) in vivo antitumor activity.

[0012] The antibody or its antigen-binding portion of this application can be used for a variety of applications, including in vitro detection of PD-1 protein in humans, monkeys or mice, and treatment of diseases associated with the PD-1 signaling pathway, such as cancer, infectious diseases and inflammatory diseases.

[0013] Therefore, in one aspect, this application relates to an isolated monoclonal antibody, or its antigen-binding portion, which binds to PD-1 and has (i) a heavy chain variable region that may comprise a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region, VH CDR2 region, and VH CDR3 region may respectively comprise (1) SEQ ID NOs: 1, 3, and 6 (X1 = D, X2 = Y); (2) SEQ ID NOs: 1, 3, and 6 (X1 = E, X2 = Y); (3) SEQ ID NOs: 1, 4, and 6 (X1 = D, X2 = W); or (4) SEQ ID NOs: 1, 4, and 6 (X1 = D, X2 = W); or (4) SEQ ID NOs: 1, 3, and 6 (X1 = D, X2 = W). NOs: 2, 5 and 7 have amino acid sequences with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; and / or (ii) a light chain variable region which may comprise a VLCDR1 region, a vL CDR2 region and a VL CDR3 region, wherein the vL CDR1 region, the vL CDR2 region and the vL CDR3 region may comprise, respectively, the same as (1) SEQ ID NOs: 8 (X1=I, X2=N), 10 and 12; (2) SEQ ID NOs: 8 (X1=I, X2=A), 10 and 12; (3) SEQ ID NOs: 8 (X1=L, X2=D), 10 and 12; or (4) SEQ ID NOs: 9, 11 and 13 have amino acid sequences with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0014] The monoclonal antibody isolated in this application or its antigen-binding portion may contain a heavy chain variable region and a light chain variable region. The heavy chain variable region contains VH CDR1, VH CDR2 and VH CDR3 regions, and the light chain variable region contains VL CDR1, VL CDR2 and VL CDR3 regions. The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 regions may respectively contain (1) SEQ ID NOs: 1, 3, 6 (X1=D, X2=Y), 8 (X1=I, X2=N), 10 and 12; (2) SEQ ID NOs: 1, 3, 6 (X1=E, X2=Y), 8 (X1=I, X2=A), 10 and 12; (3) SEQ ID NOs: 1, 4, 6 (X1=D, X2=W), 8 (X1=L, X2=D), 10 and 12; or (4) SEQ ID NOs: 2, 5, 7, 9, 11 and 13 having amino acid sequences with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0015] The heavy chain variable region may contain SEQ ID NOs: 14, 15 (X1=S, X2=M, X3=K; X1=T, X2=M, X3=K; X1=S, X2=V, X3=K; X1=S, X2=M, X3=T; X1=T, X2=V, X3=T), 16 (X1=V, X2=S, X3=I; X1=I, X2=G, X3=I; X1=I, X2=S, X3=M). Amino acid sequences 17 (X1=R, X2=A, X3=V; X1=K, X2=V, X3=V; X1=K, X2=A, X3=R), 18, 19, or 42 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. The amino acid sequence of SEQ ID NO: 14 may be encoded by the nucleotide sequence of SEQ ID NOs: 30 or 31. The amino acid sequences of SEQ ID NOs: 16 (X1=I, X2=S, X3=M), 18, and 19 may be encoded by the nucleotide sequences of SEQ ID NOs: 32, 36, and 38, respectively.

[0016] The light chain variable region may contain an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NOs: 20, 21 (X1=N; X1=A), 22, or 23. The amino acid sequence of SEQ ID NO: 20 may be encoded by the nucleotide sequence of SEQ ID NOs: 33 or 34. The amino acid sequences of SEQ ID NOs: 21 (X1=N), 22, and 23 may be encoded by the nucleotide sequences of SEQ ID NOs: 35, 37, and 39, respectively.

[0017] The isolated monoclonal antibody or its antigen-binding portion may contain a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region may respectively contain (1) SEQ ID NOs: 14 and 20; (2) SEQ ID NOs: 15(X1=S, X2=M, X3=K) and 21(X1=N); (3) SEQ ID NOs: 15(X1=T, X2=M, X3=K) and 21(X1=N); (4) SEQ ID NOs: 15(X1=S, X2=V, X3=K) and 21(X1=N); (5) SEQ ID NOs: 15(X1=S, X2=M, X3=T) and 21(X1=N); (6) SEQ ID NOs: 15(X1=T, X2=V, X3=T) and 21(X1=N); (7) SEQ ID NOs: 14 and 20; (8) SEQ ID NOs: 15(X1=T, X2=V, X3=T) and 21(X1=N); (9) SEQ ID NOs: 10 and 20. SEQ ID NOs: 16 (X1=V, X2=S, X3=I) and 21 (X1=N); (8) SEQ ID NOs: 16 (X1=I, X2=G, X3=I) and 21 (X1=N); (9) SEQ ID NOs: 16 (X1=I, X2=S, X3=M) and 21 (X1=N); (10) SEQ ID NOs: 17 (X1=R, X2=A, X3=V) and 21 (X1=N); (11) SEQ ID NOs: 17 (X1=K, X2=V, X3=V) and 21 (X1=N); (12) SEQ ID NOs: 17 (X1=K, X2=A, X3=R) and 21 (X1=N); (13) SEQ ID NOs: 42 and 21 (X1=A); (14) SEQ ID NOs: 18 and 22; or (15) SEQ ID NOs: 19 and 23 having amino acid sequences with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0018] The monoclonal antibody isolated in this application or its antigen-binding portion may contain a heavy chain and a light chain linked by disulfide bonds. The heavy chain may contain a heavy chain variable region and a heavy chain constant region, and the light chain may contain a light chain variable region and a light chain constant region. The C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region. The heavy chain variable region and the light chain variable region may contain the aforementioned amino acid sequence.

[0019] The heavy chain constant region can have reduced FcR binding affinity, such as a genetically modified human IgG1 or IgG2 constant region, or a human IgG4 constant region having, for example, the amino acid sequence shown in SEQ ID NO.: 24, or a functional fragment thereof. The heavy chain constant region can also be an IgG1 constant region having normal or enhanced FcR binding affinity. The light chain constant region can be a human κ constant region having, for example, the amino acid sequence shown in SEQ ID NO.: 25. The amino acid sequences of SEQ ID NOs: 24 and 25 can be encoded by the nucleotide sequences of SEQ ID NOs: 40 and 41, respectively.

[0020] In some embodiments, the antibody of this application may comprise two heavy chains and two light chains, or be composed of two heavy chains and two light chains, wherein each heavy chain may include the aforementioned heavy chain constant region, heavy chain variable region, or CDR sequence, and each light chain may include the aforementioned light chain constant region, light chain variable region, or CDR sequence. In other embodiments, the antibody of this application or its antigen-binding portion may be a single-chain variable fragment (scFv) antibody, or an antibody fragment such as Fab or F(ab')2 fragment.

[0021] This application also provides bispecific molecules that may comprise the antibody of this application or its antigen-binding portion, linked to a second functional group (e.g., a second antibody) having a binding specificity different from that of the antibody or its antigen-binding portion. This application also provides immunoconjugates, such as antibody-drug conjugates, that may comprise the antibody of this application or its antigen-binding portion, linked to a therapeutic agent such as a cytotoxin. On the other hand, the antibody of this application or its antigen-binding portion may be formulated as part of a chimeric antigen receptor (CAR). Immune cells are also provided that may comprise chimeric antigen receptors, such as T cells and NK cells. The antibody of this application or its antigen-binding portion may also be encoded by or used in combination with an oncolytic virus.

[0022] This application also provides a nucleic acid molecule encoding the antibody or its antigen-binding portion thereof, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector. A method for preparing a PD-1 antibody or its antigen-binding portion using the host cell of this application is provided, comprising the steps of: (i) expressing the antibody or its antigen-binding portion in the host cell, and (ii) isolating the antibody or its antigen-binding portion from the host cell or its cell culture.

[0023] This application provides compositions comprising the antibody or its antigen-binding moiety thereof, an immunoconjugate, a bispecific molecule, an immune cell, an oncolytic virus, a nucleic acid molecule, an expression vector or host cell, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may also comprise a therapeutic agent for treating a specific disease, such as an anticancer agent, an anti-infective agent, or an anti-inflammatory agent.

[0024] On the other hand, this application provides a method for treating diseases related to PD-1 signaling, which may include administering a therapeutically effective amount of the composition of this application to a subject.

[0025] The disease can be a tumor or cancer. Tumors or cancers include, but are not limited to, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer (BC), colorectal cancer (CRC) with microsatellite instability or mismatch repair deficiency (MSI-H / dMMR), hepatocellular carcinoma (HCC), classical Hodgkin lymphoma (cHL), melanoma, head and neck squamous cell carcinoma (HNSCC), cervical cancer, gastric and gastroesophageal cancer, colonic adenocarcinoma, and all advanced solid tumors with MSI-H / dMMR. The composition may comprise the antibody of this application or its antigen-binding moiety (with a heavy chain constant region having weak FcR binding), a bispecific molecule, a nucleic acid molecule, or an expression vector. In some embodiments, at least one other anticancer antibody may also be administered, such as a VISTA antibody, a PD-L1 antibody, a LAG-3 antibody, and / or a CTLA-4 antibody. In another embodiment, the antibody or its antigen-binding portion thereof is administered together with a cytokine (e.g., IL-2 and / or IL-21) or a co-stimulatory antibody (e.g., CD137 antibody and / or GITR antibody). In another embodiment, the antibody or its antigen-binding portion thereof is administered together with a chemotherapeutic agent, which may be a cytotoxic agent such as epirubicin, oxaliplatin, and / or 5-fluorouracil (5-FU). The antibody or its antigen-binding portion thereof may be, for example, mouse-derived, chimeric, or humanized. In some embodiments, the subject is a human.

[0026] The disease can be an infectious disease, including, but not limited to, those caused by viruses, bacteria, or fungi. In some embodiments, the infectious disease is sepsis, HIV infection, simian immunodeficiency virus infection, HBV infection, or HCV infection. The composition comprises the antibody of this application or its antigen-binding moiety (having a heavy chain constant region with weak FcR binding), a bispecific molecule, a nucleic acid molecule, or an expression vector. In some embodiments, the subject is further administered an anti-infective agent, such as an antiviral agent, an antibacterial agent, or an antifungal agent. The antibody of this application or its antigen-binding moiety can be, for example, mouse-derived, chimeric, or humanized. In some embodiments, the subject is a human.

[0027] The disease can be an inflammatory disease. Inflammatory diseases include, but are not limited to, rheumatoid arthritis, colitis, lupus-like nephritis, systemic lupus erythematosus, and psoriasis. The compositions of this application comprise the antibody of this application or its antigen-binding moiety (having a heavy chain constant region with strong FCR binding), an immunoconjugate, a bispecific molecule, a CAR-carrying immune cell, a nucleic acid molecule, or an expression vector. In some embodiments, the compositions of this application are administered to inflamed tissue via local delivery. The antibody of this application can be, for example, a mouse-derived, chimeric, or humanized antibody. In some embodiments, the subject is a human.

[0028] On the other hand, this application provides a method for modulating or enhancing an immune response in a subject in need, comprising administering the composition of this application to the subject to modulate / enhance the subject's immune response. The composition comprises an antibody of this application or its antigen-binding moiety (having a heavy chain constant region with weak FcR binding), a bispecific molecule, a nucleic acid molecule, or an expression vector. The antibody of this application or its antigen-binding moiety may be, for example, mouse-derived, chimeric, or humanized. In some embodiments, the subject is a human.

[0029] Other features and advantages of the present disclosure will become apparent from the following specific description and embodiments, which should not be construed as limiting. All documents, Genbank entries, patents, and published patent applications cited in this application are expressly included herein by reference.

[0030] Therefore, the objective of this application is not to include any previously known products, processes for manufacturing such products, or methods for using such products, thereby the applicant reserves the right and hereby publicly declares a waiver of any previously known products, processes, or methods. It should be further noted that this application is not intended to include any products, processes, or methods of manufacturing or using products that do not meet the requirements of written description and implementability of the USPTO (35 U.S. SC § 112, paragraph 1) or EPO (EPC, section 83), thereby the applicant reserves the right and hereby publicly declares a waiver of any previously described products, processes for manufacturing such products, or methods of using such products. In the implementation of this invention, compliance with EPC section 53(c) and EPC regulations 28(b) and (c) is advantageous. All rights are expressly reserved to make express waivers of any embodiments of the subject matter of any of the applicant's granted patents in any prior application of this application or any other family or any third party. Nothing herein should be construed as a commitment.

[0031] It should be noted that in this application, particularly in the claims and / or paragraphs, terms such as “comprising” or “including” may have the meanings given by U.S. patent law; for example, they may mean “included in”; and terms such as “substantially composed of” or “substantially constituted of” have the meanings given by U.S. patent law, for example, they allow the presence of elements not expressly stated, but exclude elements present in the prior art or elements that affect the basic or novel characteristics of the invention. Attached Figure Description

[0032] The following is a detailed description of specific embodiments provided by way of example, but it is not intended to limit this application to the specific implementations described herein. A better understanding can be obtained by referring to the accompanying drawings.

[0033] Figure 1A-1B The binding affinity of mouse-derived antibodies E1A9C8A7 and E2G4E10B7 (A), and E1G5D1H1 (B) to human PD-1 is shown in the capture ELISA.

[0034] Figure 2 The binding affinity of mouse-derived antibodies E1A9C8A7, E2G4E10B7, and E1G5D1H1 to mouse PD-1 is shown in indirect ELISA.

[0035] Figures 3A-3B The binding affinity of mouse antibodies E1A9C8A7 and E2G4E10B7 (A) and E1G5D1H1 (B) to monkey PD-1 in indirect ELISA is shown.

[0036] Figures 4A-4B The binding affinity of mouse antibodies E1A9C8A7 and E2G4E10B7 (A) and E1G5D1H1 (B) to GS-J2 / PD-1 cells expressing human PD-1 was shown in cell-based binding FACS assays.

[0037] Figure 5 The binding affinity of mouse-derived antibodies E1A9C8A7 and E2G4E10B7 to 293F-mouse PD-1 cells expressing mouse PD-1 was demonstrated in cell-based binding FACS assays.

[0038] Figures 6A-6B This demonstrates the ability of mouse-derived antibodies E1A9C8A7 and E2G4E10B7 (A), and E1G5D1H1 (B) to block human PD-1-human PD-L1 binding in competitive ELISA.

[0039] Figures 7A-7BThis demonstrates the ability of mouse-derived antibodies E1A9C8A7 and E2G4E10B7 (A), and E1G5D1H1 (B) to block the binding of human PD-1 to human PD-L1 on the cell surface in a cell-based blocked FACS assay.

[0040] Figure 8 This demonstrates the ability of mouse-derived antibodies E1A9C8A7 and E2G4E10B7 to block the binding of mouse PD-L1 to mouse PD-1 on the cell surface in a cell-based blocked FACS assay.

[0041] Figures 9A-9B This demonstrates the ability of mouse-derived antibodies E1A9C8A7 and E2G4E10B7 (A), and E1G5D1H1 (B) to block pembrolizumab-PD-1 binding in competitive ELISA.

[0042] Figure 10 This demonstrates the ability of mouse-derived antibodies E1A9C8A7, E2G4E10B7, and E1G5D1H1 to reverse PD-1-PD-L1 interaction-induced luciferase expression reduction in cell-based functional assays.

[0043] Figure 11 This demonstrates the ability of chimeric E1A9C8A7 antibodies to reverse PD-1-PD-L1 interaction-induced luciferase expression reduction in cell-based functional assays.

[0044] Figure 12 The binding affinity of humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 to human PD-1 is shown in the capture ELISA.

[0045] Figure 13 The binding affinity of humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 to monkey PD-1 in indirect ELISA is shown.

[0046] Figure 14 The binding affinity of humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 to GS-J2 / PD-1 cells expressing human PD1 was demonstrated in cell-based binding FACS assays.

[0047] Figure 15 This demonstrates the ability of humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 to block human PD-1-PD-L1 binding in competitive ELISA assays.

[0048] Figure 16This demonstrates the ability of humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 to block pembrolizumab-PD-1 binding in competitive ELISA.

[0049] Figure 17 This demonstrates the ability of humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 to block the binding of PD-1 to PD-L1 on the cell surface in cell-based closed FACS assays.

[0050] Figure 18 This demonstrates the ability of humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 to reverse PD-1-PD-L1 interaction-induced luciferase expression reduction in cell-based functional assays.

[0051] Figures 19A-19B The results of protein thermal migration experiments for humanized antibodies huE1A9C8A7-V5(A) and huE1A9C8A7-V8(B) are shown.

[0052] Figure 20 The binding affinity of humanized antibodies huE1A9C8A7-V8 and huE1A9C8A7-V8-3 to GS-J2 / PD-1 cells expressing human PD1 was demonstrated in cell-based binding FACS assays.

[0053] Figure 21 This demonstrates the ability of humanized antibodies huE1A9C8A7-V8 and huE1A9C8A7-V8-3 to reverse PD-1-PD-L1 interaction-induced luciferase expression reduction in cell-based functional assays.

[0054] Figure 22 The changes in tumor volume in mice treated with mouse-derived antibody E2G4E10B7 or the control are shown. Detailed Implementation

[0055] To ensure easier understanding of this application, some terms are defined first. Other definitions are given throughout the detailed description.

[0056] The term "PD-1" or "PD1" refers to programmed cell death protein 1, also known as CD279. The term "PD-1" can include variants, isoforms, homologs, orthologs, and paralogs. For example, an antibody specific to human PD-1 protein may, in some cases, cross-react with PD-1 protein from species other than humans, such as monkeys. In other embodiments, an antibody specific to human PD-1 protein may be completely specific to human PD-1 protein and exhibit no cross-reactivity with other species or other types, or may cross-react with PD-1 from some other species but not all others.

[0057] The term “human PD-1” refers to a PD-1 protein with an amino acid sequence derived from humans, such as the amino acid sequence of human PD-1 with NCBI reference number NP_005009.2 (Orth MF et al., (2020), CancerImmunol.Immunother.69(7):1353-1362). The term “monkey PD-1” or “cynomolgus monkey PD-1” refers to a PD-1 protein with an amino acid sequence derived from monkeys, such as the amino acid sequence of NCBI reference number XP_001107830.1 (McGary CSet et al., (2017), Immunity 47(4):776-788). The term “mouse PD-1” refers to the PD-1 protein having an amino acid sequence derived from mice, such as the amino acid sequence of mouse PD-1 with NCBI reference number NP_032824.1 (Lai X et al., (2020) PLoS ONE 15(4): e0231499).

[0058] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., PD-1) through at least one antigen-binding site, where the antigen-binding site is typically located within the variable region of the immunoglobulin molecule. As used herein, the term includes intact polyclonal antibodies, intact monoclonal antibodies, single-chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs), light chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, fusion proteins containing an antigen-binding site, and any other modified immunoglobulin molecule containing an antigen-binding site (e.g., a dual variable domain immunoglobulin molecule), provided that the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, mouse-derived antibodies, chimeric antibodies, humanized antibodies, and humanized antibodies. Antibodies can be any of the five major immunoglobulin classes—IgA, IgD, IgE, IgG, and IgM—or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on their heavy chain constant domains, respectively designated α, δ, ε, γ, and μ. Different types of immunoglobulins have different well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated to other molecules, including but not limited to toxins and radioisotopes. Unless otherwise explicitly stated, the term "antibody" as used herein includes the "antigen-binding portion" of a complete antibody. IgG can be a glycoprotein comprising two heavy chains (H) and two light chains (L) internally linked by disulfide bonds. Each heavy chain can be composed of a heavy chain variable region (abbreviated as V). H It consists of a heavy chain constant region and a heavy chain constant region. The heavy chain constant region can be composed of C H1 CH2 and C H3 These three structural domains constitute the structure. Each light chain can be composed of a light chain variable region (abbreviated as V). L It consists of a light chain constant region and a light chain constant region. The light chain constant region can be composed of C L This is composed of a single structural domain. V H and V L This can be further subdivided into highly variable regions, called complementary determinant regions (CDR), interspersed with more conservative regions called framework regions (FR). Each V H and V L It consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system.

[0059] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more fragments on an antibody that retain the ability to specifically bind to an antigen (e.g., the PD-1 protein). It has been shown that the antigen-binding function of an antibody can be exercised by fragments of a full-length antibody. Examples of binding fragments included in the term "antigen-binding portion" of an antibody include (i) the Fab fragment, which is composed of V... L V H C L and C H1 (ii) F(ab′)2 segments, which may contain divalent segments formed by two Fab segments connected by disulfide bonds in the hinge region; (iii) segments composed of V H and C m (iv) Fd fragment composed of structural domains; V of antibody single arm L and V H Fv segments composed of structural domains; (v) composed of V H The dAb fragment composed of domains (Ward et al., (1989) Nature 341: 544-546); (vi) a separated complementarity-determining region (CDR); and (viii) a nanobody containing a heavy chain variable region with a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, V L and V H Encoded by different genes, they can be linked together via synthetic linkers through recombination, whereby the synthetic linkers allow them to be prepared as single protein chains, wherein V L and V HPairing forms monovalent molecules (referred to as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding portion" of antibody. These antibody fragments are obtained using common techniques known to those skilled in the art, and the fragments are applied and screened in the same manner as intact antibodies.

[0060] The term "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to the PD-1 protein and is substantially free of antibodies that specifically bind to proteins other than PD-1). However, isolated antibodies that specifically bind to the human PD-1 protein may exhibit cross-reactivity with other antigens, such as PD-1 proteins from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0061] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that individual antibodies comprising this group are identical, except for a small number of potentially naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). Monoclonal antibodies are highly specific, targeting a single antigenic site. Unlike polyclonal antibody formulations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized through hybridoma culture, free from contamination by other immunoglobulins. The modifier "monoclonal" refers to the characteristic that the antibody is derived from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to the invention can be produced by a variety of techniques, including, for example, the hybridoma method.

[0062] As used herein, the term "mouse-derived antibody" is intended to include antibodies whose backbone and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a mouse germline immunoglobulin sequence. The mouse-derived antibodies of this application may contain amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced through in vitro random or point mutations or through in vivo somatic mutations). However, as used herein, the term "mouse-derived antibody" is not intended to include antibodies with a CDR sequence derived from another mammalian species embedded in the mouse backbone sequence.

[0063] The term "chimeric antibody" refers to an antibody prepared by combining non-human genetic material with human genetic material. Or, more generally, a chimeric antibody is an antibody containing genetic material from one species as well as genetic material from another species.

[0064] The term "humanized antibody" as used in this article refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to naturally occurring antibody variants in humans.

[0065] The term "isotype" refers to an antibody class (such as IgM or IgG1) encoded by a gene in the heavy chain constant region.

[0066] The phrases “antibody that recognizes antigen” and “antibody that is specific to antigen” are used interchangeably with the term “antibody that specifically binds to antigen” in this article.

[0067] As used herein, an antibody that "specifically binds to human PD-1" refers to an antibody that binds to human PD-1 protein (and possibly PD-1 proteins from one or more non-human species) but substantially does not bind to non-PD-1 proteins. Preferably, the antibody has "high affinity," i.e., K... D The value is 5.0 × 10 -8 M or less, more preferably 1.0 × 10 -8 M or less, preferably 5.0×10 -9 Below M, it binds to human PD-1 protein.

[0068] As used herein, the term "basically non-binding" protein or cell refers to a protein or cell that does not bind to itself, or does not bind to it with high affinity, i.e., with K... D 1×10 -6 M or higher, preferably 1×10 -5 M or higher, preferably 1×10 -4 M or higher, preferably 1×10 -3 M or higher, preferably 1×10 -2 M and above, binding proteins or cells.

[0069] For IgG antibodies, the term "high affinity" refers to an antibody's KD (kinematic density) for the target antigen being 1.0 × 10⁻⁶. -7 M or less, more preferably 1.0 × 10 -8 M or less, more preferably 1.0 × 10 -9 M or less, more preferably 1.0 × 10 -10 Below M. However, for other antibody isotypes, "high affinity" binding can vary. For example, for IgM isotypes, "high affinity" binding refers to the antibody's K... D 10 -6 M or less, more preferably 10 -7 M or less, or even more preferably 10-8 Below M.

[0070] The term "K" used in this article assoc "or "K a "K" refers to the binding rate of a specific antibody-antigen interaction, while the term "K" used in this article... dis "or "K d "K" refers to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "K" is... D "Refers to K" d With K a The ratio (i.e., K) d / K a The dissociation constant obtained is expressed as molar concentration (M). The Kc of the antibody can be determined using methods known in the art. D Value. Used to determine antibody K. D The preferred method is to use surface plasmon resonance, preferably using a biosensor system such as Biacore. TM system.

[0071] The term "EC" 50 The half-maximal effect concentration (WMP) is the antibody concentration that elicits an intermediate response between the baseline and the maximum value after a specific exposure time.

[0072] The term "IC" 50 "Half-maximal inhibitory concentration" (WMC), also known as the antibody concentration that inhibits a specific biological or biochemical function by 50% relative to the absence of an antibody, is the concentration of an antibody that inhibits a specific biological or biochemical function by 50%.

[0073] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammalians, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, are preferred.

[0074] The term "therapeutic effective amount" refers to the amount of the antibody or antigen-binding portion of this application sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., chronic inflammation) and / or reduce the severity of the disease or condition. Therapeutic effective amount is understood in the context of the disease being treated, where a person skilled in the art can readily determine the actual effective amount.

[0075] Several aspects of this application are described in further detail below.

[0076] The antibody or its antigen-binding portion of this application specifically binds to human, monkey, and / or mouse PD-1, and compared with prior art PD-1 antibodies such as nivolumab and pembrolizumab, has i) comparable (if not better) binding affinity / ability to human, monkey, and mouse PD-1, ii) comparable (if not better) activity in blocking PD-1-PD-L1 interactions, and / or iii) comparable (if not better) in vivo antitumor activity. The antibody or its antigen-binding portion of this application may be mouse-derived, chimeric, or humanized.

[0077] The antibodies or antigen-binding moieties of this application are characterized structurally and chemically as follows. The amino acid sequence IDs of the heavy / light chain variable regions and CDRs of the antibodies or antigen-binding moieties of this application are summarized in Table 1 below; some antibodies share the same VH or VL. The heavy chain constant region of the antibody may be the human IgG4 heavy chain constant region having, for example, the amino acid sequence shown in SEQ ID NO: 24, or other heavy chain constant regions with weak FcR binding affinity, for the treatment of tumors and infectious diseases or for enhancing immune responses. The heavy chain constant region may also be, for example, the human IgG1 constant region with high FcR binding affinity. The light chain constant region of the antibody may be the human κ constant region having, for example, the amino acid sequence shown in SEQ ID NO: 25. The antibodies of this application may also contain a human λ light chain constant region.

[0078] The heavy chain variable region CDR and light chain variable region CDR in Table 1 are determined by the Kabat numbering system. However, as is well known in the art, the CDR region can also be determined based on the heavy chain / light chain variable region sequence using other systems such as Chothia, IMGT, AbM, or Contact numbering systems / methods.

[0079] V, which binds to other PD-1 antibodies against human PD-1 H and V L The sequence (or CDR sequence) can be associated with the V of the PD-1 antibody disclosed herein. H and V L Sequence (or CDR sequence) "mixing and matching". Preferably, when V H and V L When chains (or CDRs within these chains) are mixed and matched, the value from a specific V H / V L Paired V H The sequence is structurally similar to V H Sequence substitution. Similarly, it is preferred to come from a specific V. H / V L Paired V L Sequence via structurally similar V L Sequence replacement.

[0080] Therefore, in one embodiment, the antibody or its antigen-binding portion of this application includes:

[0081] (a) The heavy chain variable region containing the amino acid sequences listed in Table 1; and

[0082] (b) A light chain variable region containing the amino acid sequence listed in Table 1, or a V of another PD-1 antibody. L This antibody specifically binds to human PD-1.

[0083] In another embodiment, the antibody or its antigen-binding portion of this application includes:

[0084] (a) CDR1, CDR2, and CDR3 of the heavy chain variable regions listed in Table 1 above; and

[0085] (b) CDR1, CDR2 and CDR3 of the light chain variable region listed in Table 1 above, or the CDR of another PD-1 antibody that specifically binds to human PD-1.

[0086] In another embodiment, the antibody or antigen binding site includes the heavy chain variable CDR2 region of the PD-1 antibody, and is bound to the CDR regions of other human PD-1 antibodies, such as the heavy chain variable regions CDR1 and / or CDR3, and / or the light chain variable regions CDR1, CDR2 and / or CDR3 of different PD-1 antibodies.

[0087]

[0088] Furthermore, as is well known in the art, the CDR3 domain, independent of the CDR1 and / or CDR2 domains, can independently determine the binding specificity of an antibody to a homologous antigen. And based on the same CDR3 sequence, it is foreseeable that multiple antibodies with the same binding specificity can be generated. See, for example, Klimka et al., British J. of Cancer 83(2):252-260 (2000); Beiboer et al., J. Mol. Biol. 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998); Barbas et al. al., J. Am. Chem. Soc. 116: 2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. USA 92: 2529-2533 (1995); Ditzel et al., J. Immunol. 157: 739-749 (1996); Berezov et al., BIAjournal 8:Scientific Review 8 (2001); Igarashi et al., J. Biochem (Tokyo) 117: 452-7 (1995); Bourgeois et al., J. Virol 72: 807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. USA 90: 4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000). See also U.S. Patents 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. Each of these references is incorporated herein by reference in its entirety.

[0089] Therefore, in another embodiment, the antibody of this application comprises CDR2 of the heavy chain variable region of a PD-1 antibody, at least CDR3 of the heavy chain and / or light chain variable region of a PD-1 antibody, or CDR3 of the heavy chain and / or light chain variable region of another PD-1 antibody, wherein the antibody can specifically bind to human PD-1. Preferably, these antibodies (a) competitively bind to PD-1; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have a binding affinity similar to that of the PD-1 antibody of this application. In another embodiment, the antibody may further comprise CDR2 of the light chain variable region of a PD-1 antibody, or CDR2 of the light chain variable region of another PD-1 antibody, wherein the antibody can specifically bind to human PD-1. In another embodiment, the antibody of this application may comprise CDR1 of the heavy chain and / or light chain variable region of a PD-1 antibody, or CDR1 of the heavy chain and / or light chain variable region of another PD-1 antibody, wherein the antibody can specifically bind to human PD-1.

[0090] In another embodiment, the antibody of this application comprises heavy chain and / or light chain variable region sequences of CDR1, CDR2, and CDR3, which differs from the variable region of the PD-1 antibody of this application in that it has one or more conserved modifications. As understood in the art, some conserved sequence modifications that do not eliminate antigen binding can be performed. See, for example, Brummell et al., (1993) Biochem 32: 1180-8; de Wildt et al., (1997) Prot. Eng. 10: 835-41; Komissarov et al., (1997) J. Biol. Chem. 272: 26864-26870; Hall et al., (1992) J. Immunol. 149: 1605-12; Kelley and O'Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10: 341-6; and Beers et al., (2000) Clin. Can. Res. 6: 2835-43.

[0091] Therefore, in one embodiment, the antibody comprises a heavy chain variable region having CDR1, CDR2, and CDR3 sequences and / or a light chain variable region having CDR1, CDR2, and CDR3 sequences, wherein:

[0092] (a) The heavy chain variable region CDR1 sequence contains the sequences listed in Table 1 above, and / or their conservative modifications; and / or

[0093] (b) The heavy chain variable region CDR2 sequence contains the sequences listed in Table 1 above, and / or their conservative modifications; and / or

[0094] (c) The heavy chain variable region CDR3 sequence contains the sequences listed in Table 1 above, and / or their conservative modifications; and / or

[0095] (d) The light chain variable regions CDR1, and / or CDR2, and / or CDR3 sequences contain sequences as listed in Table 1 above, and / or their conservative modifications; and

[0096] (e) This antibody specifically binds to human PD-1.

[0097] The antibody of this application has one or more of the above-mentioned functional characteristics, such as high binding affinity to human PD-1 and blocking activity against PD-1-PD-L1 binding.

[0098] In various embodiments, the antibody may be, for example, a mouse-derived, human-derived, humanized, or chimeric antibody.

[0099] As used herein, the term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing such amino acid sequences. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitution refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains have been defined in the art. These groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of this application can be replaced by other amino acid residues in the same side chain group, and the modified antibody can be tested for its retained function (i.e. the function described above) using the functional assay described herein.

[0100] The antibody of this application can be used with one or more V antibodies containing the PD-1 antibody of this application. H / V L Antibodies with specific sequences are used as starting materials to prepare modified antibodies. Antibodies can be modified by altering one or two variable regions (i.e., V...). H and / or V LThe antibody may be modified by modifying one or more residues in the constant region (e.g., in one or more CDR regions and / or one or more backbone regions). Additionally, and optionally, the antibody may be engineered by modifying residues in the constant region to, for example, alter the antibody's effector function.

[0101] In some implementations, CDR implantation can be used to engineer the variable regions of antibodies. Antibodies primarily interact with target antigens through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences within the CDRs are more diverse than those outside the CDRs among individual antibodies. Because the CDR sequence is responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of specific natural antibodies can be expressed by constructing expression vectors that incorporate CDR sequences from specific natural antibodies into the backbone sequences of different antibodies with different properties. (See, for example, Riechmann et al., (1998) Nature 332: 323-327; Jones et al., (1986) Nature 321: 522-525; Queen et al., (1989) Proc. Natl. Acad. See also USA 86: 10029-10033; US Patents 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0102] Therefore, another embodiment of this application relates to isolated monoclonal antibodies or their antigen-binding portions, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences having the sequences described above in this application, and the light chain variable region comprises CDR1, CDR2, and CDR3 sequences having the sequences described above in this application. Although these antibodies contain the V of the monoclonal antibody of this application... H and V L CDR sequences can contain different backbone sequences.

[0103] These backbone sequences can be obtained from publicly available DNA databases or references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes are available in the VBase human germline sequence database (available at www.mrc-cpe.cam.ac.uk / vbase), as well as in Kabat et al., (1991), ibid.; Tomlinson et al., (1992) J Mol. Biol. 227: 776-798; and Cox et al., (1994) Eur. J Immunol. 24: 827-836; the contents of which are explicitly incorporated herein by reference. As another example, germline DNA sequences for human heavy and light chain variable region genes are available in the Genbank database. For example, the heavy chain germline sequences in the following HCo7HuMAb mice can be obtained from the attached Genbank accession numbers 1-69 (NG-0010109, NT-024637 & BC070333), 3-33 (NG-0010109 & NT-024637), and 3-7 (NG-0010109 & NT-024637). As another example, the following heavy chain germline sequences from Hco12 HuMAb mice are obtained from Genbank accession numbers 1-69 (NG-0010109, NT-024637 & BC070333), 5-51 (NG-0010109 & NT-024637), 4-34 (NG-0010109 & NT-024637), 3-30.3 (CAJ556644) & 3-23 (AJ406678).

[0104] Antibody protein sequences were compared with compiled protein sequence databases using a sequence similarity search method known in the art, called space BLAST (Altschul et al., (1997), ibid.).

[0105] The preferred backbone sequences used for the antibodies in this application are those that are structurally similar to the backbone sequences used for the antibodies in this application. H The CDR1, CDR2, and CDR3 sequences can be inserted into a backbone region that has the same sequence as the germline immunoglobulin gene from which the backbone sequence is derived, or the CDR sequence can be inserted into a backbone region that contains one or more mutations compared to the germline sequence. For example, it has been found that, in some cases, mutated residues in the backbone region are beneficial to maintain or enhance the antigen-binding capacity of the antibody (see, for example, U.S. Patents 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0106] Another type of variable region modification is to modify V H and / or V L Amino acid residues within the CDR1, CDR2, and / or CDR3 regions are mutated to improve one or more binding properties (e.g., affinity) of the target antibody. Mutations can be introduced through point mutations or PCR-mediated mutations, and their effects on antibody binding or other target functional properties can be evaluated using in vitro or in vivo assays known in the art. Preferably, conserved modifications (known in the art) are introduced. Mutations can be amino acid substitutions, additions, or deletions, but substitution is preferred. Furthermore, typically no more than one, two, three, four, or five residues are altered in the CDR regions.

[0107] Therefore, in another embodiment, this application provides an isolated PD-1 monoclonal antibody or its antigen-binding moiety, which includes a heavy chain variable region comprising: (a) V H CDR1 region, which contains the sequence of this application, or contains one, two, three, four or five amino acid substitutions, deletions or additions; (b) V H CDR2 region, which contains the sequence of this application, or contains one, two, three, four or five amino acid substitutions, deletions or additions; (c)V H The CDR3 region contains the sequence of this application, or contains one, two, three, four, or five amino acid substitutions, deletions, or additions; (d)V L The CDR1 region contains the sequence of this application, or contains one, two, three, four, or five amino acid substitutions, deletions, or additions; (e)V L The CDR2 region contains the sequence of this application, or contains one, two, three, four, or five amino acid substitutions, deletions, or additions; and (f)V L The CDR3 region may contain the sequence of this application, or contain one, two, three, four or five amino acid substitutions, deletions or additions.

[0108] The genetically modified antibodies of this application include, for example, those used to improve antibody performance, in V H and / or V L Antibodies are antibodies with genetically modified backbone residues. Typically, these backbone modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "reverse mutate" one or more backbone residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutations may contain backbone residues that differ from the germline sequence of the resulting antibody. These residues can be identified by comparing the antibody backbone sequence with the germline sequence of the resulting antibody.

[0109] Another type of backbone modification involves mutating one or more residues in the backbone region, or even one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also known as "deimmunization," and is described in more detail in US Patent Publication 20030153043.

[0110] In addition, or as an alternative to modifications within the backbone or CDR region, the antibodies of this application can be genetically modified to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cytotoxicity. Furthermore, the antibodies of this application can be chemically modified (e.g., by attaching one or more chemical groups to the antibody), or modified to alter their glycosylation, again to alter one or more functional properties of the antibody.

[0111] In one implementation, C H1 The hinge region is modified, altering, for example, by increasing or decreasing, the number of cysteine ​​residues in the hinge region. This method is further described in U.S. Patent 5,677,425. [The text then abruptly shifts to a seemingly unrelated topic:] Changing C... H1 The number of cysteine ​​residues in the hinge region can, for example, promote the assembly of light and heavy chains or increase or decrease the stability of the antibody.

[0112] In another embodiment, the Fc hinge region of the antibody is mutated to reduce the antibody's biological half-life. More specifically, one or more amino acid mutations are introduced into the C13 region of the Fc hinge segment. H2 -C H3 The antibody exhibits weakened SpA binding compared to binding to the native Fc-hinge domain of Staphylococcus aureus protein A (SpA). This method is described in more detail in U.S. Patent 6,165,745.

[0113] In another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered, for example, to increase the antibody's affinity for the antigen. Such glycosylation modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that location. Such deglycosylation can increase the antibody's affinity for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0114] Alternatively or alternatively, antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced fucose residues or antibodies with an increased bisecting GlcNac structure. These altered glycosylation forms have been shown to increase or decrease the ADCC activity of the antibody. Such glycosylation modifications can be accomplished, for example, by expressing the antibody in a host cell with an altered glycosylation system. Cells with altered glycosylation systems are known in the art and can be used as host cells for expressing the recombinant antibody of this application to prepare glycosylated antibodies. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosylation gene FUT8 (α(1,6)-fucosylation), resulting in antibodies expressed in Ms704, Ms705, and Ms709 cell lines lacking fucose in their glycosides. Two alternative vectors were used to target and disrupt the FUT8 gene in CHO / DG44 cells to prepare Ms704, Ms705, and Ms709 FUT8- / - cell lines (see US Patent 20040110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene (encoding fucosyltransferase), in which antibodies expressed exhibit low fucosylation by reducing or eliminating the α-1,6 bond-related enzyme. EP 1,176,195 also describes a cell line with low or no activity in adding fucosylate to the N-acetylglucosamine binding region of antibody-binding cells, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 03 / 035835 describes a CHO variant cell line, Lec13 cells, which has a reduced ability to add fucose to the Asn(297)-linked sugar, resulting in hypofucosylation of antibodies expressed in host cells (see Shields et al., (2002) J. Biol. Chem. 277: 26733-26740). Antibodies with altered glycosylation profiles can also be prepared in eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies with altered glycosylation profiles can be prepared in plant cells such as duckweed. Methods for preparing antibodies in plant systems are described in U.S. Patent Application filed August 11, 2006, corresponding to Alston & Bird LLP Attorney Record No. 040989 / 314911. Fucosidases can be used to cleave fucose residues from antibodies, such as α-L-fucosidase to remove fucose residues from antibodies (Tarentino et al., (1975) Biochem. 14: 5516-23).

[0115] Another modification to the antibodies included in this application is polyethylene glycol (PEG) oxidation. Antibodies can be PEGylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as an active ester or aldehyde derivative of PEG, under conditions that attach one or more PEG groups to the antibody or antibody fragment. Preferably, PEGylation is carried out by an acylation or alkylation reaction with an active PEG molecule (or a similar reactive water-soluble polymer). The term “polyethylene glycol” as used herein is intended to include any form of PEG used to derive other proteins, such as mono(C1-C1) PEG. 10 Alkoxy- or aryloxy polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody requiring PEGylation is a deglycosylated antibody. Methods for PEGylation of proteins are known in the art and can be applied to the antibodies of this application. See, for example, EPO 154 316 and EP 0 401 384.

[0116] The antibodies of this application can be characterized by a variety of physical properties to detect and / or distinguish their different classifications.

[0117] For example, antibodies may contain one or more glycosylation sites in the variable region of the light or heavy chain. These glycosylation sites may increase the immunogenicity of the antibody or cause changes in the antibody's pK due to alterations in antigen binding (Marshallet et al. (1972) Annu Rev Biochem 41: 673-702; Gala and Morrison (2004) J Immunol 172: 5489-94; Wallick et al. (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al. (1985) Nature 316: 452-7; Mimura et al., (2000) Mol Immunol 37: 697-706). Glycosylation is known to occur in motifs containing NXS / T sequences. In some cases, PD-1 antibodies that do not contain variable region glycosylation are preferred. This can be achieved by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues within the glycosylation region.

[0118] In a preferred embodiment, the antibody does not contain an asparagine isomer site. Deamidation of asparagine may occur at the NG or DG sequence, resulting in the formation of isoaspartic residues, which introduce linkages into the polypeptide chain and reduce its stability (isoaspartic effect).

[0119] Each antibody will have a unique isoelectric point (pI), which generally falls within the pH range of 6–9.5. The pI of IgG1 antibodies typically falls within the pH range of 7–9.5, while the pI of IgG4 antibodies generally falls within the pH range of 6–8. It is speculated that antibodies with pI values ​​outside the normal range may exhibit some structural variations and instability under in vivo conditions. Therefore, PD-1 antibodies with pI values ​​within the normal range are preferred. This can be achieved by selecting antibodies with pI values ​​within the normal range or by mutating charged surface residues.

[0120] On the other hand, this application provides nucleic acid molecules encoding the variable regions or CDRs of the heavy and / or light chains of the antibody of this application. Nucleic acids can be present in intact cells, cell lysates, or in partially or substantially purified forms. When purified from other cellular components or other contaminants such as other cellular nucleic acids or proteins using standard techniques, the nucleic acid is "isolated" or "in a substantially purified state." The nucleic acid of this application can be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0121] The nucleic acids used in this application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, further described below), the cDNA encoding the light and heavy chains of the hybridoma-prepared antibody can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleotides encoding such antibodies can be recovered from the gene library.

[0122] Preferred nucleic acid molecules of this application include V encoding a PD-1 monoclonal antibody. H and V L Those sequences or CDRs. Once the encoded V is obtained... H and V L DNA fragments that can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the gene encoding V... H or V L The DNA fragment is operatively linked to another DNA fragment encoding a different protein (such as an antibody constant region or a flexible linker). In this context, the term "operatively linked" means that two DNA fragments are joined together such that the amino acid sequences encoded by both fragments are within the frame.

[0123] Encoding V H The isolated DNA from the region can be operatively linked to encode V. H DNA and coding heavy chain constant regions (CH1 C H2 and C H3 Another DNA molecule is transformed into a full-length heavy chain gene. The sequences of human heavy chain constant regions are known in the field, and DNA fragments including these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but IgG1 or IgG4 constant regions are most preferred. For Fab fragment heavy chain genes, encoding V... H The DNA of this region can be operatively coupled with DNA that encodes only the heavy chain C. Hi Another DNA molecule is linked to the constant region.

[0124] Encoding V L The isolated DNA from the region can be operatively linked to encode V. L DNA and encoding the light chain constant region C L Another DNA molecule is transformed into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant regions are known in the field, and DNA fragments including these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant regions may be κ and λ constant regions.

[0125] To create the scFv gene, which encodes V H and V L The DNA fragment can be operatively linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as a continuous single-stranded protein, where V... L and V H The regions are connected by this flexible joint (see, for example, Bird et al., (1988) Science 242: 423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., (1990) Nature 348: 552-554).

[0126] The monoclonal antibodies (mAbs) of this application can be prepared using somatic cell hybridization (hybridoma) techniques known in the art, as described in Kohler and Milstein (1975) Nature 256:495. Other methods for preparing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are known in the art. See, for example, U.S. Patents 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the contents of which are incorporated herein by reference in their entirety.

[0127] The antibodies of this application can also be generated in host cell transfection tumors using, for example, a combination of recombinant DNA technology and gene transfection methods known in the art (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding a partial or full-length light and heavy chain, obtained by standard molecular biotechnology, is inserted into one or more expression vectors, thereby operatively linking the gene to transcriptional and translational regulatory sequences. In this context, the term "operatively linking" refers to linking an antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene.

[0128] The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Such regulatory sequences have been described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that guide high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses such as the adenovirus major late promoter (AdMLP), and polyomaviruses. Alternatively, non-viral regulatory sequences, such as ubiquitin promoters or β-globin promoters, may be used. In addition, regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and long terminal repeats of human T-cell leukemia virus type I (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472). Expression vectors and expression control sequences compatible with the host cells used for expression are selected.

[0129] Antibody light chain genes and antibody heavy chain genes can be inserted into the same or different expression vectors. In a preferred embodiment, a full-length antibody gene of any antibody isotype is constructed by inserting the variable region into an expression vector that already encodes the heavy chain constant region and light chain constant region of the desired isotype, thereby V H C in part of the carrier H Partially operable connection, V L C in part of the carrier L Partially operably linked. Additionally, or furthermore, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector, allowing the signal peptide to be linked within the frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0130] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of this application may carry other sequences, such as sequences that regulate vector replication in host cells (e.g., replication origin) and selectable marker genes. Selectable marker genes facilitate selection in host cells introduced with the vector (see, for example, U.S. Patents 4,399,216; 4,634,665 and 5,179,017). For example, typically, selectable marker genes confer resistance in host cells introduced with the vector to drugs such as G418, hygromycin, or methotrexate. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for DHFR host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0131] For the expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. The term "transfection" is intended to encompass various commonly used techniques for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-glucan transfection. Although expressing the antibodies of this application in prokaryotic or eukaryotic host cells is theoretically feasible, expression in eukaryotic cells is most preferred, particularly in mammalian host cells, because these eukaryotic cells, especially mammalian cells, are more likely than prokaryotic cells to assemble and secrete appropriately folded and immunologically active antibodies.

[0132] Preferred mammalian host cells for expressing the recombinant antibodies of this application include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells used with DHFR selectable markers, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, and DHFR selectable markers described, for example, in RJ Kaufman and PASharp (1982) J. Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. Particularly when using NSO myeloma cells, another preferred expression system is the GS gene expression system, described in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, antibodies are prepared by culturing the host cells for a period sufficient for antibody expression, or preferably by secreting the antibodies into the host cell culture medium. The antibodies are then recovered from the culture medium using standard protein purification methods.

[0133] On the other hand, this disclosure relates to bispecific molecules, which may comprise one or more antibodies of this application linked to at least one other functional molecule, such as another peptide or protein (e.g., a ligand of another antibody or receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. Therefore, as used herein, a “bispecific molecule” includes molecules with three or more specificities.

[0134] In this embodiment, in addition to FcR binding specificity and PD-1 binding specificity, the bispecific molecule also possesses a third specificity. This third specificity can be against PD-1.

[0135] Bispecific molecules can appear in a variety of different forms and sizes. At one end of the size spectrum, bispecific molecules retain the traditional antibody form, except that they have two binding arms, each with different specificities, rather than two binding arms with the same specificity. At the other extreme are bispecific molecules consisting of two single-chain antibody fragments (scFv's) linked by peptide chains, the so-called Bs(scFv)2 construct. Bispecific molecules of intermediate size consist of two different F(ab) fragments linked by peptide linkers. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic cell hybridization, or chemical methods. See, for example, Kufer et al., ibid.; Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al., Immunology Today, 21(8), 391-397 (2000), and the references cited therein.

[0136] In other aspects, the present invention provides diagnostic methods, compositions, and kits. In one embodiment, the antibody of the present invention or its antigen-binding site is used to determine the presence and quantity of PD-1 in tissues. In one embodiment, the diagnosis indicates prognosis and / or guides treatment and / or follow-up therapy. For example, the PD-1 signaling pathway has been identified as a target for the treatment of infectious diseases and tumors. In one embodiment, the antibody of the present invention or its antigen-binding site is used in a diagnostic kit or method to determine the prognosis and appropriate treatment and follow-up for infectious diseases and / or PD-1-related tumors or cancers.

[0137] The antibody of this application can bind to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include anti-inflammatory agents and anticancer agents. In an ADC, the antibody and the therapeutic agent preferably bind via a cleavable linker, such as a peptide linker, a disulfide linker, or a hydrazone linker. More preferably, the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. The ADC can be prepared as described in U.S. Patents 7,087,600; 6,989,452 and 7,129,261; PCT Publications WO 02 / 096910; WO 07 / 038,658; WO 07 / 051,081; WO 07 / 059,404; WO 08 / 083,312 and WO 08 / 103,693; U.S. Patent Publications 20060024317; 20060004081 and 20060247295.

[0138] This article also provides chimeric antigen receptors (CARs) comprising anti-PD-1 scFvs, which may contain the CDRs and heavy / light chain variable regions described herein.

[0139] Anti-PD-1 CARs may include (a) an extracellular antigen-binding domain that may contain an anti-PD-1 scFv; (b) a transmembrane domain; and (c) an intracellular signal transduction domain.

[0140] CARs may contain a signal peptide at the N-terminus of the extracellular antigen-binding domain that guides nascent receptors into the endoplasmic reticulum, and a hinge peptide at the N-terminus of the extracellular antigen-binding domain that facilitates receptor binding. Preferably, the CAR contains a major intracellular signal transduction domain and one or more co-stimulatory signal transduction domains in its intracellular signal transduction domain. The primary and most effective major intracellular signal transduction domain is the CD3-ζ cytoplasmic domain containing ITAM, whose phosphorylation induces T cell activation. The co-stimulatory signal transduction domain may be derived from co-stimulatory proteins such as CD28, CD137, and OX40.

[0141] CARs can also be supplemented with factors that enhance T cell proliferation, persistence, and anti-tumor activity, such as cytokines and co-stimulatory ligands.

[0142] Genetically modified immune effector cells are also provided, which may contain the CARs provided herein. In some embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In some embodiments, the immune effector cells are T cells.

[0143] Oncolytic viruses preferentially infect and kill cancer cells. The antibodies or antigen-binding sites of this disclosure can be used in combination with oncolytic viruses. Alternatively, oncolytic viruses encoding the antibodies or antigen-binding sites of this disclosure can be introduced into the human body.

[0144] On the other hand, this application provides a pharmaceutical composition comprising an antibody or its antigen-binding portion formulated with a pharmaceutically acceptable carrier, an immunoconjugate, a bispecific molecule, an immune cell carrying a chimeric antigen receptor, an oncolytic virus, a nucleic acid molecule, an expression vector, and / or a host cell. The composition may optionally contain one or more other pharmaceutically active ingredients, such as antitumor agents, anti-infective agents, or immunomodulatory agents. The pharmaceutical composition of this application may be administered in combination therapy with, for example, antitumor agents, anti-infective agents, or immunomodulatory agents.

[0145] Pharmaceutical compositions may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are discussed in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the contents of which are incorporated herein by reference.

[0146] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that may inactivate it. As used herein, the term "parenteral administration" refers to a non-enteral and non-topical route of administration, typically by injection, including but not limited to intravenous, intramuscular, intra-articular, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Alternatively, the antibody of this application may be administered via non-parenteral routes, such as topical, epidermal, or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical application.

[0147] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0148] The amount of active ingredient that can be formulated into a single dosage form with a carrier material will vary depending on the therapeutic subject and the specific route of administration, and is generally the amount of the composition that produces the therapeutic effect. Basically, in percentage terms, this amount is about 0.01% to about 99%, preferably about 0.1% to about 70%, and most preferably about 1% to about 30% of the active ingredient when combined with a pharmaceutically acceptable carrier.

[0149] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, rapid infusions can be administered, multiple fractions can be administered over time, or the dose can be proportionally reduced or increased depending on the severity of the treatment condition. Particularly advantageous are dose-unit parenteral compositions formulated for ease of administration and uniform dosing. As used herein, dose-unit refers to physically discontinuous units suitable for a single dose to the therapeutic subject; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect when combined with the desired pharmaceutical carrier. Alternatively, antibodies can be administered as sustained-release formulations, in which case the required dosing frequency is reduced.

[0150] For administration of the composition, the dosage range can be about 0.0001-100 mg / kg, typically 0.01-5 mg / kg. For example, the dosage can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg, or in the range of 1-10 mg / kg. Exemplary treatment regimens require administration once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every three months, or once every three to six months. The optimal dosage regimen for the PD-1 antibody of this application is intravenous administration of the antibody at 1 mg / kg or 3 mg / kg body weight using one of the following dosing schedules: (i) six injections every four weeks, followed by one injection every three months; (ii) once every three weeks; (iii) one injection at 3 mg / kg body weight, followed by one injection at 1 mg / kg body weight every three weeks. In embodiments, the dosage is adjusted to ensure that the antibody concentration in the blood is about 1-1000 μg / ml, and in some methods 25-300 μg / ml.

[0151] The therapeutically effective dose of the PD-1 antibody of this application preferably causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of damage or disability caused by disease suffering. For example, in the treatment of tumor-bearing subjects, the therapeutically effective dose preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and more preferably at least about 80% compared to untreated subjects. The therapeutically effective dose of the antibody can reduce tumor size or alleviate symptoms in subjects, typically humans, or other mammals.

[0152] The pharmaceutical composition may be a controlled-release dosage form, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, for example, Sustained and Controlled Release Drug Delivery Systems. JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0153] Pharmaceutical compositions can be administered via medical devices, such as (1) needle-free subcutaneous injection devices (e.g., U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 and 4,596,556); (2) microinfusion pumps (U.S. Patent 4,487,603); (3) transdermal drug delivery devices (U.S. Patent 4,486,194); (4) bolus injection devices (U.S. Patents 4,447,233 and 4,447,224); and (5) permeation devices (U.S. Patents 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0154] In some embodiments, the monoclonal antibodies of this application or their antigen-binding sites can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibodies of this application can cross the blood-brain barrier, they can be formulated in liposomes, which may also additionally contain targeting functional groups to enhance selective delivery to specific cells or organs. See, for example, U.S. Patents 4,522,811; 5,374,548; 5,416,016; and 5,399,331; VVRanade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al., (1995) FEBS Lett. 357:140; M. Owais et al., (1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al., (1995) Am. J. Physiol. 1233:134; Schreier et al., (1994) J. Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

[0155] The compositions of this application have many in vitro and in vivo uses, relating to, for example, the treatment of infectious diseases and tumors associated with the PD-1 signaling pathway.

[0156] This application provides a method for treating diseases related to the PD-1 signaling pathway, which may include administering a therapeutically effective amount of the composition of this application to a subject.

[0157] The disease can be a tumor or cancer. Tumors or cancers include, but are not limited to, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer (BC), colorectal cancer (CRC) with microsatellite instability or mismatch repair deficiency (MSI-H / dMMR), hepatocellular carcinoma (HCC), classical Hodgkin lymphoma (cHL), melanoma, head and neck lymphoma (HNSCC), cervical cancer, gastric and gastroesophageal cancer, and all advanced solid tumors with MSI-H / dMMR. The composition comprises the antibody of this application or its antigen-binding site (having a heavy chain constant region with weak FcR binding), a bispecific molecule, a nucleic acid molecule, or an expression vector. In some embodiments, the subject is a human.

[0158] The disease can be an infectious disease, including, but not limited to, those caused by viruses, bacteria, or fungi. In some embodiments, the infectious disease is sepsis, HIV infection, simian immunodeficiency virus infection, HBV infection, or HCV infection. The composition comprises an antibody of the present application or its antigen-binding moiety (having a heavy chain constant region with weak FcR binding), a bispecific molecule, a nucleic acid molecule, or an expression vector.

[0159] The disease can be an inflammatory disease. Inflammatory diseases include, but are not limited to, rheumatoid arthritis, colitis, lupus-like nephritis, systemic lupus erythematosus, and psoriasis. The compositions of this application comprise the antibody of this application or its antigen-binding moiety (having a heavy chain constant region with strong FCR binding), an immunoconjugate, a bispecific molecule, a CAR-containing immune cell, a nucleic acid molecule, or an expression vector. In some embodiments, the compositions of this application are administered to inflamed tissue via local delivery.

[0160] On the other hand, this application provides a method for modulating or enhancing the immune response of a subject, comprising administering the composition of this application to the subject to modulate / enhance the subject's immune response. The composition comprises an antibody of this application or its antigen-binding moiety (having a heavy chain constant region with weak FcR binding), a bispecific molecule, a nucleic acid molecule, or an expression vector.

[0161] In one aspect, this application provides a combination therapy in which the pharmaceutical composition of this application is administered together with one or more other agents that can effectively improve PD-1-related infectious diseases. The agent may be an anti-infective agent, such as an antiviral agent, an antibacterial agent, or an antifungal agent. In some embodiments, the subject is a human being.

[0162] In another aspect, this application provides combination treatment methods, wherein the pharmaceutical composition of this application is co-administered with one or more other antibodies effective in inhibiting tumor growth in a subject. In one embodiment, this application provides a method for inhibiting tumor growth in a subject, which may include administering the pharmaceutical composition of this application and one or more other antibodies, such as VEGF antibodies, OX40 antibodies, TIM-3 antibodies, CD137 antibodies, GITR antibodies, LAG-3 antibodies, and PD-L1 antibodies to the subject. In some embodiments, the subject is a human. PD-1 signaling pathway blockade may also be combined with standard cancer treatment. For example, PD-1 signaling pathway blockade may be combined with CTLA-4 blockade and a chemotherapy regimen. For example, a chemotherapeutic agent may be administered with a PD-1 antibody, and the chemotherapeutic agent may be a cytotoxic agent. For example, epirubicin, oxaliplatin, and 5-FU may be administered to a patient receiving PD-1 therapy. Optionally, the combined use of PD-1 antibodies with one or more other antibodies (e.g., CTLA-4 antibodies and / or PD-L1 antibodies) can also be combined with immunogens such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), and cells transfected with genes encoding immunostimulatory factors (He et al., (2004) J. Immunol. 173: 4919-28). Non-limiting examples of tumor vaccines that can be used include melanoma antigen peptides, such as gp100 peptide, MAGE antigen, Trp-2, MART1, and / or tyrosinase, or tumor cells transfected with the cytokine GM-CSF. Other therapeutic regimens that can be combined with PD-1 antibodies include, but are not limited to, administration of interleukin-2 (IL-2), radiotherapy, surgery, or hormone suppression.

[0163] The combination of therapeutic agents discussed herein can be administered concurrently as single components in a pharmaceutically acceptable carrier, or concurrently as separate compositions of each drug in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0164] In addition, if a combination therapy with more than one dose is required, the order of administration can be reversed or kept in the same order at each administration point. Sequential administration can be combined with concurrent administration or any combination thereof.

[0165] Although the present application and its advantages have been described in detail, it should be understood that various changes, substitutions and modifications may be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0166] This application will be further illustrated in the following embodiments, which should not be construed as further limiting. All figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0167] Example

[0168] Example 1. Preparation of mouse-derived PD-1 monoclonal antibody using hybridoma technology

[0169] immunity

[0170] Mice were immunized according to the method described in E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998. Recombinant human PD-1 protein with a C-terminal human IgG1 Fc tag (Cat#PD1-H5257, Acrobiosystems Inc., containing the extracellular domain of human PD-1) was used as an immunogen. Internally prepared human PD-1-Fc protein (amino acid sequence SEQ ID NO: 26) was used for antiserum titer determination and screening for hybridomas secreting antigen-specific antibodies.

[0171] For primary immunization, the dose consists of 22.5 μg recombinant human PD-1-Fc protein per mouse per injection; for booster immunization, it is 25 μg per mouse per injection. To enhance the immune response, Freund's complete adjuvant and Freund's incomplete adjuvant (Sigma, St. Louis, Mo., USA) are used for primary and booster immunizations, respectively. In brief, the immunogen is prepared at a concentration of 0.23–0.35 mg / ml in PBS or saline. The calculated amount of immunogen is added to a microcentrifuge tube containing the required amount of adjuvant, and the mixture is gently vortexed for 2 minutes to form a water-in-oil emulsion. The adjuvant-antigen emulsion is then aspirated into a suitable syringe and injected into the animal. A total of 22.5 or 25 μg of immunogen is injected in volumes of 150–200 μl. Each animal is immunized, followed by 3–4 booster immunizations based on antiserum titer. Before hybridoma fusion, animals that showed good titers as detected by ELISA were given a final booster injection via intraperitoneal injection.

[0172] Hybridoma fusion and screening

[0173] Cells from the mouse myeloma cell line (SP2 / 0-Ag14, ATCC#CRL-1581) were cultured and brought to the logarithmic growth phase before hybridoma fusion. Spleen cells from immunized mice were aseptically prepared and fused with mouse myeloma cells according to the method described in Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 256:495-497 (1975). The fused "hybrid cells" were then seeded in 96-well plates in DMEM / 20% FCS / HAT medium. Viable hybridoma colonies were observed under a microscope 7–10 days post-fusion. Two weeks later, a capture ELISA was performed on the supernatant of each well using internally prepared biotin-labeled human PD-1-Fc protein. Positive hybridomas secreting antibodies binding to human PD-1-Fc protein were selected and transferred to 24-well plates. By limiting dilution, hybridoma clones producing antibodies exhibiting high specificity for human PD-1 and high PD-1-PD-L1 blocking activity were subcloned to ensure the monoclonal origin of the cell lines, followed by purification of the monoclonal antibodies. In short, the protein A agarose column (Cat#AA0273, bestchrom (Shanghai) Biosciences) was washed with 5-10 column volumes of PBS buffer. The cell supernatant from the hybridomas of this application was passed through the column and then washed with PBS buffer until the protein absorbance reached baseline. The column was eluted with elution buffer (0.1M glycine-HCl, pH 2.7) and immediately collected into tubes containing neutral buffer (1M Tris-HCl, pH 9.0). The fraction containing immunoglobulins was concentrated and dialyzed overnight in PBS at 4°C. Subsequently, the functional activity of the purified mouse-derived monoclonal antibodies was characterized in vitro and in vivo as described below.

[0174] Example 2. Determination of binding of mouse-derived PD-1 monoclonal antibody using BIACORE surface plasmon resonance technology. Affinity

[0175] The binding affinity and binding kinetics of the purified mouse monoclonal PD-1 antibody (mAb) produced in Example 1 were characterized using a Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA). Nivolumab (Bristol-Myers Squibb) and Pembrolizumab (Merck) was used as a positive control.

[0176] In summary, using a standard amine conjugation kit provided by Biacore (GE Healthcare, Pittsburgh, PA, USA), goat anti-mouse IgG (Cat#BR100838, GE Healthcare, Mouse Antibody Capture Kit) was covalently linked to a CM5 chip (carboxymethyldextrose coated chip, Cat#BR-1005-30, GE Healthcare) via a primary amine. Unreacted groups on the biosensor surface were blocked with ethanolamine. Affinity assays for positive controls were performed using a Protein G chip (Cat#29-1793-15, GE Healthcare). The PD-1 antibody and two positive controls were flowed through the chip at a concentration of 2 μg / ml and a flow rate of 10 μl / min. Then, serially diluted recombinant human PD-1-his protein (Cat#10377-H08H, Sinobiological Inc.), monkey PD-1-his protein (Cat#PD1-C5223, Acro biosystems Inc.), and recombinant mouse PD-1-his protein (Cat#50124-M08H, Sino Biological Inc.) (2-fold serially diluted on HBS-EP) + Buffer (provided by Biacore, starting at 200 nM) was flowed through the chip at a flow rate of 30 μl / min. Antigen-antibody binding kinetics were observed for 2 minutes, and dissociation kinetics for 10 minutes. Binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAcore evaluation software. K was determined. D K a and K d The values ​​are summarized in Tables 2-1 and 2-2 below.

[0177] Table 2-1. Binding affinity of mouse-derived PD-1 monoclonal antibody to human and monkey PD-1

[0178]

[0179] Table 2-2. Binding affinity of mouse-derived PD-1 monoclonal antibody to mouse PD-1

[0180]

[0181] All mouse-derived antibodies in this application bind specifically to human PD-1 with a binding affinity higher than that of nivolumab and pembrolizumab, and bind to monkey PD-1 with a binding affinity comparable to or higher than that of nivolumab and pembrolizumab. Furthermore, E1A9C8A7 and E2G4E10B7 bind specifically to mouse PD-1, while nivolumab and pembrolizumab do not bind.

[0182] Example 3. Binding activity of mouse-derived PD-1 antibody

[0183] The binding activity of the mouse-derived PD1 antibody to PD-1 was further tested by capture ELISA, indirect ELISA, and flow cytometry (FACS).

[0184] 3.1 Capturing ELISA

[0185] In summary, 96-well microplates were coated with 100 μl of 2 μg / ml F(ab′)2 fragment-specific affinity-purified goat anti-mouse IgG (Cat#115-005-072, Jackson Immuno Research) or goat anti-human IgG (Cat#109-005-097, Jackson Immuno Research, for positive and negative controls) in PBS solution and incubated overnight at 4°C. The plates were washed once with wash buffer (PBST, PBS + 0.05% Tween-20) and then blocked with 200 μl / well blocking buffer (PBST containing 5% w / v skim milk) at 37°C for 2 hours. The plates were washed again and incubated at 37°C for 40 minutes with serially diluted PD-1 antibody, pembrolizumab, and negative control hIgG (human immunoglobulin for intravenous injection (pH4), Hualan Biological Engineering Co., Ltd.) (5-fold diluted in PBST containing 2.5% skim milk, starting concentration 66.7 nM), followed by 4 washes. The plates containing the captured PD-1 antibody were then incubated with biotin-labeled human PD-1-Fc protein (internally prepared, SEQ ID NO: 26, 110 ng / mL in PBST containing 2.5% skim milk, 100 μl / well), followed by 4 washes and incubation at 37°C for 40 minutes with streptavidin-peroxidase (1:10000 diluted in PBST, Cat#016-030-084, Jackson Immuno Research, 100 μl / well). After final washing, the plate was incubated with 100 μl / well of TMB (Cat#TMB-S-002, Innoreagents) at room temperature. The reaction was terminated with 50 μl / well of 1M H₂SO₄ after 3–10 minutes. The absorbance was read using a microplate reader in dual-wavelength mode, with 450 nm for TMB and 630 nm as the reference wavelength. OD(450–630) values ​​were plotted against antibody concentration. The data were analyzed using GraphpadPrism software to determine the EC50 values. 50 Value. Result as follows Figure 1A and 1B As shown.

[0186] 3.2 Indirect ELISA

[0187] The cross-reactivity of the PD-1 antibody of this application with monkey PD-1 protein and mouse PD-1 protein was detected.

[0188] In summary, 96-well microplates were coated with 100 μl of 2 μg / ml mouse PD-1-his (Cat#50124-M08H, Sino Biological Inc.) or 2 μg / ml monkey PD-1-his (Cat#PD1-C5223, Acro biosystems Inc.) in carbonate / bicarbonate buffer (pH 9.6) and incubated at 37°C for 2 hours. The plates were washed once with wash buffer (PBST, PBS + 0.05% Tween-20) and then blocked at 37°C with 200 μl of blocking buffer (PBST containing 5% w / v skim milk) for 2 hours. The plates were washed again and incubated at 37°C for 40 minutes with 100 μl / well serially diluted PD-1 antibody or control (starting concentration 66.7 nM, 5-fold serially diluted in PBST containing 2.5% skim milk). The ELISA plate was washed four times and incubated at 37°C for 40 minutes with either peroxidase-adapted Fcγ fragment-specific goat anti-mouse IgG (1:5000 diluted in PBST buffer, Cat#115-035-071, Jackson Immuno Research Laboratories, Inc., 100 μl / well) or peroxidase-adapted F(ab′)2 fragment-specific goat anti-human IgG (Cat#109-036-098, Jackson Immuno Research, for control). After the final wash, the plate was incubated at room temperature with 100 μl / well of TMB (Cat#TMB-S-002, Innoreagents). The reaction was terminated with 50 μl / well of 1MH2SO4 after 3–10 minutes. The absorbance was read using a dual-wavelength reader, with 450 nm for TMB and 630 nm as the reference wavelength. Plot OD (450-630) values ​​against antibody concentration. Analyze the data using Graphpad Prism software to obtain EC5 values. 50 Value. Result as follows Figure 2 and Figures 3A-3B As shown.

[0189] 3.3 Cell-based binding FACS

[0190] The binding activity of mouse-derived PD-1 antibody to mouse PD-1 expressed on the cell surface was detected by flow cytometry (FACS). 293F-mouse PD-1 cells, prepared internally by BioSens, expressing full-length mouse PD-1 (SEQ ID NO.: 27) on the cell membrane, were used. 293F cells were transfected with the PCMV-TP plasmid containing a mouse PD-1 coding sequence inserted between the EcoRI and XbaI sites, according to the instructions of the Lipofectamine 3000 transfection reagent (Thermo Fisher), to prepare 293F-mouse PD-1 cells.

[0191] The binding activity of the mouse-derived PD-1 antibody of this application to human PD-1 expressed on the cell surface was detected by flow cytometry (FACS) using the GS-J2 / PD-1 cell line (Genscript) expressing human PD-1 on the cell surface.

[0192] Specifically, 293F-mouse PD-1 cells and GS-J2 / PD-1 cells were collected from cell culture flasks, washed twice, and resuspended in PBS (FACS buffer) containing 2% v / v fetal bovine serum. Then, in 96-well plates, 2 × 10⁶ cells per well were cultured. 5 Cells were incubated on ice with 100 μl of PD1 antibody at various concentrations or a control for 40 minutes. Cells were washed twice with FACS buffer, followed by the addition of 100 μl / well of R-phycoerythrin affinity-purified F(ab′)2 fragment goat anti-mouse IgG (H+L) for mouse monoclonal antibody (1°C, 1000 dilution in FACS buffer, Cat#115-116-146, Jackson Immuno Research) or R-phycoerythrin affinity-purified goat anti-human IgG, Fcγ fragment specific (Cat#109-115-098, Jackson Immuno Research) for control. After incubation at 4°C in the dark for 40 minutes, cells were washed three times and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument, and graphs were plotted against antibody concentration. Data analysis was performed using Graphpad Prism software to obtain EC50 values. 50 Value. Result as follows Figures 4A-4B and Figure 5 As shown.

[0193] from Figure 1A-1B and Figures 4A-4B It can be seen that the mouse-derived PD1 antibody of this application has a Bmax (maximum binding) that is comparable to or higher than that of pembrolizumab, and an EC50 that is comparable to or slightly lower. 50 It specifically binds to human PD-1.

[0194] The antibody in this application binds to monkey PD-1, such as Figures 3A-3B As shown, compared to pembrolizumab, it has a slightly lower Bmax and a higher EC50. 50 .

[0195] according to Figure 2 and Figure 5 Mouse-derived antibodies E1A9C8A7 and E2G4E10B7 specifically bind to mouse PD-1, while pembrolizumab does not bind.

[0196] Example 4. Blocking activity of mouse-derived PD1 antibody against PD-1-PD-L1 or PD-1-control reference material

[0197] 4.1 Ligand Blocking ELISA

[0198] In a competitive ELISA assay, the activity of the PD1 antibody in blocking PD-L1-PD1 binding was determined.

[0199] In summary, 96-well microplates were coated with 100 μl of 2 μg / mL human PD-L1-Fc protein (internal preparation, SEQ ID NO: 28) in carbonate / bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. The plates were washed with washing buffer (PBS + 0.05% w / v Tween-20, PBST) and then blocked with PBST containing 5% w / v skim milk at 37°C for 2 hours. The PD1 antibody or control of this application was diluted with biotin-labeled human PD-1-Fc protein (internal preparation, SEQ ID NO: 26, 137.5 ng / mL, in PBST containing 2.5% skim milk) at an initial concentration of 80 nM, serially diluted 4-fold, and incubated at room temperature for 40 minutes. After washing the plates, the antibody / human PD-1-Fc mixture was added to the human PD-L1-Fc-coated plates, 100 μl / well. After incubation at 37°C for 40 minutes, the plates were washed with washing buffer. Then, 100 μl / well of streptavidin peroxidase (1:10000 diluted in PBST buffer, Cat#016-030-084, Jackson Immunoresearch) was added to the plate and incubated at 37°C for 40 minutes to detect biotin-labeled human PD-1-Fc. The plate was washed again with washing buffer. Finally, TMB was added, and the reaction was terminated with 1M H2SO4. The absorbance was read in a microplate reader using a dual-wavelength mode, with 450 nm for TMB and 630 nm as the reference wavelength. The OD (450-630) values ​​were then plotted against the antibody concentration. The data were analyzed using Graphpad Prism software to derive the IC50. 50 Value. Result as follows Figures 6A-6B As shown.

[0200] 4.2 Cell-based ligand-blocking FACS

[0201] The activity of the antibody in this application in blocking the binding of human PD-1-Fc protein to cell surface PD-L1 was evaluated by flow cytometry (FACS) using the GS-C2 / PD-L1 cell line (Genscript) expressing human PD-L1 on the cell surface.

[0202] The PD1 antibody and control of this application were diluted with biotin-labeled human PD-1-Fc solution (internally prepared, SEQ ID NO: 26, 137.5 ng / mL in FACS buffer) at an initial concentration of 6.67 nM, serially diluted 3-fold, and incubated at room temperature for 40 minutes. GS-C2 / PDL1 cells in the logarithmic growth phase were collected from cell culture flasks, washed twice, and resuspended in PBS (FACS buffer) containing 2% v / v fetal bovine serum. GS-C2 / PD-L1 cells were then cultured in 96-well plates at 0.6 × 10⁶ wells. 5 Cells / well were incubated with 100 μl / well of antibody / PD1-Fc mixture at 4°C for 40 min. Cells were washed twice with FACS buffer, and 100 μl / well of R-phycoerythrin-streptavidin (1:500 diluted in FACS buffer, Cat#016-110-084, Jackson Immunoresearch) was added, and incubated at 4°C in the dark for 40 min. Cells were washed twice and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACSCanto II-HTS instrument. Data analysis was performed using Graphpad Prism software to determine the IC50 values. 50 Value. Result as follows Figures 7A-7B As shown.

[0203] Using the above-mentioned 293F-mouse PD-1 cells, the activity of PD1 antibody in blocking the binding of mouse PD-L1 to mouse PD-1 on the cell surface was evaluated by flow cytometry (FACS).

[0204] In short, 293F-mouse PD-1 cells were collected from cell culture flasks, washed twice, and resuspended in PBS (FACS buffer) containing 2% v / v fetal bovine serum. 1×10⁻⁶ cells were added to each well of a 96-well plate. 5Cells / well were added to 100 μL / well of FACS buffer containing different concentrations of PD-1 antibody or control, starting at 80 nM and serially diluted 3-fold, and incubated at 4°C for 40 min. The plates were washed twice with FACS buffer and then incubated at 4°C for 40 min with 100 μL / well of recombinant mouse PD-L1-hFc protein (Cat#50010-M03H, Sinobiological Inc., 1 μg / mL in FACS buffer). The plates were washed twice with FACS buffer, and then 100 μL / well of R-phycoerythrin affinity-purified goat anti-human IgG, Fcγ fragment specific (1°C, 1000 dilution in FACS buffer, Cat#109-115-098, Jackson Immunoresearch) was added, and the plates were incubated at 4°C in the dark. Cells were washed twice and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument. Data analysis was performed using Graphpad Prism software to obtain IC values. 50 Value. Result as follows Figure 8 As shown.

[0205] 4.3 Control standard to block ELISA

[0206] In a competitive ELISA assay, the PD1 antibody blocking control standard of this application was measured. The ability of pembrolizumab to bind to human PD-1-Fc protein. Briefly, 100 μL of 2 μg / mL pembrolizumab in PBS was coated onto 96-well microplates and incubated at 37°C for 2 hours. ELISA plates were washed once with washing buffer (PBST, PBS + 0.05% Tween-20) and then blocked at 37°C with 200 μL / well blocking buffer (PBST containing 5% w / v skim milk) for 2 hours. During blocking, the PD-1 antibody or control was diluted with biotin-labeled human PD-1-Fc protein (internally prepared, SEQ ID NO: 26, 11 ng / mL in PBST containing 2.5% skim milk) at an initial concentration of 80 nM, serially diluted 4-fold, and incubated at room temperature for 40 minutes. After rinsing the plates, the antibody / PD-1 protein mixture was added to the control standard-coated plates at 100 μL / well. After incubation at 37°C for 40 minutes, wash the plate with wash buffer, add 100 μl / well of streptavidin-peroxidase, and incubate at 37°C for 40 minutes. Wash the plate again with wash buffer. Finally, add TMB and terminate the reaction with 1M H2SO4. Read the absorbance using a microplate reader in dual-wavelength mode, with 450 nm for TMB and 630 nm as the reference wavelength. Plot the OD(450-630) values ​​against the antibody concentration. Analyze the data using GraphpadPrism software to determine the IC50. 50Value. Result as follows Figures 9A-9B As shown.

[0207] from Figures 6A-6B As can be seen from 7A-7B, the PD-1 antibody of this application can block the binding of human PD-1 to human PD-L1 with an activity comparable to or slightly lower than that of pembrolizumab. According to... Figure 8 Antibodies E1A9C8A7 and E2G4E10B7 blocked the binding of mouse PD-L1 to mouse PD-1 on the cell surface, exhibiting higher blocking activity, unlike pembrolizumab, which cannot bind to mouse PD-1.

[0208] Figures 9A-9B As shown, the PD-1 antibody of this application can block the binding of PD-1 to pembrolizumab, indicating that these antibodies are likely to bind to the same or similar epitopes as pembrolizumab.

[0209] Example 5. Cell-based functional assay of mouse-derived PD-1 antibody

[0210] In cell-based functional assays, two genetically modified cell lines were used: GS-J2 / PD-1 (Genscript, effector cell line) stably expressing human PD-1 and a luciferase reporter gene driven by the NFAT response element (NFAT-RE), and GS-C2 / PD-L1 (Genscript, antigen-presenting cell line) stably expressing human PD-L1 and a genetically modified cell surface antigen peptide / major histocompatibility complex (MHC). The activity of the antibody in this application blocking the interaction between cell membrane PD-1 and cell membrane PD-L1 was evaluated. When these two cell lines were co-cultured, T-cell receptor (TCR)-NFAT-mediated luciferase expression in GS-J2 / PD-1 cells may have been inhibited or reduced by the PD-1-PD-L1 interaction.

[0211] Cell-based functional assays were performed as follows. Briefly, 1.0 × 10⁶ cells were added to 20 μL of RPMI 1640 culture medium (Cat#: 12430-054, Gibco) supplemented with 10% FBS (Cat#10099-141, Gibco). 4 GS-C2 / PD-L1 cells in logarithmic growth phase were seeded in 384-well cell culture plates (Cat#3765, Coming). The following day, serially diluted PD-1 antibody or controls (including internally prepared CD22 antibody as a negative control) were prepared in assay buffer (RPMI 1640 + 1% FBS), starting at 300 nM and serially diluted 5-fold. After discarding the culture medium, 20 μl of diluted PD1 antibody and 20 μl of GS-J2 / PD-1 effector cells (7.5*10⁻⁶ cells) were added to each well of the 384-well plate. 5 / mL). The plates were co-incubated at 37°C for 6 hours. Luciferase assay reagent (30 μL / well, Cat#E6120, Promega) was added to the plates. After 10 minutes, the plates were analyzed using a Tecan Infinite 200Pro microplate reader. The luminescence signal was analyzed using Graphpad Prism software to obtain EC. 50 value.

[0212] Experimental results are as follows Figure 10 As shown.

[0213] It can be seen that all PD1 antibodies in this application can reverse the reduction in luciferase expression induced by PD-1-PD-L1 interaction in GS-J2 / PD-1 cells more effectively than nivolumab, but their activity is comparable to that of pembrolizumab.

[0214] Example 6. Preparation and characterization of chimeric antibodies

[0215] The heavy / light chain variable region of PD-1 mAb in this application was sequenced, and the sequence IDs are summarized in Table 1.

[0216] The heavy and light chain variable regions of mouse PD1 mAb E1A9C8A7 were cloned into the human IgG4 heavy chain constant region (SEQ ID NO.: 24) and the human κ light chain constant region (SEQ ID NO.: 25) in a form within the readable frame, respectively, wherein the C-terminus of the variable region was connected to the N-terminus of the respective constant region.

[0217] Vectors containing nucleotides encoding the variable region of the heavy chain linked to the constant region of the human IgG4 heavy chain, and vectors containing nucleotides encoding the variable region of the light chain linked to the constant region of the human κ light chain, were transiently transfected into 50 ml of 293F suspension cell culture containing 1 mg / mL PEI at a light / heavy chain construct ratio of 1.1:1.

[0218] After 6 days in shake flasks, the cell supernatant containing the chimeric antibody was collected, and the chimeric antibody was then purified from the cell supernatant. Following the experimental procedures described in the above embodiments, the purified chimeric antibody was tested in the BIAcore affinity assay and cell-based functional assays.

[0219] The results are shown in Table 3 and Figure 11 As shown.

[0220] The binding affinity data in Table 3 show that the chimeric antibody E1A9C8A7 has a high binding affinity for human PD-1.

[0221] according to Figure 11The chimeric antibody E1A9C8A7 was able to reverse the reduction in luciferase expression induced by PD-1-PD-L1 interaction in GS-J2 / PD-1 cells more effectively than nivolumab, but its activity was comparable to that of pembrolizumab.

[0222] Table 3. Binding affinity of chimeric PD1 antibodies

[0223]

[0224] Example 7. Humanization of PD1 monoclonal antibody E1A9C8A7

[0225] The mouse-derived PD-1 antibody E1A9C8A7 was humanized and further studied. Humanization was performed using the well-established CDR transplantation technique detailed below.

[0226] To select a receptor framework for the humanization of the mouse antibody E1A9C8A7, the light and heavy chain variable region sequences of the mouse antibody E1A9C8A7 were aligned in the human immunoglobulin gene database. The human lineage with the highest homology was selected as the receptor framework for humanization. The heavy / light chain variable region CDRs of the mouse antibody were inserted into the selected framework, and further reversion mutations were performed on the residues in the framework to obtain more candidate heavy / light chain variable regions. A total of 11 exemplary humanized E1A9C8A7 antibodies were obtained, namely huE1A9C8A7-V1-huE1A9C8A7-V11, and their heavy / light chain variable region sequence IDs are listed in Table 1.

[0227] Vectors containing nucleotides encoding the humanized heavy chain variable region linked to the human IgG4 heavy chain constant region (SEQ ID NO: 24) and vectors containing nucleotides encoding the humanized light chain variable region linked to the human κ light chain constant region (SEQ ID NO: 25) were transiently transfected into 50 ml of 293F suspension cell culture containing 1 mg / mL PEI at a light / heavy chain construct ratio of 1.1:1.

[0228] Example 8. Characterization of humanized E1A9C8A7 antibody

[0229] Six days after shaking, cell supernatant containing humanized E1A9C8A7 antibody was collected, and its binding affinity to human PD-1 was determined using the Octet system (Fortebio, Octet RED 96).

[0230] In summary, the AHC biosensor (anti-human IgG Fc capture, ForteBio) was pre-soaked in 10 mM glycine (pH 1.5) for 3 seconds, followed by immersion in wells containing flow buffer (PBST containing 0.5% w / v BSA) for 3 seconds. This soaking and immersion process was repeated three times. Subsequently, the sensor was immersed in cell supernatant containing humanized PD-1 antibody and HBS-EP. + 2 μg / mL of chimeric E1A9C8A7 antibody and HBS-EP + The sensor was immersed in wells containing 2 μg / mL nivolumab for 200 seconds, then in wells containing flow buffer for 1 minute. A new baseline was established after 100 seconds in another well containing flow buffer. Next, the sensor was immersed in wells containing serially diluted human PD-1-his protein (Cat#: PD1-H5221, Acro biosystems Inc., starting concentration 100 nM, 2-fold serial dilution) for 120 seconds, then in baseline wells for 4 minutes. Finally, the sensor was pre-soaked in 10 mM glycine (pH 1.5) for 30 seconds, then in wells containing flow buffer for 30 seconds. Binding and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio Data Analysis 8.1 evaluation software. K was determined. a K d and K D The values ​​are summarized in Table 4 below.

[0231] Table 4. Binding affinity of humanized E1A9C8A7 mAb

[0232]

[0233] Data shows that all cell supernatants containing humanized PD1 antibodies, except for huE1A9C8A7-V3, have similar human PD-1 binding affinity to chimeric antibodies, but higher than that of nivolumab.

[0234] The humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 were purified as described above and detected in BIAcore, capture ELISA, indirect ELISA, competitive ELISA, cell-based binding FACS, cell-based ligand blocking FACS, and cell-based functional assays according to the experimental procedures described in the foregoing embodiments (with minor modifications as described below).

[0235] For capture ELISA, 100 μL of 2 μg / mL Fcγ fragment-specifically affinity purified goat anti-human IgG (Cat#109-005-098, Jackson Immunoresearch) was used instead of F(ab')2 fragment-specifically affinity purified goat anti-mouse IgG to coat 96-well plates. Biotin-labeled human PD-1-his protein (internally prepared, SEQ ID NO: 29) was used instead of biotin-labeled human PD-1-Fc, 39.2 ng / mL, in PBST containing 2.5% skim milk, 100 μL / well.

[0236] For indirect ELISA, humanized antibodies were purified using peroxidase affinity purification of the F(ab′)2 fragment goat anti-human IgG, Fcγ fragment specific (Cat#109-036-098, Jackson Immunoresearch), 100 μl / well.

[0237] For BIAcore, goat anti-human IgG (Cat#BR100839, GE Healthcare, Human Antibody Capture Kit) was covalently linked to the CM5 chip.

[0238] For the control reference blocking ELISA, biotin-labeled human PD-1-his protein (internally prepared, SEQ ID NO: 29, 15.7 ng / mL in PBST containing 2.5% skim milk) was used instead of the biotin-labeled human PD-1-Fc solution, 100 μl / well.

[0239] For cell-based binding FACS, humanized antibodies were purified using R-phycoerythrin affinity-purified Fcγ fragment-specific goat anti-human IgG (Cat#109-115-098, Jackson ImmunoResearch), diluted 1:1000 in FACS buffer, 100 μl / well.

[0240] The thermal stability of the humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 was also tested. In short, Tm (melting temperature) was determined by protein thermal migration assay using the GloMelt™ Thermal Migration Protein Stability Kit (Cat#33022-T, Biotium). In short, GloMelt... TMThe dye was melted and brought to room temperature. The vial containing the dye was vortexed and centrifuged. Then, 10× dye was prepared by adding 5 μL of 200× dye to 95 μL of PBS. 2 μL of 10× dye and 10 μg of humanized antibody were added, and PBS was added to a total reaction volume of 20 μL. The tube containing the dye and antibody was simply rotated and placed in a real-time PCR thermal cycler (Roche, LightCycler 480II) set to the melting curve program with the parameters in Table 5.

[0241] Table 5. Melt Profile Program Parameters

[0242] Initial hold 25℃ NA 30s Melting curve 25-99℃ 0.1℃ / s NA

[0243] The results are shown in Tables 6-1 and 6-2. Figure 12-18 and Figures 19A-19B As shown.

[0244] According to the data, the humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 showed higher levels of human (e.g., nivolumab and pembrolizumab) than nivolumab and pembrolizumab. Figure 12 Table 6-1) and monkey PD-1 (e.g. Figure 13 (Table 6-1) Binding affinity / activity. Humanized antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 also specifically bind to mouse PD1, while nivolumab and pembrolizumab do not bind (Table 6-1).

[0245] Table 6-1. Binding affinity / capacity of humanized mAbs huE1A9C8A7-V5 and huE1A9C8A7-V8

[0246]

[0247] *Not tested.

[0248] Table 6-2. Functional activities of humanized mAbs huE 1A9C8A7-V5 and huE 1A9C8A7-V8

[0249]

[0250] *Not tested.

[0251] Furthermore, in the competition for ELISA ( Figure 15 ) and cell-based blocking FACS detection ( Figure 17In this study, antibodies huE1A9C8A7-V5 and huE1A9C8A7-V8 exhibited blocking activity against PD-1-PD-L1 binding or interaction comparable to nivolumab and pembrolizumab. In cell-based functional assays, antibodies huE1A9C8A7-V8 and huE1A9C8A7-V5 were able to reverse PD-1-PD-L1 interaction-induced reduction in luciferase expression with activity comparable to nivolumab. Figure 18 ).

[0252] Example 9. Preparation and characterization of huE1A9C8A7-V8-3 antibody

[0253] Deamidation occurs at asparagine residues, particularly those in the CDR, leading to antibody degradation. Aspartic acid residues, especially those immediately following glycine residues, are prone to isomerization, another common cause of antibody degradation. To avoid deamidation and / or isomerization of amino acids during the production, storage, and in vivo metabolism of the antibodies in this application, the CDR sequence can be optimized to achieve better chemical stability.

[0254] The humanized antibody huE1A9C8A7-V8 was modified in the heavy chain variable region CDR3 and the light chain variable region CDR1. The antibody with CDR modification was named huE1A9C8A7-V8-3, purified as described above, and tested in BIAcore, cell-based binding FACS, and cell-based functional assays according to the operating procedures of the aforementioned embodiments.

[0255] The results are shown in Tables 7-1 and 7-2. Figure 20 and 21 As shown.

[0256] Table 7-1. Binding affinity of huE1A9C8A7-V8-3 with human and monkey PD-1

[0257]

[0258] Table 7-2. Binding affinity of huE1A9C8A7-V8-3 antibody to mouse PD1

[0259]

[0260] According to Tables 7-1 and 7-2, huE1A9C8A7-V8-3 showed a slightly higher PD-1 binding affinity to mice, humans, and monkeys than huE1A9C8A7-V8. Figure 20 As shown, these two antibodies appear to exhibit comparable human PD-1 binding activity.

[0261] In addition, from Figure 21It can be seen that huE1A9C8A7-V8 and huE1A9C8A7-V8-3 can reverse the reduction in luciferase expression induced by PD-1-PD-L1 interaction in GS-J2 / PD-1 cells more effectively than nivolumab, but their activity is comparable to that of pembrolizumab.

[0262] Example 10. In vivo antitumor efficacy of mouse-derived E2G4E10B7 antibody

[0263] The in vivo antitumor activity of mouse-derived E2G4E10B7 antibody was detected in C57BL / 6 mice. In short, 2 × 10⁻⁶ E2G4E10B7 antibodies were subcutaneously injected into the right axilla of C57BL / 6 mice. 6 Mouse colon cancer MC38 cells (Cat#HYC3401, Obio Technology (Shanghai) Corp., Ltd.) were used. Tumor volume was measured using electronic calipers twice a week for 3 weeks and calculated as (length × width²) / 2. Tumors were classified as tumor volume when the average volume reached approximately 30-150 mm². 3 Twenty-four tumor-bearing mice were randomly divided into three groups, with the day of grouping designated as day 1. On days 1, 4, 8, 11, 15, and 18, the mice were injected via tail vein with a solvent (Dulbecco phosphate-buffered saline, also known as DPBS), mouse-derived E2G4E10B7 antibody, and InVivoMAb anti-mouse PD-1 antibody (Cat#BE0146, BioXCell, also known as CD279), at a dose of 10 mg / kg.

[0264] Mice in the solvent group and the other two groups were sacrificed on days 15 and 22, respectively, and tumors were collected and weighed. Tumor growth rate (T / C% = (V of each treatment group) was measured. t (Tumor volume measured on day t) / V0(Tumor volume measured on day 1) / (Control group V) t (V0)×100%) and tumor growth inhibition rate (IR) Tv % = 100% - T / C%.

[0265] Tumor-bearing animals tolerated all treatments well. (From Table 8 and...) Figure 22 It can be seen that the 10 mg / kg mouse-derived E2G4E10B7 antibody showed significantly better efficacy against mouse PD-1 than the 10 mg / kg InVivoMAb.

[0266] Table 8. Antitumor efficacy of mouse-derived E2G4E10B7 antibody

[0267]

[0268] aTumor volume on day 15 was compared with the solvent control group using Student's t-test. *P≤0.05 and **P<0.01.

[0269] b T / C% and IR on day 15 Tv The percentage was compared with the InVivoMAb anti-mouse PD-1 group using Student's t-test, and *P≤0.05 and **P<0.01.

[0270] Although this application has been described above in conjunction with one or more embodiments, it should be understood that this application is not limited to those embodiments, and the specification is intended to cover all alternatives, modifications, and equivalents that may be included within the spirit and scope of the appended claims. All references cited herein are further incorporated herein by reference in their entirety.

[0271] The sequences in this application are summarized below.

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280] ***

[0281] The preferred embodiments of the invention have been described in detail in this application. It should be understood that the invention as defined in the foregoing paragraphs is not limited to the specific details listed in the foregoing description, and many obvious modifications can be made without departing from the spirit and scope of the invention. sequence list <110> Bio-Sens Biotechnology (Nanjing) Co., Ltd. <120> PD-1 binding antibodies and their uses <130> 55532 00039 <150> US 63 / 072,421 <151> 2020-08-31 <160> 42 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Mouse, chimeric and humanized E1A9C8A7 antibodies and mouse E2G4E10B7 VH CDR1 <400> 1 Asp Tyr Val Met His 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 in mouse E1G5D1H1 <400> 2 Asp Phe Tyr Met Tyr 1 5 <210> 3 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 of mouse, chimeric and humanized E1A9C8A7 antibodies <400> 3 Val Ile Ser Thr Tyr His Ile Pro Ala Val Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 4 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 in mouse E2G4E10B7 <400> 4 Val Ile Ser Thr His Phe Gly Asp Gly Val Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 5 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 in mouse E1G5D1H1 <400> 5 Thr Ile Ser Asn Ser Gly Thr Gln Thr Tyr Tyr Leu Asp Ser Val Lys 1 5 10 15 Gly <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Mouse, chimeric and humanized E1A9C8A7 antibodies and mouse E2G4E10B7 VH CDR3 <220> <221> Other features <222> (5)..(5) <223> Xaa can be Asp or Glu <220> <221> Other features <222> (7)..(7) <223> Xaa can be Tyr or Trp <400> 6 Glu Val Tyr Gly Xaa Ser Xaa Tyr Phe Asp Val 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 in mouse E1G5D1H1 <400> 7 Asp Tyr Tyr Arg Tyr Asp Ser Trp Tyr Phe Asp Val 1 5 10 <210> 8 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Mouse, chimeric and humanized E1A9C8A7 antibodies and mouse E2G4E10B7 VL CDR1 <220> <221> Other features <222> (6)..(6) <223> Xaa can be Ile or Leu <220> <221> Other features <222> (10)..(10) <223> Xaa can be Asn, Ala, or Asp. <400> 8 Arg Ser Ser Gln Ser Xaa Val His Ser Xaa Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 9 <211> 15 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 in mouse E1G5D1H1 <400> 9 Arg Ala Ser Glu Ser Val Asp Phe Tyr Gly Thr Ser Leu Met Gln 1 5 10 15 <210> 10 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Mouse, chimeric and humanized E1A9C8A7 antibodies and mouse E2G4E10B7 VL CDR2 <400> 10 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 11 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 in mouse E1G5D1H1 <400> 11 Ala Ala Ser Asn Val Glu Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Mouse, chimeric and humanized E1A9C8A7 antibodies and mouse E2G4E10B7 VL CDR3 <400> 12 Phe Gln Gly Ser His Val Pro Tyr Thr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 in mouse E1G5D1H1 <400> 13 Gln Gln Ser Arg Lys Val Pro Trp Thr 1 5 <210> 14 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Mice and VH with chimeric E1A9C8A7 antibody <400> 14 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Arg Pro Gly Val 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30 Val Met His Trp Met Lys Gln Ser His Gly Glu Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr His Ile Pro Ala Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Leu Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Tyr Gly Asp Ser Tyr Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 15 <211> 120 <212> PRT <213> Artificial sequence <220> <223> VH of huE1A9C8A7-V1 - huE1A9C8A7-V5 <220> <221> Other features <222> (30)..(30) <223> Xaa can be Ser or Thr <220> <221> Other features <222> (37)..(37) <223> Xaa can be Met or Val <220> <221> Other features <222> (74)..(74) <223> Xaa may be Lys or Thr <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Xaa Asp Tyr 20 25 30 Val Met His Trp Xaa Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr His Ile Pro Ala Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Xaa Ser Thr Ser Thr Val Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Tyr Gly Asp Ser Tyr Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 16 <211> 120 <212> PRT <213> Artificial sequence <220> <223> VH of huE1A9C8A7-V6 to huE1A9C8A7-V8 <220> <221> Other features <222> (20)..(20) <223> Xaa can be Val or Ile <220> <221> Other features <222> (44)..(44) <223> Xaa can be either Ser or Gly <220> <221> Other features <222> (48)..(48) <223> Xaa can be Ile or Met <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Xaa Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Xaa Leu Glu Trp Xaa 35 40 45 Gly Val Ile Ser Thr Tyr His Ile Pro Ala Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Tyr Gly Asp Ser Tyr Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 17 <211> 120 <212> PRT <213> Artificial sequence <220> <223> huE1A9C8A7-V9 - huE1A9C8A7-V11's VH <220> <221> Other features <222> (67)..(67) <223> Xaa can be Arg or Lys <220> <221> Other features <222> (68)..(68) <223> Xaa can be Ala or Val <220> <221> Other features <222> (72)..(72) <223> Xaa can be Val or Arg <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr His Ile Pro Ala Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Xaa Xaa Thr Met Thr Xaa Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Tyr Gly Asp Ser Tyr Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 18 <211> 120 <212> PRT <213> Artificial sequence <220> <223> VH of mouse E2G4E10B7 <400> 18 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Met His Trp Val Arg Gln Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr His Phe Gly Asp Gly Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Tyr Gly Asp Ser Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 19 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH of mouse E1G5D1H1 <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Asn Ser Gly Thr Gln Thr Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Tyr Arg Tyr Asp Ser Trp Tyr Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 20 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL of mouse and chimeric E1A9C8A7 <400> 20 Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> twenty one <211> 112 <212> PRT <213> Artificial sequence <220> <223> VL of huE1A9C8A7-V1 - huE1A9C8A7-V11 and huE1A9C8A7-V8-3 <220> <221> Other features <222> (33)..(33) <223> Xaa can be either Asn or Ala <400> twenty one Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Xaa Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> twenty two <211> 112 <212> PRT <213> Artificial sequence <220> <223> VL of mouse E2G4E10B7 <400> twenty two Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Leu Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> twenty three <211> 111 <212> PRT <213> Artificial sequence <220> <223> VL of mouse E1G5D1H1 <400> twenty three Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Gln Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Phe Tyr 20 25 30 Gly Thr Ser Leu Met Gln Trp Phe Gln Leu Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Ser Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe Cys Gln Gln Ser Arg 85 90 95 Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys 100 105 110 <210> twenty four <211> 327 <212> PRT <213> Artificial sequence <220> <223> heavy chain constant regions of chimeric and humanized antibodies <400> twenty four Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 25 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Light chain constant regions of chimeric and humanized antibodies <400> 25 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 26 <211> 382 <212> PRT <213> Artificial sequence <220> <223> Human PD1-Fc protein <400> 26 Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro 35 40 45 Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln 50 55 60 Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Glu 130 135 140 Asn Leu Tyr Phe Gln Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr 145 150 155 160 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 165 170 175 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 180 185 190 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 195 200 205 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 210 215 220 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 225 230 235 240 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 245 250 255 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 260 265 270 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 275 280 285 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 290 295 300 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 305 310 315 320 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 325 330 335 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 340 345 350 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 355 360 365 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 27 <211> 294 <212> PRT <213> Artificial Sequence <220> <223> murine PD-1-his <400> 27 Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln 1 5 10 15 Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp 20 25 30 Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala 35 40 45 Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met 50 55 60 Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala 65 70 75 80 Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln 85 90 95 Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp 100 105 110 Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val 130 135 140 Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro 145 150 155 160 Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile Gly Ile Met Ser Ala 165 170 175 Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp Ala Leu Ala Val 180 185 190 Phe Cys Ser Thr Ser Met Ser Glu Ala Arg Gly Ala Gly Ser Lys Asp 195 200 205 Asp Thr Leu Lys Glu Glu Pro Ser Ala Ala Pro Val Pro Ser Val Ala 210 215 220 Tyr Glu Glu Leu Asp Phe Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro 225 230 235 240 Thr Ala Cys Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly 245 250 255 Leu Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu Gln 260 265 270 Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 His His His His His His 290 <210> 28 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Human PDL1-Fc protein <400> 28 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110 Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 115 120 125 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 130 135 140 Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 145 150 155 160 Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr 165 170 175 Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 180 185 190 Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 195 200 205 Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 29 <211> 151 <212> PRT <213> Artificial Sequence <220> <223> Human PD1-his <400> 29 Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro 35 40 45 Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln 50 55 60 Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln His 130 135 140 His His His His His His His 145 150 <210> 30 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH of mouse E1A9C8A7 <400> 30 caggtccagc tgcagcagtc tggggctgag gtggtgaggc ctggggtctc agtgaggatt 60 tcctgcaagg gttctggcta cacattctct gattatgtta tgcactggat gaagcagagt 120 catggagaga gcctagagtg gattggggtt atcagtactt accatattcc tgctgtctac 180 aaccagaagt tcaagggcaa ggccacaatg actgtagaca aatcctccag tacagtctat 240 ctggaacttg ccagactgac atctgaggat tctgccatct attactgtgc aagagaggtc 300 tatggtgact cttactactt cgatgtctgg ggcgcgggga ccacggtcac cgtctcctca 360 <210> 31 <211> 361 <212> DNA <213> Artificial Sequence <220> <223> VH of chimeric E1A9C8A7 <400> 31 caagtgcagc tgcagcagag cggcgccgaa gtggtgagac ccggcgtgag cgtgaggatc 60 agctgcaagg gcagcggcta taccttcagc gactacgtga tgcactggat gaagcagagc 120 cacggcgagt ctctggagtg gatcggagtg atcagcacct accacatccc cgccgtgtac 180 aaccagaagt tcaagggcaa ggccaccatg accgtggaca agagcagcag caccgtgtat 240 ctggagctgg ctagactgac ctccgaggat agcgccatct actactgcgc cagagaggtg 300 tacggcgata gctactactt cgacgtgtgg ggcgccggaa ccacagtgac cgtgagcagc 360 g 361 <210> 32 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH of huE1A9C8A7-V8 <400> 32 caagtgcagc tggtgcagag cggagccgaa gtggtgaaac ccggcgccag cgtgaagatc 60 agctgcaagg ccagcggcta caccttcacc gactacgtga tgcactgggt gagacaagcc 120 cccggccagt ctctggagtg gatgggcgtg atcagcacct accacatccc cgccgtgtac 180 aaccagaagt tcaagggcaa ggccaccatg accgtggaca ccagcaccag caccgtgtat 240 ctggagctgt cctctctgag gagcgaggac accgccgtgt actactgcgc cagagaggtg 300 tacggcgaca gctactactt cgacgtgtgg ggccaaggca ccacagtgac cgtgagcagc 360 <210> 33 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> VL of mouse E1A9C8A7 <400> 33 gatgttttga tgacccaaat tccactctcc ctgcctgtta gtcttggaga tcaagtctcc 60 atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120 tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagg tctggggagtt tattactgct ttcaaggttc acatgttccg tacacgttcg gaggggggac caagctgga ataaaa <210> 34 <211> 336 <212> DNA <213> The snowstorm <220> <223> Order E1A9C8A7VL <400> 34 gacgtgctga tgacccagat ccctctgtct ctgcccgtgt ctctggggaga tcaagtgagc atcagctgca gatccagcca gagcatcgtg cacagcaacg gcaacaccta tctggagtgg tatctgcaga agcccggcca gagccccag ctgctgatct acaaggtctc caatagattc tccggcgtgc ccgatagatt cagcggaagc ggcagcggca ccgacttcac actgaagatc tccagagtgg aggccgagga tctgggcgtg tactactgct ttcaaggcag ccacgtgccc 300 tacacctttg gcggcggcac caaactggag atcaag <210> 35 <211> 336 <212> DNA <213> The snowstorm <220> <223> huE1A9C8A7-V1 - huE1A9C8A7-V11VL <400> 35 gacatcgtga tgacccagac acctctgtct ctgcccgtga cacccggcga gcccgccagc atcagctgca gaagcagcca gagcatcgtg cacagcaacg gcaacaccta tctggagtgg tatctgcaga agcccggcca gagccctcag ctgctgatct acaaggtgtc caatagattc agcggcgtgc ccgacagatt cagcggcagc ggagcggca cagacttcac actgaagatc tccagagtgg aggccgagga cgtggggcgtg tactactgct tccaaggcag ccacgtgcct 300 tacaccttcg gcggcggcac caaggtggag atcaag <210> 36 <211> 360 <212> DNA <213> The snowstorm <220> <223> Block E2G4E10B7VH <400> 36 caggtccagc tgcagcagtc tggggctgag ctggtgaggc ctggggtctc agtgaagatt tcctgcaagg gttctggcta cacattcact gattatgtta tgcactgggt gaggcagagc 120 180. catgcaaaga gtctagagtg gattggagtt atcagtactc actttggtga tggtgtttac 240. aaccagaagt tcaagggcaa ggccacaatg actgtagaca aatcctccag cacagcctat atggaacttg ccagactgac atctgaggat tctgccatct attactgtgc aagagaggtc 300 tatggtgact cttggtactt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca 360 <210> 37 <211> 336 <212> DNA <213> Artificial sequence <220> <223> VL of mouse E2G4E10B7 <400> 37 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta catagtgatg gaaacaccta tttagaatgg 120 tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagactgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300 tacacgttcg gaggggggac caagctggaa ataaaa 336 <210> 38 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH of mouse E1G5D1H1 <400> 38 gaagtgcagt tggtggagtc tgggggaggc ttagtgaagc ctggagggtc tctgaagctc 60 tcctgtgcag cctctggatt cactttcagt gacttttaca tgtattgggt tcgccagact 120 accattagtaata gtggtactca aacctactat 180 ctagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa taacctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt actattgtgc tagagactac 300 tataggtacg acagctggta cttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360 tca 363 <210> 39 <211> 333 <212> DNA <213> Artificial sequence <220> <223> VL of mouse E1G5D1H1 <400> 39 gacattgtat taacccaatc tccagcttct ttggctgtgt ctcaagggca gagagccacc 60 atctcctgca gagccagtga aagtgttgat ttttatggca caagtttaat gcagtggttc 120 caactgaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa cgtcgaatct 180 ggagtccctg ccaggtttag tggcagtggg tctgggacaa acttcagcct caacatccat 240 cctgtggagg aggatgatat tgccatgtat ttctgtcagc aaagtaggaa ggttccgtgg 300 acattcggtg gaggcaccaa ccttgaaatc aaa 333 <210> 40 <211> 984 <212> DNA <213> Artificial sequence <220> <223> Heavy chain constant region of chimeric and humanized antibodies <400> 40 gccagcacaa agggcccttc cgtgtttccc ctggccccct gcagcaggag cacctctgag 60 tccaccgccg ccctgggctg tctggtgaag gactactttc ccgagcccgt gaccgtgagc 120 tggaattccg gcgccctgac atccggcgtg cacaccttcc ccgccgtgct gcagtcctcc 180 ggcctgtaca gcctgagctc cgtggtgaca gtgccttcct cctccctggg caccaagacc 240 tacacatgta atgtggatca caagcccagc aacacaaagg tggataagag agtggagtcc 300 aagtacggcc ctccttgccc tccctgtcct gccccagagt tcctgggcgg cccctctgtg 360 ttcctgttcc cccctaagcc caaggacaca ctgatgatct ccaggacccc tgaggtgacc 420 tgcgtggtgg tggacgtgag ccaggaggac cctgaggtgc agttcaattg gtacgtggat 480 ggcgtggagg tgcacaatgc caagacaaag cccagagagg agcagtttaa ttccacatac 540 agggtggtgt ccgtgctgac cgtgctgcac caggattggc tgaacggcaa ggagtacaag 600 tgtaaggtga gcaacaaggg cctgccttcc tccatcgaga agacaatcag caaggccaag 660 ggccagccta gggagcccca ggtgtacaca ctgcctccca gccaggagga gatgaccaag 720 aaccaggtga gcctgacctg cctggtgaag ggcttctacc ctagcgacat cgccgtggag 780 tgggagtcca acggccagcc cgagaataac tacaagacaa caccccccgt gctggattcc 840 gatggcagct tctttctgta ctccaggctg accgtggata agagcaggtg gcaggagggc 900 aatgtgttca gctgctccgt gatgcacgag gccctgcaca atcactacac ccagaagagc 960 ctgtccctga gcctgggcaa gtga 984 <210> 41 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Light chain constant region of chimeric and humanized antibody <400> 41 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg ttag 324 <210> 42 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH of huE1A9C8A7-V8-3 <400> 42 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Met 35 40 45 Gly Val Ile Ser Thr Tyr His Ile Pro Ala Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Val Tyr Gly Glu Ser Tyr Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120

Claims

1. A monoclonal antibody or its antigen-binding portion thereof, capable of specifically binding to PD-1, comprising: i) Heavy chain variable regions, which include VH CDR1, VH CDR2, and VH CDR3 regions; and ii) The light chain variable region, which includes VL CDR1, VL CDR2, and VL CDR3 regions. The amino acid sequences of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 regions are shown in SEQ ID NOs: 1, 3, 6, 8, 10, and 12, respectively. in, The 5th and 7th amino acid residues of SEQ ID NO: 6 are E and Y, respectively, and the 6th and 10th amino acid residues of SEQ ID NO: 8 are I and A, respectively.

2. The monoclonal antibody or its antigen-binding portion as claimed in claim 1, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are as shown in SEQ ID NOs: 42 and 21, respectively, wherein the 33rd amino acid residue of SEQ ID NO: 21 is A.

3. The monoclonal antibody or its antigen-binding portion as described in claim 1, wherein it is an IgG1, IgG2 or IgG4 isotype.

4. The monoclonal antibody or its antigen-binding portion as claimed in claim 1, comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 24 and is linked to the heavy chain variable region, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 25 and is linked to the light chain variable region.

5. The monoclonal antibody or its antigen-binding portion as described in claim 1, wherein it is mouse-derived, chimeric, or humanized.

6. A nucleic acid molecule encoding the monoclonal antibody of claim 1 or its antigen-binding portion thereof.

7. An expression vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the expression vector of claim 7.

9. A pharmaceutical composition comprising a monoclonal antibody or its antigen-binding portion as described in any one of claims 1-5, a nucleic acid molecule as described in claim 6, an expression vector as described in claim 7, or a host cell as described in claim 8, and a pharmaceutically acceptable vector.

10. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for treating a disease associated with the PD-1 signaling pathway, wherein the disease is a solid tumor.

11. The use as described in claim 10, wherein the solid tumor is non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer (BC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), melanoma, head and neck squamous cell carcinoma (HNSCC), cervical cancer, or gastric cancer.

12. The use as described in claim 11, wherein the colorectal cancer is colon cancer.

13. The use as described in claim 11, wherein the gastric cancer is gastroesophageal cancer.

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