Antibodies that bind to CTLA4 and their uses

Abstract

The present application relates to an isolated monoclonal antibody or its antigen-binding portion that specifically binds to human CTLA4. Also provided are nucleic acid molecules encoding the antibody or its antigen-binding portion, expression vectors, host cells, and methods for expressing the antibody or its antigen-binding portion. The present application also provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising the antibody or its antigen-binding portion, as well as treatment methods using the CTLA4 antibody or its antigen-binding portion.

Description

[0001] Related Applications and Incorporation by Reference

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 008,931, filed April 13, 2020.

[0003] The above-mentioned applications, all documents cited therein or cited during their prosecution ("application citations"), all documents cited or mentioned herein (including but not limited to all documents, patents, published patent applications cited herein) ("herein citations"), all documents cited or mentioned in the herein citations, and any manufacturer's manuals, instructions, product specifications and product sheets mentioned herein or in any document incorporated by reference herein are incorporated herein by reference and may be used in the practice of the present invention. More specifically, all references are incorporated by reference as if each document was specifically and individually incorporated by reference. Any Genbank sequences mentioned in this disclosure are incorporated by reference as of the earliest effective filing date of this disclosure. Field of the Invention

[0004] The present application generally relates to isolated monoclonal antibodies, such as mouse, chimeric or humanized monoclonal antibodies, or antigen-binding portions thereof, which specifically bind to human CTLA4 with high affinity and functionality. Also provided are nucleic acid molecules encoding the antibody or its antigen-binding portion, expression vectors, host cells, and methods for expressing the antibody or its antigen-binding portion. The application also provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising the antibody or its antigen-binding portion, as well as diagnostic or therapeutic methods using the CTLA4 antibody or its antigen-binding portion of the present application. Background Art

[0005] Immune checkpoints regulate the immune system, preventing it from indiscriminately attacking cells. Among the immune checkpoints, cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed cell death receptor 1 (PD-1) are two very important ones that provide inhibitory signals in immune responses. For example, it has been reported that CTLA4 prevents potential self-reactive T cells at the initial stage of naive T cell activation, and PD-1 has been found to regulate activated T cells in the subsequent stage. These two inhibitory immune checkpoint pathways are also manipulated by tumor cells to escape the attack of the immune system (Buchbinder EI, and Desai A. (2016) Am J Clin Oncol. 39 (1): 98-106).

[0006] CTLA4 is a CD28 homolog that is primarily located in the intracellular compartment of resting naive T cells. It competes with CD28 for binding to CD80 / CD86 with a much higher affinity than CD80 / CD86 (Chambers CA et al., (2001) Annu Rev Immunol. 19: 565-594). After TCR binds to antigens presented by the major histocompatibility complex (MHC) on the surface of antigen-presenting cells (APCs), the CD28-CD80 / CD86 interaction is crucial for T cell activation, and sufficient CD28-CD80 / CD86 binding leads to T cell proliferation, survival, and differentiation (Buchbinder EI, and Desai A. (2016) Am J Clin Oncol. 39(1): 98-106). When there is a stimulatory signal of TCR-TCR and CD28-CD80 / 86 interaction, CTLA4 is transferred to the cell surface (Linsley PS et al., (1996) Immunity. 4: 535-543), and the binding of CTLA4 to CD80 / CD86 does not generate a stimulatory signal, or even generates an inhibitory signal to counteract the stimulatory signal of the binding of TCR-TCR and CD28-CD80 / 86 (Chambers CA et al., (2001) Annu Rev Immunol. 19: 565-594; Egen JG et al., (2002) Nat Immunol. 3: 611-618; Parry RV et al., (2005) Mol Cell Biol. 25: 9543-9553; Fallarino F et al., (2006) Mol Cell Biol. 35: 9553-9564). et al., (1998) J Exp Med. 188: 205-210; Masteller EL et al., (2000) 164: 5319-5327). Therefore, the relative amount of CD28-CD80 / 86 binding compared to CTLA4-CD80 / 86 binding determines whether T cells will undergo activation or anergy (Krummel MF, and Allison JP. (1995) J Exp Med. 182: 459-465). In addition, CTLA4 can trigger a reverse signal through CD80 / CD86, directing indoleamine-2,3-dioxygenase, resulting in tryptophan metabolism and T cell proliferation inhibition (Boasso A et al., (2005) Blood 105: 1574-1581).

[0007] CTLA4 is also expressed on non-T cells, normal cells, and neoplastic cells (Laurent S et al., (2010) Hum Immunol 71:934-941; Contardi E et al., (2005) Int J Cancer 117:538-550). Persistent CTLA4 expression in neoplastic cells promotes the progression of hematological and solid tumors (Pistillo MP et al., (2003) Blood 101:202-209; Kosmaczewska A et al., (2005) Leukemia 19:301-304). Blockade of the CTLA4 pathway has been shown to effectively slow tumor growth (Leach DR et al., (1996) 271:1734-1736; Hirano F et al., (2005) Cancer Res. 65:1089-1096). CTLA4 antibody, ipilimumab It has been approved for the treatment of melanoma, colorectal cancer, hepatocellular carcinoma, malignant pleural mesothelioma, non-small cell lung cancer and renal cell carcinoma. Tisitumomab, another CTLA4 antibody, is in clinical trials for the treatment of mesothelioma, melanoma and intestinal cancer, for example. CTLA4 antibodies can also be used in combination with PD-1 antibodies and / or other anti-tumor agents. For example, AGEN1884, also a CTLA4 antibody, is being tested in clinical trials in combination with PD-1 antibodies for the treatment of cervical cancer, angiosarcoma, muscle-invasive bladder cancer and soft tissue sarcomas (including synovial sarcoma, nerve sheath tumors and phyllodes tumors) (National Cancer Institute, U.S.A.).

[0008] Studies have further shown that CTLA4 is upregulated in chronic infections such as human immunodeficiency virus (HIV), and that CTLA4 therapy alone or in combination with PD-1 antibodies interferes with HIV persistence in clinical trials (Thomas A Rasmussen et al., (2021) Clinical Infectious Diseases ciaa1530; Colston E et al., (2018) PLoS One 13(6):e0198158). Ipilimumab and nivolumab are also in Phase II trials for the treatment of Epstein-Barr virus (HHV-4) infection. In addition, preclinical studies are exploring the effects of ipilimumab in graft-versus-host disease (GVHD), peripheral nerve damage, and neurofibromatosis type I (von Recklinghausen disease).

[0009] Efforts are currently underway to discover more potent or more desirable CTLA4 binding sites.

[0010] The citation or identification of any document in this application does not constitute an admission that such document is prior art to this application. Summary of the Invention

[0011] The present application provides an isolated monoclonal antibody, such as a mouse, human, chimeric or humanized monoclonal antibody, or an antigen-binding portion thereof, which binds to CTLA4 (e.g., human CTLA4 and monkey CTLA4) and has comparable (if not higher) CTLA4 binding affinity, comparable (if not higher) blocking activity for CTLA4-CD80 / CD86 interaction, and comparable activity in promoting T cell responses compared to prior art CTLA4 antibodies such as ipilimumab.

[0012] The antibodies or antigen-binding portions thereof of the present application can be used in many applications, including the detection of CTLA4 protein and the treatment and prevention of CTLA4-related diseases, such as tumors and infectious diseases.

[0013] Thus, in one aspect, the present application relates to an isolated monoclonal antibody (e.g., a mouse, chimeric, or humanized antibody), or an antigen-binding portion thereof, that binds to CTLA4 and has i) a heavy chain variable region that can comprise a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region, the VH CDR2 region, and the VH CDR3 region can comprise the sequences corresponding to (1) SEQ ID NOs: 1, 2, and 3; (2) SEQ ID NOs: 7, 8, and 9; (3) SEQ ID NOs: 1, 2, and 13; (4) SEQ ID NOs: 16, 17, and 18; (5) SEQ ID NOs: 22, 23, and 24; (6) SEQ ID NOs: 28, 29, and 30; (7) SEQ ID NOs: 22, 34, and 24; (8) SEQ ID NOs: 36, 37, and 38; (9) SEQ ID NOs: NOs: 42, 43 and 44; or (10) SEQ ID NOs: 48, 49 and 50 having an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; and or ii) a light chain variable region, which may comprise a VL CDR1 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 respectively comprise an amino acid sequence identical to (1) SEQ ID NOs: 4, 5 and 6; (2) SEQ ID NOs: 10, 11 and 12; (3) SEQ ID NOs: 14, 5 and 15; (4) SEQ ID NOs: 19, 20 and 21; (5) SEQ ID NOs: 25, 26 and 27; (6) SEQ ID NOs: 31, 32 and 33; (7) SEQ ID NOs: 35, 26 and 27; (8) SEQ ID NOs: 39, 40 and 41; (9) SEQ ID NOs: 45, 46 and 47; or (10) SEQ ID NOs: 51, 52 and 53 having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

[0014] The antibodies or antigen-binding portions thereof of the present application may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region may comprise a VH CDR1 region, a VH CDR2 region and a VH CDR3 region, and the light chain variable region may comprise a VL CDR1 region, a VL CDR2 region and a VL CDR3 region, wherein the VH CDR1 region, the VH CDR2 region, the VH CDR3 region, the VL CDR1 region, the VL CDR2 region and the VL CDR3 region may comprise the following: (1) SEQ ID NOs: 1, 2, 3, 4, 5 and 6; (2) SEQ ID NOs: 7, 8, 9, 10, 11 and 12; (3) SEQ ID NOs: 1, 2, 13, 14, 5 and 15; (4) SEQ ID NOs: 16, 17, 18, 19, 20 and 21; (5) SEQ ID NOs: 22, 23, 24, 25, 26 and 27; (6) SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37; NOs: 28, 29, 30, 31, 32 and 33; (7) SEQ ID NOs: 22, 34, 24, 35, 26 and 27; (8) SEQ ID NOs: 36, 37, 38, 39, 40 and 41; (9) SEQ ID NOs: 42, 43, 44, 45, 46 and 47; or (10) SEQ ID NOs: 48, 49, 50, 51, 52 and 53 having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, wherein the antibody or antigen-binding portion thereof binds to CTLA4.

[0015] The heavy chain variable region of the antibodies or antigen-binding portions thereof of the present application can comprise an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 54, 55 (X1=A, X2=V; X1=A, X2=A; X1=G, X2=V; X1=G, X2=A), 58, 59 (X1=P, X2=A, X3=D; X1=L, X2=A, X3=N; X1=L, X2=A, X3=D; X1=L, X2=S, X3=N), 62, 64, 66, 68, 70, 72, 74 or 76, wherein the antibodies or antigen-binding portions thereof bind to CTLA4. The amino acid sequence of SEQ ID NO: 54 can be encoded by the nucleotide sequence of SEQ ID NOs: 80 or 81. The amino acid sequence of SEQ ID NO: 58 can be encoded by the nucleotide sequence of SEQ ID NOs: 86 or 87. The amino acid sequences of SEQ ID NOs: 55 (X1=G, X2=A) and 59 (X1=P, X2=A, X3=D) can be encoded by the nucleotide sequences of SEQ ID NOs: 82 and 88, respectively.

[0016] The light chain variable region of the antibodies or antigen-binding portions thereof of the present application can comprise an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 56, 57 (X1=S, X2=M, X3=R, X4=Y; X1=S, X2=V, X3=T, X4=F; X1=V, X2=V, X3=T, X4=F), 60, 61 (X1=T, X2=V, X3=F; X1=V, X2=P, X3=F; X1=V, X2=P, X3=Y), 63, 65, 67, 69, 71, 73, 75 or 77, wherein the antibodies or antigen-binding portions thereof bind to CTLA4. The amino acid sequence of SEQ ID NO: 56 may be encoded by the nucleotide sequence of SEQ ID NO: 83 or 84. The amino acid sequence of SEQ ID NO: 60 may be encoded by the nucleotide sequence of SEQ ID NO: 89 or 90. The amino acid sequences of SEQ ID NOs: 57 (X1=S, X2=V, X3=T, X4=F) and 61 (X1=V, X2=P, X3=Y) may be encoded by the nucleotide sequences of SEQ ID NOs: 85 and 91, respectively.

[0017] The antibodies or antigen-binding portions thereof of the present application may comprise a heavy chain variable region and a light chain variable region, which may respectively comprise the following: (1) SEQ ID NOs: 54 and 56; (2) SEQ ID NOs: 55 (X1=A, X2=V) and 57 (X1=S, X2=M, X3=R, X4=Y); (3) SEQ ID NOs: 55 (X1=A, X2=A) and 57 (X1=S, X2=M, X3=R, X4=Y); (4) SEQ ID NOs: 55 (X1=G, X2=V) and 57 (X1=S, X2=M, X3=R, X4=Y); (5) SEQ ID NOs: 55 (X1=G, X2=A) and 57 (X1=S, X2=M, X3=R, X4=Y); (6) SEQ ID NOs: NOs: 55 (X1=A, X2=V) and 57 (X1=S, X2=V, X3=T, X4=F); (7) SEQ ID NOs: 55 (X1=A, NOs: 55 (X1=G, X2=V) and 57 (X1=S, X2=V, X3=T, X4=F); (9) SEQ ID NOs: 55 (X1=G, NOs: 55 (X1=A, X2=V) and 57 (X1=V, X2=V, X3=T, X4=F); (11) SEQ ID NOs: 55 (X1=A, X2=A) and 57 (X1=V, X2=V, X3=T, X4=F); (12) SEQ ID NOs: 55 (X1=G, IDNOs: 55 (X1=G, X2=A) and 57 (X1=V, NOs: 59 (X1=L, X2=A, X3=N) and 61 (X1=T, X2=V, X3=F); (17) SEQ ID NOs: 59 (X1=L, NOs: 59 (X1=L, X2=S, X3=N) and 61 (X1=T, X2=V, X3=F); (19) SEQ ID NOs: 59 (X1=P,(20) SEQ ID NOs: 59 (X1=L, X2=A, X3=N) and 61 (X1=V, X2=P, X3=F); (21) SEQ ID NOs: 59 (X1=L, NOs: 59 (X1=L, X2=S, X3=N) and 61 (X1=V, X2=P, X3=F); (23) SEQ ID NOs: 59 (X1=P, NOs: 59 (X1=L, X2=A, X3=N) and 61 (X1=V, X2=P, X3=Y); (25) SEQ ID NOs: 59 (X1 = L, X2 = A, X3 = D) and 61 (X1 = V, X2 = P, X3 = Y); (26) SEQ ID NOs: 59 (X1 = L, X2 = S, X3 = N) and 61 (X1 = V, X2 = P, X3 = Y); (27) SEQ ID NOs: 62 and 63; (28) SEQ ID NOs: 64 and 65; (29) SEQ ID NOs: 66 and 67; (30) SEQ ID NOs: 68 and 69; (31) SEQ ID NOs: 70 and 71; (32) SEQ ID NOs: 72 and 73; (33) SEQ ID NOs: 74 and 75; or (34) SEQ ID NOs: NOs: 76 and 77 have amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

[0018] The isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain and a light chain connected by a disulfide bond. The heavy chain may comprise a heavy chain variable region and a heavy chain constant region, and the light chain may comprise a light chain variable region and a light chain constant region, wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region. The heavy chain variable region and the light chain variable region may comprise the aforementioned amino acid sequences, and the antibody or antigen-binding portion thereof binds to CTLA4. The heavy chain constant region may be an IgG1, IgG2, or IgG4 heavy chain constant region, such as a human IgG4 heavy chain constant region having the amino acid sequence set forth in SEQ ID NO.:78. The heavy chain constant region, such as an Fc fragment, may be genetically engineered to have reduced or enhanced FcR binding affinity. The light chain constant region may be a kappa constant region, such as a human kappa constant region having the amino acid sequence set forth in SEQ ID NO.:79. The amino acid sequences of SEQ ID NOs:78 and 79 may be encoded by the nucleotide sequences of SEQ ID NOs:92 and 93, respectively.

[0019] In some embodiments, the antibody of the present 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 comprise the above-mentioned heavy chain constant region, heavy chain variable region or CDR sequence, and each light chain may comprise the above-mentioned light chain constant region, light chain variable region or CDR sequence, wherein the antibody binds to CTLA4. The antibody of the present application or its antigen-binding portion thereof may be a full-length antibody, such as an IgG1, IgG2 or IgG4 isotype. In other embodiments, the antibody of the present application or its antigen-binding portion thereof may be a single-chain variable fragment (scFv) antibody or antibody fragment, such as a Fab or F(ab')2 fragment.

[0020] The application also provides bispecific molecules, which may include the antibody or its antigen-binding portion thereof of the present application, connected to a second functional group (e.g., a second antibody) having a binding specificity different from that of the present antibody or its antigen-binding portion thereof. The application also provides immunoconjugates, such as antibody-drug conjugates, which may include the antibody or its antigen-binding portion thereof of the present application, connected to a therapeutic agent such as a cytotoxic agent. On the other hand, the antibody or its antigen-binding portion thereof of the present application can be prepared as a part of a chimeric antigen receptor (CAR). Immune cells comprising the antigen chimeric receptor, such as T cells and NK cells, are also provided. The antibody or its antigen-binding portion thereof of the present application may also be encoded by an oncolytic virus or used together with an oncolytic virus.

[0021] The present application also relates to nucleic acid molecules encoding the antibodies or antigen-binding portions thereof of the present application, as well as expression vectors that may contain the nucleic acids, and host cells that may contain the expression vectors. Also provided are methods for preparing CTLA4 antibodies or antigen-binding portions thereof using host cells, which may include the steps of: (i) expressing the antibodies in the host cells and (ii) isolating the antibodies from the host cells or cell cultures thereof.

[0022] Compositions are also provided, which may include antibodies or antigen-binding portions thereof, immunoconjugates, bispecific molecules, oncolytic viruses, CARs, CAR-T cells, nucleic acid molecules, expression vectors or host cells of the present application, and pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical composition may also include therapeutic agents such as anticancer agents.

[0023] In another aspect, the present application provides a method for modulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion thereof of the present application, or optionally a nucleic acid molecule capable of expressing the same in the subject, thereby modulating the immune response in the subject. Preferably, the antibody or antigen-binding portion thereof of the present application enhances, stimulates or increases the immune response in the subject.

[0024] On the other hand, the present application provides a method for inhibiting tumor growth in a desired subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion thereof, or optionally a nucleic acid molecule capable of expressing these in the subject. In some embodiments, the method comprises administering a bispecific molecule, immunoconjugate, CAR-T cell, or an oncolytic virus encoding or carrying an antibody of the present application. The tumor may be a solid tumor or a hematologic tumor. In some embodiments, the tumor is a solid tumor, including but not limited to, melanoma, colorectal cancer, hepatocellular carcinoma, pleural mesothelioma, lung cancer (e.g., non-small cell lung cancer), renal cell carcinoma, cervical cancer, angiosarcoma, malignant pleural stromal tumor, metastatic transitional urothelial carcinoma, ureteral cancer, urethral cancer, urinary tract cancer, head and neck cancer, squamous cell tumor, transitional cell carcinoma (urothelial carcinoma), esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, GE junction adenocarcinoma, anal cancer, bile duct cancer, dysgerminoma, endometrial cancer, fallopian tube cancer, germ cell tumors, myelodysplastic syndrome, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, peritoneal cancer, prostate cancer, salivary gland cancer, sarcoma, triple negative breast cancer (TNBC), or muscle invasive bladder cancer. In some embodiments, at least one other anti-cancer antibody can be administered together with the present application antibody or its antigen-binding portion thereof, such as VISTA antibody, PD-1 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody, STAT3 antibody and / or ROR1 antibody. In another embodiment, the antibody or its antigen-binding portion thereof of the present application is administered together with a cytokine (such as IL-2, IL-21, GM-CSF and / or IL-4) or a co-stimulatory antibody (such as CD137 and / or GITR antibody). In other embodiments, the antibody or its antigen-binding portion thereof of the present application 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 of the present application may be, for example, mouse source, human source, chimeric or humanized.

[0025] On the other hand, the present application provides a method for treating or alleviating an infectious disease in a desired subject, comprising administering to the subject a therapeutically effective amount of the present composition. The infectious disease can be a disease caused by a viral, bacterial, fungal, or mycoplasma infection. In certain embodiments, the infectious disease is caused by chronic HIV infection or HHV-4 infection. In certain embodiments, the subject can further administer at least one anti-infective agent, such as an antiviral, antibacterial, antifungal, or antimycoplasma agent.

[0026] Based on the following specific description and examples, other features and advantages of the current disclosure will become clearer, and specific description and examples should not be interpreted as restrictive. The contents of all documents, Genbank records, patents and published patent applications cited in this application are expressly included in this article by reference.

[0027] Furthermore, it is the intention of this application not to include within this application any previously known product, process for making such product, or method for using such product, and the applicants reserve the right, and hereby disclose a disclaimer, to any previously known product, process, or method. It should be further noted that this application does not intend to include within its scope any product, process, or method for making such product or method for using such product that does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (EPC, Section 83), and the applicants reserve the right, and hereby disclose a disclaimer, to any previously described product, process for making such product, or method for using such product. In practicing this invention, compliance with Section 53(c) and Section 28(b) and (c) of the EPC is advantageous. All rights are expressly reserved to expressly disclaim any embodiment of the subject matter of any of the applicant's issued patents in the same family of this application or any other family of this application or any third party prior application. Nothing herein should be construed as a commitment.

[0028] It should be noted that in this application, especially in the claims and / or paragraphs, terms such as "comprising", "including", etc. may have the meanings assigned by U.S. patent law; for example, they may mean "containing", etc.; and terms such as "essentially consisting of..." or "essentially composed of..." have the meanings assigned by U.S. patent law, for example, allowing the existence of elements not explicitly stated, but excluding elements existing in the prior art or elements that affect the basic or novel characteristics of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following detailed description is given by way of example but is not intended to limit the present application to the specific embodiments described, and can be better understood in conjunction with the accompanying drawings.

[0030] Figure 1A and 1B The binding ability of mouse-derived antibodies D1H4, D1A7, and D1B6 (A), and C1G4, D1H3, D1B8, D1D5, D2A4, C1D1, and D1G6 (B) to human CTLA4 in capture ELISA is shown.

[0031] Figure 2A and 2BShown are the abilities of mouse-derived antibodies D2A4, C1D1, C1G4, D1B6, and D1D5 (A), D1H3, D1A7, D1G6, D1H4, and D1B8 (B) to block binding of a control benchmark to human CTLA4 in a competition ELISA.

[0032] Figure 3A and 3B Shown are the abilities of mouse-derived antibodies D1H4, D1A7, and D1B6 (A), C1G4, D1H3, D1B8, D1D5, D2A4, C1D1, and D1G6 (B) to block CTLA4 binding to cell surface CD80 / CD86 in a cell-based blocking FACS assay.

[0033] Figure 4 Mouse-derived antibodies D2A4, C1D1, C1G4, D1B6, D1D5, D1H3, D1H4, D1G6, D1B8, and D1A7 were shown to block CTLA4-CD80 binding and induce IL-2 release in cell-based functional assays.

[0034] Figures 5A to 5E The binding of chimeric antibodies C1G4 (A), D1B6 (B), C1D1 (C), D1D5 (D), and D1B8 (E) to human CTLA4 in a capture ELISA is shown.

[0035] Figure 6A and 6B Shown are the abilities of chimeric antibodies C1G4, D1B6 and C1D1 (A), D1D5 and D1B8 (B) to block CTLA4 binding to cell surface CD80 / CD86 in a cell-based blocking FACS assay.

[0036] Figure 7A and 7B Shown are the binding abilities of humanized antibodies huC1D1-V8 (A) and huD1D5-V9 (B) to human CTLA4 in capture ELISA.

[0037] Figure 8A and 8B Shown are the abilities of humanized antibodies huC1D1-V8 (A) and huD1D5-V9 (B) to block control benchmark-human CTLA4 binding in a competition ELISA.

[0038] Figure 9A and 9B Shown are the abilities of humanized antibodies huC1D1-V8 (A) and huD1D5-V9 (B) to block CTLA4 binding to cell surface CD80 / CD86 in a cell-based blocking FACS assay.

[0039] Figure 10Humanized antibodies huC1D1-V8 and huD1D5-V9 were shown to block CTLA4-CD80 binding and induce IL-2 release in cell-based functional assays.

[0040] Figure 11A and 11B The results of protein thermal shift assays of humanized antibodies huC1D1-V8 (A) and huD1D5-V9 (B) are shown. DETAILED DESCRIPTION

[0041] To ensure easier understanding of this application, some terms are first defined. Other definitions are given throughout the detailed description.

[0042] The term "CTLA4" refers to cytotoxic T lymphocyte-associated antigen 4. The term "CTLA4" includes variants, isoforms, homologs, orthologs, and paralogs. For example, an antibody specific for human CTLA4 protein may, in some cases, cross-react with CTLA4 proteins from species other than humans, such as monkeys. In other embodiments, an antibody specific for human CTLA4 protein may be completely specific for human CTLA4 protein and exhibit no cross-reactivity with other species or other types, or may cross-react with CTLA4 from certain other species but not all other species.

[0043] The term "human CTLA4" refers to a CTLA4 protein having an amino acid sequence from a human, such as the human CTLA4 amino acid sequence of Genbank Accession No. NP_005205. The terms "monkey or macaque CTLA4" and "mouse CTLA4" refer to monkey and mouse CTLA4 sequences, respectively, such as those having the amino acid sequences of Genbank Accession Nos. NP_001038204.1 and NP_033973.2, respectively.

[0044] The term "immune response" refers to the process by which cells, such as lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by these cells or the liver (including antibodies, cytokines, and complement) induce the selective damage, destruction, or elimination from the human body of invading pathogens, cells or tissues infected by pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0045] The term "antibody" as used herein refers to an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., CTLA4) through at least one antigen binding site, wherein the antigen binding site is generally located within the variable region of the immunoglobulin molecule. As used herein, the term includes complete polyclonal antibodies, complete monoclonal antibodies, single-chain Fv (scFv) antibodies, heavy chain antibodies (HCAb), light chain antibodies (LCAb), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, fusion proteins comprising an antigen binding site of an antibody, and any other modified immunoglobulin molecule containing an antigen binding site (e.g., a dual variable domain immunoglobulin molecule), as long as the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, chimeric antibodies, humanized antibodies, and human 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 the characteristics of their heavy chain constant domains, which are referred to as α, δ, ε, γ, and μ, respectively. Different classes 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 expressly stated, the term "antibody" as used herein includes the "antigen-binding portion" of an intact antibody. IgG is a glycoprotein that can comprise two heavy chains (H) and two light chains (L) that are internally linked by disulfide bonds. Each heavy chain can be composed of a heavy chain variable region (abbreviated as V H ) and a heavy chain constant region. The heavy chain constant region may be composed of C H1 、C H2 and C H3 Each light chain can be composed of a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region may be composed of C L This domain is composed of V H and V L The V domain can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions termed framework regions (FRs). H and V L It is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the traditional complement system.

[0046] As used herein, the term "antigen-binding portion" of an antibody (or simply "partial antibody") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CTLA4 protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) Fab fragments, which consist of V L 、V H 、C L and C m (ii) F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments connected by a disulfide bond in the hinge region; (iii) H and C H1 (iv) an Fd fragment consisting of a V domain of an antibody single arm; L and V H Fv fragment composed of V H (vi) isolated complementarity determining regions (CDRs); and (viii) nanobodies, which contain a heavy chain variable region and two constant domains. In addition, although the two domains of the Fv fragment, V L and V H , encoded by different genes, which can be connected by a synthetic linker through a recombinant method, wherein the synthetic linker allows them to be prepared as a single protein chain, wherein V L and V H The fragments are paired to form monovalent molecules (termed 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 encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained by conventional techniques known to those skilled in the art, and the fragments are screened for applications in the same manner as intact antibodies.

[0047] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to a CTLA4 protein is substantially free of antibodies that specifically bind to proteins other than CTLA4). However, an isolated antibody that specifically binds to a human CTLA4 protein may have cross-reactivity with other antigens, such as CTLA4 proteins from other species. In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0048] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0049] The term "mouse-derived antibody" as used herein is intended to include antibodies whose framework and CDR regions are derived from the variable regions of mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from mouse germline immunoglobulin sequences. The mouse-derived antibody of the present application may include amino acid residues that are not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo). However, the term "mouse-derived antibody" as used herein is not intended to include antibodies in which the CDR sequences derived from another mammalian species germline are implanted in the mouse framework sequence.

[0050] The term "chimeric antibody" refers to an antibody created by combining non-human genetic material with human genetic material. More generally, a chimeric antibody is one that contains genetic material from one species with genetic material from another.

[0051] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase similarity to naturally occurring antibody variants in humans.

[0052] The term "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

[0053] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0054] As used herein, an antibody that "specifically binds to human CTLA4" refers to an antibody that binds to human CTLA4 protein (and possibly CTLA4 protein from one or more non-human species) but does not substantially bind to non-CTLA4 proteins. Preferably, the antibody binds with "high affinity," i.e., with a K D The value is 5.0x10 -8 M or less, more preferably 1.0x10 -8 M or less, more preferably 7.0x10 -9 Below M, binds to human CTLA4 protein.

[0055] As used herein, the term "does not substantially bind" a protein or cell means that the protein or cell does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., with a K D 1x10 -6 M or more, more preferably 1x10 -5 M or more, more preferably 1x10 -4M or more, more preferably 1x10 -3 M or more, more preferably 1x10 -2 M and above, bound to proteins or cells.

[0056] The term "high affinity" for IgG antibodies refers to the K D 1.0x10 -6 M or less, more preferably 5.0x10 -8 M or less, even more preferably 1.0x10 -8 M or less, even more preferably 7.0x10 -9 M or less, even more preferably 1.0x10 -9 M or less. However, for other antibody isotypes, "high affinity" binding may vary. For example, "high affinity" binding for an IgM isotype refers to an antibody with a K of D is 10 -6 M or less, more preferably 10 -7 M or less, even more preferably 10 - 8 Below M.

[0057] The term "K assoc ” or “K a ” refers to the association rate of a specific antibody-antigen interaction, and the term “K dis ” or “K d " refers to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "K D " refers to the d With K a The ratio (K d / K a ) is obtained, expressed as a molar concentration (M). The K of an antibody can be determined using methods well known in the art. D Used to determine the antibody K D A preferred method is to use surface plasmon resonance, preferably using a biosensor system such as Biacore TM system.

[0058] The term "EC 50 ”, also known as half-maximal effect concentration, is the antibody concentration that elicits a response intermediate between baseline and maximum values after a specific exposure time.

[0059] The term "IC 50 ", also known as half-maximal inhibitory concentration, is the concentration of an antibody that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.

[0060] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows and horses, are preferred.

[0061] The term "therapeutically effective amount" refers to an amount of the antibody of the present application sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer) and / or reduce the severity of the disease or condition. A therapeutically effective amount is understood in the context of the disease being treated, wherein those skilled in the art can readily discern the actual effective amount.

[0062] Various aspects of the application are described in further detail in the following subsections.

[0063] The antibodies of the present application, or antigen-binding portions thereof, specifically bind to human CTLA4 with binding affinity / ability comparable to (if not better than) previously described CTLA antibodies, such as ipilimumab.

[0064] The antibodies or antigen-binding portions thereof of the present application block the binding of CTLA4 to CD80 / CD86 with activity comparable to or greater than that of previously described CTLA antibodies, such as ipilimumab. The antibodies or antigen-binding portions thereof of the present application promote T cell responses that are suppressed by CTLA4-CD80 / CD86 binding.

[0065] The antibodies or antigen-binding portions thereof of the present application are mouse, chimeric, or humanized.

[0066] The antibodies or antigen-binding portions thereof of the present application are structurally and chemically characterized as described below and in the Examples. The amino acid sequence ID numbers of the heavy and light chain variable regions of the antibodies are summarized in Table 1 below; some antibodies have the same VH or VL. The heavy chain constant region of the antibody can be, for example, a human IgG4 heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 78, and the light chain constant region of the antibody can be a human kappa constant region having the amino acid sequence set forth in SEQ ID NO: 79. These antibodies may also comprise a mouse IgG4 heavy chain constant region and a mouse kappa constant region.

[0067] The heavy chain variable region CDRs and light chain variable region CDRs in Table 1 are identified by the Kabat numbering system. However, as is known in the art, CDR regions can also be identified based on the heavy chain / light chain variable region sequences by other systems such as Chothia, IMGT, AbM or Contact numbering systems / methods.

[0068] V of other CTLA4 antibodies that bind to human CTLA4 H and V LThe sequence (or CDR sequence) can be combined with the V sequence of the CTLA4 antibody of the present disclosure. H and V L Sequences (or CDR sequences) are "mixed and matched". Preferably, when V H and V L When chains (or CDRs within those chains) are mixed and matched, H / V L Paired V H The sequence is replaced by the structurally similar V H Sequence replacement. Similarly, preferably from a specific V H / V L Paired V L The sequence is similar to the structure of V L Sequence replacement.

[0069]

[0070]

[0071] Therefore, in one embodiment, the antibody or antigen-binding portion thereof of the present application comprises:

[0072] (a) a heavy chain variable region comprising the amino acid sequence listed in Table 1; and

[0073] (b) a light chain variable region comprising the amino acid sequence listed in Table 1, or a V region of another CTLA4 antibody L , wherein the antibody specifically binds to human CTLA4.

[0074] In another embodiment, the antibody or antigen-binding portion thereof of the present application comprises:

[0075] (a) CDR1, CDR2, and CDR3 of the heavy chain variable region listed in Table 1; and

[0076] (b) CDR1, CDR2, and CDR3 of the light chain variable region listed in Table 1, or the CDRs of another CTLA4 antibody, wherein the antibody specifically binds to human CTLA4.

[0077] In another embodiment, the antibody or antigen-binding portion thereof comprises the heavy chain variable CDR2 region of a CTLA4 antibody in combination with the CDR regions of another antibody that binds to human CTLA4, e.g., heavy chain variable region CDR1 and / or CDR3, and / or light chain variable region CDR1, CDR2 and / or CDR3 of a different CTLA4 antibody.

[0078] In addition, as is 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 cognate 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; 6,090,38; 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.

[0079] Thus, in another embodiment, the antibody of the present application comprises the CDR2 of the heavy chain variable region of the CTLA4 antibody, at least the CDR3 of the heavy chain and / or light chain variable region of the CTLA4 antibody, or the CDR3 of the heavy chain and / or light chain variable region of another CTLA4 antibody, wherein the antibody is capable of specifically binding to human CTLA4. Preferably, these antibodies (a) compete for binding to CTLA4; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have a binding affinity similar to that of the CTLA4 antibody of the present application. In another embodiment, the antibody may further comprise the CDR2 of the light chain variable region of the CTLA4 antibody, or the CDR2 of the light chain variable region of another CTLA4 antibody, wherein the antibody is capable of specifically binding to human CTLA4. In another embodiment, the antibody of the present application may comprise the CDR1 of the heavy chain and / or light chain variable region of the CTLA4 antibody, or the CDR1 of the heavy chain and / or light chain variable region of another CTLA4 antibody, wherein the antibody is capable of specifically binding to human CTLA4.

[0080] In another embodiment, the antibody of the present application comprises a heavy chain and / or light chain variable region sequence comprising CDR1, CDR2, and CDR3 sequences that differ from the variable region of the CTLA4 antibody of the present application in that it has one or more conservative modifications. As is understood in the art, some conservative sequence modifications can be made that do not eliminate antigen binding. See, e.g., 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.

[0081] Thus, in one embodiment, the antibody comprises a heavy chain variable region comprising a CDR1, CDR2, and CDR3 sequence and / or a light chain variable region comprising a CDR1, CDR2, and CDR3 sequence, wherein:

[0082] (a) the heavy chain variable region CDR1 sequence comprises the sequence listed in Table 1 above, and / or conservative modifications thereof; and / or

[0083] (b) the heavy chain variable region CDR2 sequence comprises the sequence listed in Table 1 above, and / or conservative modifications thereof; and / or

[0084] (c) the heavy chain variable region CDR3 sequence comprises the sequence listed in Table 1 above, and conservative modifications thereof; and / or

[0085] (d) the light chain variable region CDR1, and / or CDR2, and / or CDR3 sequences comprise the sequences listed in Table 1 above, and / or conservative modifications thereof; and

[0086] (e) The antibody specifically binds to human CTLA4.

[0087] The antibodies or antigen-binding portions thereof of the present application have one or more of the above-mentioned functional characteristics, such as high binding affinity to human CTLA4 and blocking activity against CTLA4-CD80 / CD86.

[0088] In various embodiments, the antibody can be, for example, a mouse, human, humanized, or chimeric antibody.

[0089] The term "conservative sequence modification" as used herein refers to an amino acid modification that does not significantly affect or change the binding properties of an antibody containing such an amino acid sequence. Such conservative modifications include amino acid replacements, additions, and deletions. Modifications can be introduced into the present application's antibodies by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid replacement 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), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar 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 the present application can be replaced with 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 assays described herein.

[0090] The antibodies of the present application can be used with one or more V H / V L The antibody can be modified by modifying one or two variable regions (i.e., V H and / or V L) (e.g., in one or more CDR regions and / or one or more framework regions) to carry out genetic modification. Additionally and alternatively, an antibody can be engineered by modifying the residues in the constant region, for example, to alter the effector functions of the antibody.

[0091] In certain embodiments, CDR implantation can be used to engineer the variable regions of antibodies. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific natural antibody can be expressed by constructing an expression vector that contains the CDR sequence from a specific natural antibody implanted into the framework sequence of a different antibody 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. Sci. USA 86:10029-10033; U.S. Patents 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0092] Therefore, another embodiment of the present application relates to an isolated monoclonal antibody or antigen-binding portion thereof, which comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3 sequence having the above-mentioned sequence of the present application, and the light chain variable region comprises a CDR1, CDR2 and CDR3 sequence having the above-mentioned sequence of the present application. Although these antibodies comprise the V sequences of the monoclonal antibodies of the present application, H and V L CDR sequences, which may contain different framework sequences.

[0093] These framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrc-cpe.cam.ac.uk / vbase), or in Kabat et al., (1991), supra; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference. As another example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences in the HCo7 HuMAb mouse 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 the Hco12 HuMAb mouse are available from Genbank Accession Nos. 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).

[0094] The antibody protein sequences were compared to compiled protein sequence databases using a sequence similarity search method known in the art as spaced BLAST (Altschul et al., (1997), supra).

[0095] Preferred backbone sequences for use in the antibodies of the present application are those that are structurally similar to the backbone sequences used in the antibodies of the present application. H The CDR1, CDR2, and CDR3 sequences can be implanted into a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence was derived, or the CDR sequences can be implanted into a framework region that contains one or more mutations compared to the germline sequence. For example, it has been found that, in some cases, it is beneficial to mutate residues in the framework region to maintain or enhance the antigen binding ability of an antibody (see, e.g., U.S. Patents 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0096] Another type of variable region modification is to modify V H and / or V L The amino acid residues in the CDR1, CDR2 and / or CDR3 regions are mutated to improve one or more binding properties (e.g., affinity) of the target antibody. Point mutations or PCR-mediated mutations can be used to introduce mutations, and the impact of these mutations on antibody binding or other target functional properties can be evaluated in vitro or in vivo assays known in the art. Preferably, conservative modifications (known in the art) are introduced. The mutations can be amino acid replacements, additions, or deletions, but are preferably replacements. In addition, the changes in the CDR regions are typically no more than one, two, three, four, or five residues.

[0097] In another embodiment, the present application provides an isolated CTLA4 monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region comprising: (a) V H CDR1 region, which comprises the sequence of the present application, or an amino acid sequence containing one, two, three, four or five amino acid substitutions, deletions or additions; (b) V H CDR2 region, which comprises the sequence of the present application, or an amino acid sequence containing one, two, three, four or five amino acid substitutions, deletions or additions; (c) V H CDR3 region, which comprises the sequence of the present application, or an amino acid sequence containing one, two, three, four or five amino acid substitutions, deletions or additions; (d) V L CDR1 region, which comprises the sequence of the present application, or an amino acid sequence containing one, two, three, four or five amino acid substitutions, deletions or additions; (e) V L A CDR2 region comprising the sequence of the present application, or an amino acid sequence containing one, two, three, four or five amino acid substitutions, deletions or additions; and (f) V L The CDR3 region comprises the sequence of the present application, or an amino acid sequence containing one, two, three, four or five amino acid substitutions, deletions or additions.

[0098] The genetically modified antibodies of the present application include, for example, to improve antibody properties, H and / or V L Antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence of the derived antibody. Generally, these framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence of the derived antibody. These residues can be identified by comparing the antibody framework sequence to the germline sequence of the derived antibody.

[0099] 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 approach is also known as "deimmunization" and is described in more detail in U.S. Patent Publication 20030153043.

[0100] In addition, or as an alternative to modifications within the framework or CDR region, the antibodies of the present 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 fixation, Fc receptor binding, and / or antibody-dependent cellular toxicity. In addition, the antibodies of the present application can be chemically modified (e.g., one or more chemical groups can be added to the antibody), or modified to alter its glycosylation, also to alter one or more functional properties of the antibody.

[0101] In one embodiment, C H1 The hinge region is modified to alter, for example, increase or decrease, the number of cysteine residues in the hinge region. This method is further described in U.S. Patent No. 5,677,425. H1 The number of cysteine residues in the hinge region can be adjusted to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0102] In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C H2 -C H3 The invention relates to a method for synthesizing an antibody that binds to a domain-linking region of the Fc-hinge domain, thereby reducing Staphylococcus aureus protein A (SpA) binding compared to SpA binding to the native Fc-hinge domain. This approach is described in more detail in U.S. Patent No. 6,165,745.

[0103] In another embodiment, the glycosylation of the antibody is modified. For example, a deglycosylated antibody (i.e., an antibody lacking glycosylation) can be prepared. Glycosylation can be altered, for example, to increase the affinity of the antibody 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 position. Such deglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0104] Additionally or alternatively, antibodies with altered glycosylation types can be prepared, such as low-fucosyl antibodies with reduced amounts of fucose residues or antibodies with increased bisecting GlcNac structures. These altered glycosylation forms have been shown to increase the ADCC activity of antibodies. 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 antibodies of the present application to prepare antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), resulting in antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lacking fucose in their sugars. Two replacement vectors were used to target the FUT8 gene in CHO / DG44 cells to generate the Ms704, Ms705, and Ms709 FUT8- / - cell lines (see U.S. Patent Publication No. 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 (which encodes a fucosyltransferase), thereby reducing or eliminating the α-1,6 linkage-related enzyme, resulting in antibodies expressed in the cell line exhibiting hypofucosylation. EP 1,176,195 also describes a cell line that has low or no activity in adding fucose to N-acetylglucosamine bound to the Fc region of an antibody, 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 Asn(297)-linked sugars, thereby resulting in hypofucosylation of antibodies expressed in the host cells (see Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Antibodies with altered glycosylation patterns can also be produced in eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies with altered glycosylation patterns can be produced in plant cells, such as duckweed. Methods for producing antibodies in plant systems are described in U.S. patent application filed August 11, 2006, attorney docket number 040989 / 314911 to Alston & Bird LLP. Fucosidase can be used to remove fucose residues from antibodies, for example, α-L-fucosidase removes fucose residues from antibodies (Tarentino et al., (1975) Biochem. 14: 5516-23).

[0105] Another modification of the antibodies herein encompassed in this application is pegylation. 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 fragment thereof is typically reacted with polyethylene glycol (PEG), such as an active ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is performed by an acylation reaction or an 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 derivatize other proteins, such as mono(C1-C1-PEG) or ... 10 ) alkoxy- or aryloxypolyethylene glycol or polyethylene glycol maleimide. In certain embodiments, the antibody to be pegylated is a deglycosylated antibody. Methods for protein pegylation are known in the art and can be applied to the antibodies of the present application. See, for example, EPO 154316 and EP 0401384.

[0106] The antibodies of the present application can be characterized by their various physical properties in order to detect and / or distinguish between different classes thereof.

[0107] For example, an antibody 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 alter the pK of the antibody due to altered antigen binding (Marshall 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 at motifs containing the N-X-S / T sequence. In some cases, it is preferred that the CTLA4 antibody does not contain variable region glycosylation. 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.

[0108] In a preferred embodiment, the antibody does not contain an asparagine isomerization site. Deamidation of asparagine may occur at the NG or DG sequence, resulting in the generation of an isoaspartic acid residue, which introduces a link to the polypeptide chain and reduces its stability (isoaspartic acid effect).

[0109] 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 generally 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 pIs outside the normal range may have some unfolded structure and instability under in vivo conditions. Therefore, CTLA4 antibodies with pI values falling within the normal range are preferred. This can be achieved by selecting antibodies with pIs within the normal range or by mutating charged surface residues.

[0110] On the other hand, the application provides nucleic acid molecules encoding the heavy chain and / or light chain variable regions or CDRs of the antibodies of the application. The nucleic acid can be present in intact cells, cell lysates, or in partially purified or substantially purified form. A nucleic acid is "isolated" or "in a substantially purified state" when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acid of the 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.

[0111] The nucleic acids of the present 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, which will be further described below), cDNAs encoding the light and heavy chains of the antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene library.

[0112] The preferred nucleic acid molecules of the present application include V encoding CTLA4 monoclonal antibody H and V L Once the sequences encoding V H and V L These DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L or V H The term "operably linked" is used herein to refer to a DNA fragment that is operably linked to another DNA fragment encoding another protein (e.g., an antibody constant region or a flexible linker). In this context, the term "operably linked" refers to two DNA fragments that are linked together so that the amino acid sequences encoded by the two DNA fragments are in frame.

[0113] Code V H The isolated DNA of the V region can be operably linked to H DNA encoding the heavy chain constant region (CH1 、C H2 and C H3 The heavy chain constant region can be converted into a full-length heavy chain gene by using another DNA molecule of IgG1). The sequences of human heavy chain constant region genes are known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but most preferably an IgG1 or IgG4 constant region. For Fab fragment heavy chain genes, the DNA encoding the VH region can be operably combined with the DNA encoding only the heavy chain C region. H1 The constant region of another DNA molecule is attached.

[0114] Code V L The isolated DNA of the V region can be operably linked to L DNA encoding the light chain constant region C L The light chain constant region is converted into a full-length light chain gene (and a Fab light chain gene) by another DNA molecule of the same sequence. The sequences of human light chain constant region genes are known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0115] To create scFv genes, the V H and V L The DNA fragment can be operably linked to another fragment encoding a flexible linker, such as encoding the amino acid sequence (Gly4-Ser)3, so that V H and V L The sequence can be expressed as a continuous single-chain protein, where V L and V H The regions are connected by this flexible linker (see, e.g., 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).

[0116] The monoclonal antibodies (mAbs) of the present application can be prepared using the somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256:495, which is well known in the art. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display technology. Chimeric or humanized antibodies are well 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 specifically incorporated herein by reference in their entirety.

[0117] The antibodies of the present application can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection technology known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biological techniques is inserted into one or more expression vectors so that the genes are operably linked to transcriptional and translational regulatory sequences. In this case, the term "operably linked" is intended to indicate that the antibody gene is linked to the vector so that the transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody gene.

[0118] 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 the antibody gene. Such regulatory sequences are 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 direct high-level protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses such as the adenovirus major late promoter (AdMLP), and polyomavirus. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or the β-globin promoter, may be used. Alternatively, regulatory elements may be composed of sequences from various sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472). Expression vectors and expression control sequences are selected that are compatible with the host cell being used.

[0119] The antibody light chain gene and the antibody heavy chain gene 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, so that V H Part and C in the carrier H Part operatively connected, V L Part and C in the carrier L Partially operably connected. In addition or additionally, the recombinant expression vector can encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector, and then the signal peptide is connected to the amino terminus of the antibody chain gene in frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0120] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the present application may carry other sequences, for example, sequences (e.g., replication origins) and selectable marker genes that regulate vector replication in host cells. Selectable marker genes promote selection of host cells into which vectors have been introduced (see, for example, U.S. Patents 4,399,216; 4,634,665 and 5,179,017). For example, generally, selectable marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate on host cells into which vectors have been introduced. Preferred selectable marker genes include dihydrofolate reductase (DHFR) genes (for dhfr host cells with methotrexate selection / amplification) and neo genes (for G418 selection).

[0121] For the expression of light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to include a variety of common techniques for introducing exogenous DNA into prokaryotic or eukaryotic host cells, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is theoretically possible to express the antibodies of the present application in prokaryotic or eukaryotic host cells, it is most preferred to express the antibodies in eukaryotic cells, most preferably in mammalian host cells, because these eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete properly folded and immunologically active antibodies than prokaryotic cells.

[0122] Preferred mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells used with the DHFR selectable marker described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220; the DHFR selectable marker is described, for example, in R.J. Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. Another preferred expression system, particularly when using NSO myeloma cells, 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 a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient for the antibody to be expressed in the host cell, or preferably for the antibody to be secreted into the culture medium in which the host cell is grown. The antibody is recovered from the culture medium using standard protein purification methods.

[0123] The antibody of the present application or its antigen-binding portion thereof can be combined with therapeutic agent to form the immunoconjugate of for example antibody-drug conjugate (ADC).Suitable therapeutic agent includes cytotoxin, alkylating agent, DNA minor groove binding agent, DNA intercalating agent, DNA cross-linking agent, histone deacetylase inhibitor, nuclear export inhibitor, proteasome inhibitor, topoisomerase I or II inhibitor, heat shock protein inhibitor, tyrosine kinase inhibitor, antibiotic and antimitotic agent.In ADC, antibody and therapeutic agent are preferably combined by cleavable linker, for example peptide linker, disulfide linker or hydrazone linker.More preferably, linker is peptide linker, for example 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. ADCs 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.

[0124] On the other hand, the present disclosure relates to bispecific molecules, which comprise one or more antibodies of the present application linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to generate bispecific molecules that bind to at least two different binding sites or target molecules. Therefore, as used herein, "bispecific molecules" include molecules with three or more specificities.

[0125] Bispecific molecules can come in a variety of different forms and sizes. At one end of the size spectrum, bispecific molecules retain the traditional antibody format, except that they have two binding arms, each with a different specificity, rather than two binding arms with the same specificity. At the other extreme are bispecific molecules composed of two single-chain antibody fragments (scFv) linked by peptide chains, the so-called Bs(scFv)2 constructs. Bispecific molecules of intermediate size include two different F(ab) fragments linked by a peptide linker. These and other forms of bispecific molecules can be prepared by genetic modification, somatic cell hybridization, or chemical methods. See, for example, Kufer et al, supra; Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al., Immunology Today, 21(8), 391-397 (2000), and references cited therein.

[0126] The present invention also provides oncolytic viruses that preferentially infect and kill cancer cells. The antibodies of the present application can be used together with oncolytic viruses. Alternatively, oncolytic viruses encoding the antibodies of the present application can be introduced into the human body.

[0127] Also provided herein are chimeric antigen receptors (CARs) comprising a CTLA4 scFv comprising the CDRs and heavy / light chain variable regions described herein.

[0128] The CTLA4 CAR may comprise (a) an extracellular antigen-binding domain comprising a CTLA4 scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0129] CAR can contain a signal peptide at the N-terminus of the extracellular antigen binding domain that directs the nascent receptor to enter the endoplasmic reticulum, and a hinge peptide at the N-terminus of the extracellular antigen binding domain that makes the receptor more easily bound. CAR preferably comprises a primary intracellular signaling domain and one or more costimulatory signaling domains in the intracellular signaling domain. The main and most effective primary intracellular signaling domain is the CD3-ζ cytoplasmic domain containing ITAM, whose phosphorylation causes T cell activation. The costimulatory signaling domain can be derived from costimulatory proteins, such as CD28, CD137, and OX40.

[0130] CARs can also be supplemented with factors that enhance T cell expansion, persistence, and anti-tumor activity, such as cytokines and co-stimulatory ligands.

[0131] Genetically modified immune effector cells are also provided, which include CAR provided herein. In some embodiments, immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMC), hematopoietic stem cells, pluripotent stem cells or embryonic stem cells. In some embodiments, immune effector cells are T cells.

[0132] On the other hand, the present application provides a pharmaceutical composition, which may include one or more antibodies or their antigen-binding portions, bispecific molecules, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors or host cells of the present application formulated with a pharmaceutically acceptable carrier. When the composition contains more than one antibody (or its antigen-binding portion, bispecific molecules, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors or host cells), the antibody or its antigen-binding portion, bispecific molecules, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors or host cells can be administered separately. The composition may optionally include one or more other pharmaceutically active ingredients, such as another antibody or drug, such as an anti-tumor drug.

[0133] The pharmaceutical composition 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 taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0134] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Based on the difference in route of administration, the active ingredient can be wrapped in a material to protect it from the influence of acid and other natural conditions that may inactivate it. The term "parenteral administration" as used herein refers to a non-enteral and non-local external administration mode of administration, usually carried out by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the antibody of the present application can be administered by non-parenteral route, such as external, epidermal or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical.

[0135] 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 concentration.

[0136] The amount of active ingredient that can be prepared into a single dosage form together with the carrier material will vary depending on the subject being treated and the specific mode of administration, and is generally an amount of the composition that produces a therapeutic effect. Basically, in percentage terms, this amount is about 0.01% to about 99% combined with a pharmaceutically acceptable carrier.

[0137] The dosage regimen is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a bolus may be administered, multiple divided doses may be administered over time, or the dose may be reduced or increased in proportion to the severity of the therapeutic situation. It is particularly advantageous to configure the parenteral composition in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable for single administration to a subject; each unit contains a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect together with the required pharmaceutical carrier. Alternatively, the antibody may be administered as a sustained-release formulation, in which case the required dosing frequency is reduced.

[0138] For administration of the composition, the dosage range may be about 0.0001-100 mg / kg.

[0139] The "therapeutically effective dose" of the present application's CTLA4 antibody or antigen-binding portion thereof, or bispecific molecule, CAR-T cell, oncolytic virus, immunoconjugate preferably causes a reduction in the severity of disease symptoms, an increase in the frequency and persistence of disease asymptomatic periods, or prevents damage or disability caused by disease torture. For example, for the treatment of tumor-bearing subjects, it is preferred that the "therapeutically effective dose" 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. A therapeutically effective amount of a therapeutic antibody can reduce tumor size in a subject, or alleviate the subject's symptoms, and the subject is typically a human, or can be another mammal.

[0140] The pharmaceutical composition can be in a controlled release dosage form, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems. JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0141] The pharmaceutical composition 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 delivery devices (U.S. Patent 4,486,194); (4) push injection devices (U.S. Patents 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Patents 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0142] In certain embodiments, the monoclonal antibodies of the present application can be formulated to ensure proper distribution in the body. For example, to ensure that the therapeutic antibodies of the present application can cross the blood-brain barrier, they can be formulated in liposomes, which can also additionally contain targeting functional groups to enhance selective delivery to specific cells or organs. See, e.g., U.S. Patents 4,522,811; 5,374,548; 5,416,016; and 5,399,331; VV Ranade (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.

[0143] The compositions of the present application have many in vitro and in vivo uses, involving, for example, the treatment of cancer and infectious diseases. The compositions can be administered to human subjects, for example, to inhibit tumor growth, or reduce or eliminate pathogens in vivo.

[0144] In view of the CTLA4 antibody or its antigen-binding portion thereof of the present application, the ability of reversing CTLA4-CD80 / CD86-mediated T cell inhibition and promoting T cell response, the present application provides a method for inhibiting tumor cell growth in a subject, including administering the composition of the present application to the subject, so that tumor growth in the subject is inhibited. Non-limiting examples of tumors that can be treated by the composition of the present application include, but are not limited to, melanoma, colorectal cancer, hepatocellular carcinoma, pleural mesothelioma, lung cancer (e.g., non-small cell lung cancer), renal cell carcinoma, cervical cancer, angiosarcoma, and muscle-invasive bladder cancer. In addition, the antibody of the present application can be used to inhibit the growth of recurrent or refractory malignant tumors.

[0145] In another aspect, the present application provides a combined treatment method, wherein the CTLA4 antibody or its antigen binding portion thereof, or bispecific molecule, CAR-T cell, oncolytic virus, immunoconjugate of the present application is co-administered with one or more other antibodies effective for suppressing tumor growth in the subject. In one embodiment, the present application provides a method for suppressing tumor growth in a subject, comprising administering a CTLA4 antibody (or its antigen binding portion thereof, or CAR-T cell, oncolytic virus, immunoconjugate) and one or more other antibodies, such as VISTA antibody, LAG-3 antibody, PD-L1 antibody and / or PD-1 antibody to the subject. In certain embodiments, the subject is a person.

[0146] CTLA4 signaling pathway blockade can also be combined with standard tumor treatments. For example, CTLA4 signaling pathway blockade can be combined with LAG-3 and / or PD-1 blockade and chemotherapy regimens. For example, a chemotherapy agent can be administered together with a CTLA4 antibody, which can be a cytotoxic agent. For example, epirubicin, oxaliplatin, and 5-FU are administered to a patient receiving CTLA4 therapy.

[0147] Alternatively, the combined use of CTLA4 antibodies and one or more other antibodies (e.g., TIM-3 antibodies and / or LAG-3 antibodies and / or PD-1 antibodies) can also be combined with immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrates), and cells transfected with genes encoding immune stimulatory 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.

[0148] Other therapies that can be combined with CTLA4 therapy include, but are not limited to, administration of interleukin-2 (IL-2), radiation therapy, surgery, or hormone suppression.

[0149] In another aspect, the present application provides a combination therapy method, wherein the CTLA4 antibody or its antigen-binding portion thereof of the present application is administered together with one or more other drugs that are effective in reducing or eliminating pathogens. In one embodiment, the present application provides a method for treating or alleviating an infectious disease in a subject, comprising administering to the subject a CTLA4 antibody or its antigen-binding portion thereof and one or more other drugs directed against pathogens, such as antiviral agents, antibacterial agents, antifungal agents, or anti-mycoplasma antibodies. In certain embodiments, the subject is human.

[0150] The combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions in which each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0151] Furthermore, if more than one dose of the combination therapy is to be administered sequentially, the order of sequential administration can be reversed or maintained in the same order at each administration time point, sequential administration can be combined with simultaneous administration, or any combination thereof.

[0152] The present application will be further illustrated in the following examples, which should not be interpreted 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.

[0153] Example

[0154] Example 1. Preparation of mouse anti-CTLA4 monoclonal antibodies by hybridoma technology

[0155] immunity

[0156] 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 CTLA4 protein with a human IgG1 Fc tag at the C-terminus (Acro biosystems, Cat# CT4-H5255) was used as an immunogen. Human CTLA4-his protein (Acro biosystems, Cat# CT4-H5229) was used for the determination of antiserum titers and the screening of hybridomas secreting antigen-specific antibodies. The immunization dose contained 25 μg of human CTLA4-Fc protein / mouse / injection for primary and booster immunizations. To increase the immune response, Freund's complete adjuvant and Freund's incomplete adjuvant (Sigma, St. Louis, Mo., USA) were used in primary and booster immunizations, respectively. Briefly, the adjuvant-antigen mixture was prepared by first gently mixing the adjuvant in a vial using a vortex mixer. Transfer the required amount of adjuvant to an autoclaved 1.5 mL microcentrifuge tube. Prepare the antigen in PBS or saline at a concentration of 0.25-0.5 mg / ml. Then add the calculated amount of antigen to the microcentrifuge tube containing the adjuvant and gently vortex for 2 minutes to mix the resulting solution to form an oil-in-water emulsion. The adjuvant-antigen emulsion is then drawn into a suitable syringe for animal injection. Inject a total of 25 μg of antigen in a volume of 100-200 μl. Immunize each animal and then perform 4-5 boosts based on the antiserum titer. Before cell fusion, animals with better titers are given a final boost by intraperitoneal injection.

[0157] Hybridoma fusion and screening

[0158] Cells of a mouse myeloma cell line (SP2 / 0-Ag14, ATCC #CRL-1581) were cultured and allowed to reach the logarithmic growth phase before cell fusion. Splenocytes from immunized mice were prepared aseptically and fused with myeloma cells according to the method described in Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibodies of predefined specificity," Nature, 256: 495-497 (1975). The fused "hybrid cells" were then plated in 96-well cell plates in DMEM / 20% FCS / HAT medium. 7-10 days after fusion, surviving hybridoma colonies were observed under a microscope. Two weeks later, the supernatant of each well was subjected to indirect ELISA using recombinant human CTLA-4-his protein. Positive hybridomas that secrete antibodies that bind to human CTLA-4-his protein were selected and transferred to 24-well plates. The hybridoma was further tested for its ability to block the binding of human CTLA-4-Fc protein to cell surface CD80 / CD86 by flow cytometry (FACS). By limiting dilution, hybridoma clones producing antibodies with high specific binding to human CTLA-4 and CTLA-4-Daudi cell blocking activity were subcloned to ensure the monoclonal origin of the cell line, and then the monoclonal antibodies were purified. Briefly, a protein A agarose chromatography column (Bestchrom (Shanghai) Biosciences, Cat#AA0273) was washed with 5-10 column volumes of PBS buffer. The cell supernatant of the hybridoma monoclonal was passed through the column, and then the column was washed with PBS buffer until the protein absorbance reached baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7) and immediately collected into a 1.5 ml tube containing neutral buffer (1 M Tris-HCl, pH 9.0). The fractions containing immunoglobulins were mixed and dialyzed in PBS at 4°C overnight. Subsequently, the functional activity of the purified monoclonal antibodies was characterized in vitro as follows.

[0159] Example 2. Binding of Mouse-Derived CTLA4 Monoclonal Antibodies Determined Using BIACORE Surface Plasmon Resonance Affinity

[0160] The binding affinity and binding kinetics of the purified mouse monoclonal CTLA4 antibody (mAb) produced in Example 1 were characterized using the Biacore T200 system (GE healthcare, Pittsburgh, PA, USA).

[0161] Table 2. Binding affinity of mouse CTLA4 antibodies

[0162]

[0163] Briefly, goat anti-mouse IgG (GE healthcare, Cat# BR100838, Mouse Antibody Capture Kit) was covalently linked to a CM5 chip (carboxymethyl dextrose coated chip, GE healthcare, Cat# BR100530) via primary amines using a standard amine coupling kit provided by Biacore (GE healthcare, Pittsburgh, PA, USA). Protein G chips (GE healthcare, Cat# 29-1793-15) were used for affinity determination of the control standard. Unreacted groups on the biosensor surface were blocked with ethanolamine. The purified CTLA4 antibody of the present application and the CTLA4 control standard (Bristol-Myers Squibb Co, Cat# NDC: 0003-2327-11, also known as or BM) was flowed through the chip at a concentration of 10 μg / ml and a flow rate of 10 μL / min. + Recombinant human CTLA4-his (Acro biosystems, Cat#CT4-H5229, starting concentration 80 nM, two-fold serial dilution) or monkey CTLA4-his protein (Acro biosystems, Cat#CT4-C5227, starting concentration 80 nM, two-fold serial dilution) in buffer (provided by Biacore) 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 were observed for 10 minutes. Association 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 values and are summarized in Table 2 below.

[0164] All mouse-derived antibodies in this application specifically bind to human and monkey CTLA4 with comparable or higher binding affinities compared to the control. Antibodies C1D1, C1G4, D1D5, D1H4, D1B8, and D1A7 exhibit the highest binding affinities to human CTLA4.

[0165] Example 3. CTLA4 Binding Activity of Mouse-Derived CTLA4 Antibodies

[0166] The binding activity of the mouse-derived CTLA4 antibody of the present application to CTLA4 was determined by capture ELISA.

[0167] Briefly, 96-well plates were coated with 2 μg / ml of F(ab′)2 fragment-specific affinity-purified goat anti-mouse IgG (Jackson Immuno Research, Cat#115-005-072) in PBS, with 100 μl per well incubated overnight at 4°C. The plates were washed once with wash buffer (PBS + 0.05% v / v Tween-20, PBST) and then blocked with 200 μl / well of blocking buffer (PBST containing 5% w / v skim milk powder) at 37°C for 2 hours. The plates were washed four times and incubated with 100 μl / well of serially diluted mouse CTLA4 antibodies of the present invention, control standards, or negative control hIgG (human immunoglobulin for intravenous injection (pH 4, Hualan Biotechnology Co., Ltd.) (5-fold dilutions in PBST containing 2.5% w / v skim milk powder, starting at a concentration of 10,000 ng / ml) at 37°C for 40 minutes, followed by four additional washes. 100 μl / well of biotinylated human CTLA4-Fc protein (Acrobiosystems, Cat#CT4-H5255, 26 ng / ml in PBST containing 2.5% skim milk powder) was added to the plate containing the captured CTLA4 antibody, incubated at 37°C for 40 minutes, washed four times, and incubated with streptavidin-conjugated HRP (1:10,000 diluted in PBST, Jackson Immuno Research, Cat#016-030-084, 100 μl / well) at 37°C for 40 minutes. After the final wash, the plate was incubated with 100 μl / well of the substrate TMB (Innoreagents, Cat#TMB-S-002). After 15 minutes at room temperature, the reaction was terminated with 50 μl / well 1M H2SO4. The absorbance of each well was read in a microplate reader using dual wavelength mode, 450 nm for TMB and 630 nm as a reference wavelength. OD (450-630) values were plotted against antibody concentration. Data were analyzed using Graphpad Prism software to determine EC values. 50 The result is as follows Figure 1A and 1B shown.

[0168] from Figures 1A-1B It can be seen that all mouse-derived CTLA4 antibodies in the present application, except D1B8, specifically bind to human CTLA4 with high binding affinity.

[0169] Example 4. Blocking Activity of Mouse-derived CTLA4 Antibodies against Reference Material and CTLA4-CD80 / 86

[0170] 4.1 Control Benchmark Blocking ELISA

[0171] The ability of the present CTLA4 antibodies to block binding of a control standard to human CTLA4 was measured in a competitive ELISA assay. Briefly, a 96-well microplate was coated with 1.0 μg / mL of the control standard in PBS, 100 μl / well, and incubated at 37°C for 2 hours. The plate was washed once with wash buffer, blocked with 200 μl of PBST containing 5% w / v nonfat dry milk, incubated at 37°C for 2 hours, and washed four times.

[0172] The CTLA4 antibody or control of the present application was diluted with biotinylated human CTLA4-Fc (Acro biosystems, Cat#CT4-H5255, 65 ng / ml in PBST containing 2.5% w / v skim milk powder) at a starting concentration of 133.33 nM. A three-fold serial dilution was performed and incubated at room temperature for 40 minutes. The antibody / human CTLA4-Fc mixture was then added to the control standard-coated plate at 100 μl / well. After incubation at 37°C for 40 minutes, the plate was washed again four times with wash buffer. 100 μl / well of streptavidin-conjugated HRP (1:10,000 dilution in PBST, Jackson Immuno Research, Cat#016-030-084, 100 μl / well) was added to the plate and incubated at 37°C for 40 minutes. Finally, the plate was washed with wash buffer. Finally, TMB was added and the reaction was terminated with 1M H2SO4. The absorbance of each well was read in a microplate reader using dual wavelength mode, 450nm for TMB and 630nm as a reference wavelength. OD (450-630) values were then plotted against antibody concentration. The data were analyzed using Graphpad Prism software to obtain IC 50 value.

[0173] 4.2 Cell-based ligand blocking FACS

[0174] The Daudi cell line expressing human CD80 and human CD86 was used. CCL-213), and the activity of the CTLA4 antibody of the present application in blocking the binding of human CTLA4-Fc protein to cell surface CD80 / CD86 was evaluated by flow cytometry (FACS).

[0175] The CTLA4 antibody of the present application, the control standard, or the negative control hIgG (human immunoglobulin for intravenous injection (pH 4), Hualan Biotechnology Co., Ltd.) was diluted with human CTLA4-Fc solution (Acro biosystems, Cat# CT4-H5255, 1 μg / mL in FACS buffer) at a starting concentration of 33.33 nM, and then diluted 2-fold in series. The cells were incubated at room temperature for 30 minutes. Daudi cells in the logarithmic growth phase were collected from the cell culture flask, washed twice, and resuspended in PBS containing 2% v / v fetal bovine serum (FACS buffer). Daudi cells were plated in a 96-well plate at 1×10 cells per well. 5 Cells were incubated with 100 μl / well antibody / CTLA4-Fc mixture at 4°C for 40 minutes. The plate was washed twice with FACS buffer, and then 100 μl / well R-phycoerythrin affinity purified Fcγ fragment-specific goat anti-human IgG (1:1000 dilution in FACS buffer, Jackson Immunoresearch, Cat#109-115-098) was added and incubated at 4°C in the dark for 40 minutes. The cells were washed twice and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS device. Data were analyzed using Graphpad Prism software to obtain IC a0 value.

[0176] The results are as follows Figures 2A-2B and 3A-3B.

[0177] from Figures 2A-2B As can be seen, most of the antibodies in the present application were able to block binding of human CTLA4 to the control standard, indicating that these antibodies in the present application bind to the same or similar epitopes as the control standard. Antibodies D2A4, D1B8, and D1H4 were unable to block binding of human CTLA4 to the control standard, indicating that D2A4, D1B8, and D1H4 may bind to different epitopes.

[0178] Figures 3A-3B The results show that most of the antibodies of the present application can block the binding of CTLA4 to cell surface CD80 / CD86 with an activity comparable to or higher than that of the control benchmark.

[0179] Example 5. Cell-based functional assay of mouse CTLA4 antibodies

[0180] The CTLA4 antibody of the present application was tested for its activity in promoting T cell response.

[0181] Briefly, 4×10 WT cells were cultured in 20 μL of RPMI1640 medium (Gibco, Cat#11875-093) supplemented with 10% FBS (Gibco, Cat#10099-141) and 5 μg / mL PHA (Sigma, Cat#L1668-5M). 4 GS-J1 cells (immortalized human T lymphocytes expressing CD28, GenScript, Cat#M00611) were plated in each well of a 384-well plate (Corning, Cat#3707). Human CTLA4-Fc (Acro biosystems, Cat#CT4-H5255) was diluted to 8 μg / mL in RPMI1640 medium supplemented with 10% FBS, and 20 μL of CTLA4-Fc was added to each well of the plate. 2×10 WT cells in 20 μL of RPMI1640 medium supplemented with 10% FBS were then added to each well of the plate. 4 GS-C1 / CD80 cells (immortalized antigen-presenting cells expressing cell surface CD80, GenScript, Cat#M00614) were then added with 20 μL of serially diluted CTLA4 antibody (starting concentration 333.33 nM, 2.5-fold serial dilution) in RPMI1640 medium supplemented with 10% FBS. The plate was placed in a 5% CO2 incubator at 37°C for 24 hours. The plate was centrifuged and the IL-2 level in the supernatant of a 384-well low-capacity microplate (Greiner, Cat#784075) was tested using a human IL-2 HTRF kit (Cisbio, Cat#62HIL02PEG). Data were analyzed using Graphpad Prism software to obtain EC values. 50 value.

[0182] The test results are as follows Figure 4 shown.

[0183] It can be seen that all antibodies in this application can promote T cell response. Compared with the control benchmark, EC 50 Slightly higher, but similar maximum IL2 release levels.

[0184] Example 6. Preparation and characterization of chimeric antibodies

[0185] The heavy and light chain variable domains of mouse CTLA4 mAbs were sequenced, and the sequence ID codes are summarized in Table 1.

[0186] The heavy and light chain variable domains of the mouse CTLA4 mAbs C1G4, D1B6, C1D1, D1D5, and D1B8 were cloned in frame into the human IgG4 heavy chain (SEQ ID NO.: 78) and human kappa light chain constant region (SEQ ID NO.: 79), respectively, with the C-termini of the variable regions linked to the N-termini of the respective constant regions.

[0187] A vector containing nucleotides encoding the heavy chain variable region linked to the human IgG4 heavy chain constant region (SEQ ID NO: 78) and a vector containing nucleotides encoding the light chain variable region linked to the human kappa light chain constant region (SEQ ID NO: 79) were transiently transfected into 200 ml of 293F suspension cell culture containing 1 mg / mL PEI at a light / heavy chain construct ratio of 1.1:1.

[0188] After 6 days in the shake flask, the cell supernatant containing the chimeric antibody was collected and the chimeric antibody was purified from the cell supernatant. Purified chimeric antibodies were tested in capture ELISA, Octet affinity test, and cell-based ligand blocking FACS according to the experimental protocol of the above example (with or without modifications) and the experimental protocol described below.

[0189] The binding affinity and binding kinetics of the purified CTLA4 chimeric antibody were characterized by the Octet system (Fortebio, Octet RED 96). Briefly, the AHC biosensor (anti-human IgG Fc capture biosensor, ForteBio) was pre-soaked with 10 mM glycine (pH 1.5) for 3 seconds and then immersed in a well containing flow buffer (PBST containing 0.5% w / v BSA) for 3 seconds. The soaking and immersion steps were repeated three times. The sensor was then immersed in HBS-EP containing 5 μg / mL chimeric CTLA4 antibody. + Solution or HBS-EP containing 5 μg / mL reference standard + The sensor was immersed in a well containing a flow buffer solution for 100 seconds and then immersed in a well containing a flow buffer for 5 minutes. In another well containing a flow buffer for 180 seconds, a new baseline was run. The sensor was then immersed in a well containing a flow buffer containing gradient dilutions of human CTLA4-his protein (Acro biosystems, Cat#CT4-H5229, starting concentration 80nM, 2-fold gradient dilution) for 100 seconds and then immersed in the baseline well for 10 minutes. Finally, the sensor was pre-soaked in 10mM glycine (pH 1.5) for 3 seconds and then immersed in a well containing a flow buffer for 3 seconds. The soaking and immersion steps were repeated three times. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio data analysis 8.1. Determine K a , Kd and K D The values are summarized in Table 3 below.

[0190] For capture ELISA, affinity purified Fc γ Fragment-specific goat anti-human IgG (Jackson ImmunoResearch, Cat# 109-005-008) was substituted for affinity-purified F(ab')2 fragment-specific goat anti-mouse IgG, 100 μl / well.

[0191] The results are shown in Table 3 and Figures 5A-5E and Figures 6A-6B shown.

[0192] As shown in Table 3, the chimeric CTLA4 antibodies C1G4, C1D1, and D1D5 specifically bound to human CTLA4 with higher binding affinities than the control benchmark.

[0193] like Figures 5A-5E and Figures 6A-6B As shown in Figure 2, the chimeric CTLA4 antibodies have similar binding capacity and ligand blocking activity to their parental mouse mAbs. In cell-based ligand blocking FACS, the chimeric CTLA4 antibodies D1B6, C1D1, and D1D5 have better binding capacity and ligand blocking activity than their parental mouse mAbs. Higher blocking activity.

[0194] Table 3. Binding affinity of chimeric antibodies to human CTLA4

[0195]

[0196] *D1B8 not tested

[0197] Example 7. Humanization of mouse-derived CTLA4 monoclonal antibodies C1D1 and D1D5

[0198] The mouse-derived CTLA4 antibodies C1D1 and D1D5 were selected for humanization and further study. Humanization of the mouse-derived antibodies was performed using the established CDR grafting technique, described in detail below.

[0199] To select suitable acceptor frameworks for the humanization of the mouse antibodies C1D1 and D1D5, the light and heavy chain variable region sequences of each mouse antibody were aligned against the human immunoglobulin gene database. The human germline with the highest homology was selected as the humanized acceptor framework. The mouse antibody heavy and light chain variable region CDRs were inserted into the selected frameworks, and further backmutations were performed on residues within the frameworks to obtain a larger number of candidate heavy and light chain variable regions. A total of 12 humanized C1D1 antibodies, huC1D1-V1 to huC1D1-V12, and 12 humanized D1D5 antibodies, huD1D5-V1 to huD1D5-V12, were obtained. The heavy and light chain variable region sequence ID numbers are shown in Table 1.

[0200] A vector containing nucleotides encoding the humanized heavy chain variable region linked to the human IgG4 heavy chain constant region (SEQ ID NO: 78) and a vector containing nucleotides encoding the humanized light chain variable region linked to the human kappa light chain constant region (SEQ ID NO: 79) were transiently transfected into 200 ml of 293F suspension cell culture containing 1 mg / mL PEI at a 1.1:1 light / heavy chain construct ratio.

[0201] Example 8. Characterization of humanized antibodies

[0202] Table 4. Binding affinity of humanized C1D1 mAbs

[0203]

[0204] Table 5. Binding affinity of humanized D1D5 mAbs

[0205]

[0206] After 6 days in the shake flask, the cell supernatant containing humanized antibody was collected and then assayed by Octet according to the above experimental protocol. + The binding affinity of the chimeric antibody and the control standard to human CTLA4 was obtained. a , K d and K D The values are summarized in Tables 4 and 5 below.

[0207] The data showed that all cell supernatants containing the humanized C1D1 antibody exhibited higher human CTLA4 binding affinity than the benchmark control, and cell supernatants containing huCTLA4 D1D5-V1 to huCTLA4 D1D5-V3 exhibited higher human CTLA4 binding affinity than the control benchmark.

[0208] Humanized antibodies huC1D1-V8 and huD1D5-V9 were purified as described above and tested in Biacore, capture ELISA, control standard blocking ELISA, cell-based ligand blocking FACS, and cell-based T cell response promotion assays according to the protocol described in the previous example (with minor modifications). In the capture ELISA assay, 2 μg / ml goat anti-human IgG (affinity purified goat anti-human IgG, F(ab')2 fragment specific, Jackson Immunoresearch, Cat#109-005-097) was used instead of goat anti-mouse IgG F(ab')2 fragment to coat 96-well microplates at 100 μl / well. In the Biacore assay, goat anti-human IgG (GE healthcare, Cat#BR100839, Human Antibody Capture Kit) was covalently linked to a CM5 chip instead of goat anti-mouse IgG, and the CM5 chip was used for the control standard instead of the Protein G chip.

[0209] Using GloMelt TM Purified antibodies were tested in a thermal stability assay to determine the Tm (melting temperature) using the Thermal Shift Protein Stabilization Kit (Biotium, Cat# 33022-T). TM The dye was melted to room temperature. The vial containing the dye was vortexed and centrifuged. Then, 10x dye was prepared by adding 5 μL 200x dye to 95 μL PBS. 2 μL 10x dye and 10 μg humanized antibody were added, and PBS was then 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) and set to the melting curve program with the parameters of Table 6.

[0210] Table 6. Melting curve program parameters

[0211] Approximate steps temperature Heating rate Keep time Initial Hold 25℃ NA 30s Melting curve 25-99℃ 0.1℃ / s NA

[0212] The results are shown in Table 7 and Figures 7A-7B 、 Figures 8A-8B 、 Figures 9A-9B 、 Figure 10 and Figures 11A-11B shown.

[0213] Table 7. Binding affinity of humanized antibodies huC1D1-V8 and huD1D5-V9

[0214]

[0215] As can be seen in Table 7, antibody huC1D1-V8 exhibited lower binding affinity for human CTLA4 and monkey CTLA4 than the parental antibody or the benchmark control. Figure 9A The results showed that the antibody effectively blocked the binding of CTLA4 to cell surface CD80 / CD86, and the blocking activity was comparable to that of the control benchmark.

[0216] As can be seen from Table 7, antibody huD1D5-V9 has comparable binding affinities to human CTLA4 and monkey CTLA4. Figure 9B The results showed that the antibody effectively blocked the binding of CTLA4 to cell surface CD80 / CD86 with a blocking ability slightly higher than that of the benchmark control.

[0217] according to Figures 8A-8B The humanized antibodies huC1D1-V8 and huD1D5-V9 of the present application can block human CTLA4-BM binding, indicating that the antibodies huC1D1-V8 and huD1D5-V9 of the present application are likely to bind to an epitope similar to BM.

[0218] like Figure 10 As shown, the humanized antibodies huC1D1-V8 and huD1D5-V9 of the present application have the activity of promoting T cell response comparable to BM.

[0219] Although the present application has been described above in conjunction with one or more embodiments, it should be understood that the application is not limited to those embodiments, and the description is intended to cover all alternatives, modifications and equivalents included within the spirit and scope of the appended claims. All references cited herein are incorporated by reference in their entirety.

[0220] The sequences in this application are summarized below.

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] ***

[0232] Although specific preferred embodiments of the present application have been described, it should be understood that the invention defined by the foregoing paragraphs is not limited to the specific details set forth in the foregoing description, and that many obvious modifications can be made without departing from the spirit and scope of the invention. Sequence Listing <110> BioScience Biotechnology (Nanjing) Co., Ltd. <120> Antibodies that bind to CTLA4 and their uses <130> 55532 00029 <150> US 63 / 008,931 <151> 2020-04-13 <160> 93 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Mouse, chimeric, and humanized C1D1, and VH CDR1 of mouse and chimeric C1G4 <400> 1 Asp Asn Trp Met Asn 1 5 <210> 2 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Mouse, chimeric, and humanized C1D1, and VH CDR2 of mouse and chimeric C1G4 <400> 2 Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp Ser 1 5 10 15 Val Lys Gly <210> 3 <211> 4 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 of mouse, chimeric, and humanized C1D1 <400> 3 Gly Met Asp Tyr 1 <210> 4 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 of mouse, chimeric, and humanized C1D1 <400> 4 Gly Ala Ser Glu Ile Ile Tyr Gly Ala Leu Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Mouse, chimeric, and humanized C1D1, and VL CDR2 of mouse and chimeric C1G4 <400> 5 Gly Ala Thr Asn Leu Ala Asp 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 of mouse, chimeric, and humanized C1D1 <400> 6 Gln Lys Ile Leu Ser Pro Pro Pro Trp Thr 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 of mouse, chimeric, and humanized D1D5 <400> 7 Asp Tyr Gly Met Ala 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 of mouse, chimeric, and humanized D1D5 <400> 8 Phe Ile Ser Asn Leu Ala Tyr Ser Val Tyr Tyr Ala Asp Thr Glu Thr 1 5 10 15 Gly <210> 9 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 of mouse, chimeric, and humanized D1D5 <400> 9 Ser Gly Leu Pro Tyr Ala Met Asp Tyr 1 5 <210> 10 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 of mouse, chimeric, and humanized D1D5 <400> 10 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 of mouse, chimeric, and humanized D1D5 <400> 11 Tyr Ile Ser Arg Leu His Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 of mouse, chimeric, and humanized D1D5 <400> 12 Gln Gln Gly Arg Met Leu Pro Trp Thr 1 5 <210> 13 <211> 4 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 of mouse C1G4 <400> 13 Gly Phe Thr Tyr 1 <210> 14 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 of mouse C1G4 <400> 14 Gly Ala Ser Glu Asn Ile Tyr Gly Gly Leu Asn 1 5 10 <210> 15 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 of mouse C1G4 <400> 15 Gln Asn Val Leu Asn Thr Pro Tyr Thr 1 5 <210> 16 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 of mouse D1A7 <400> 16 Ser Ser Lys Leu Gly Val Gly 1 5 <210> 17 <211> 16 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 of mouse D1A7 <400> 17 His Ile Trp Trp Asn Asp Asp Asn Tyr Tyr Val Pro Ser Leu Lys Ser 1 5 10 15 <210> 18 <211> 12 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 of mouse D1A7 <400> 18 Val Pro Tyr Tyr Thr Thr Gln Pro Trp Phe Ala Tyr 1 5 10 <210> 19 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 of mouse D1A7 <400> 19 Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala 1 5 10 <210> 20 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 of mouse D1A7 <400> 20 Asn Thr Glu Thr Leu Ala Asp 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 of mouse D1A7 <400> twenty one Gln His Leu Trp Ser Thr Pro Trp Thr 1 5 <210> twenty two <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 of mouse D1B6 and mouse D1G6 <400> twenty two Thr Tyr Val Leu Asn 1 5 <210> twenty three <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 of mouse D1B6 <400> twenty three Tyr Phe Asn Pro Tyr Asn Asp Gly Ile Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> twenty four <211> 10 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 of mouse D1B6 and mouse D1G6 <400> twenty four Phe Glu Gly Gly Gly Tyr Ala Met Asp Tyr 1 5 10 <210> 25 <211> 15 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 of mouse D1B6 <400> 25 Arg Ala Ser Gln Ser Val Gly Thr Ser Arg Asn Thr Tyr Ile His 1 5 10 15 <210> 26 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 of mouse D1B6 and mouse D1G6 <400> 26 Tyr Ala Ser Asp Leu Glu Ser 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 of mouse D1B6 and mouse D1G6 <400> 27 Gln His Ser Trp Glu Ile Pro Tyr Thr 1 5 <210> 28 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 of mouse D1B8 <400> 28 Asp Tyr Tyr Ile Asn 1 5 <210> 29 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 of mouse D1B8 <400> 29 Trp Ile Tyr Pro Gly Asn Asn Asn Thr Arg Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 30 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 of mouse D1B8 <400> 30 Tyr Tyr Phe Asp Tyr 1 5 <210> 31 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 of mouse D1B8 <400> 31 Arg Ala Ser Gln Glu Ile Thr Gly Tyr Leu Ser 1 5 10 <210> 32 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 of mouse D1B8 <400> 32 Ala Ala Ser Thr Leu Asp Ser 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 of mouse D1B8 <400> 33 Leu Gln Tyr Ala Ser Tyr Pro Arg Thr 1 5 <210> 34 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 of mouse D1G6 <400> 34 Tyr Phe Asn Pro Tyr Asn Asp Ala Ile Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 35 <211> 15 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 of mouse D1G6 <400> 35 Arg Ala Ser Gln Ser Val Gly Thr Ser Ser Ser Tyr Ser Tyr Ile His 1 5 10 15 <210> 36 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 of mouse D1H3 <400> 36 Ser Tyr Trp Leu His 1 5 <210> 37 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 of mouse D1H3 <400> 37 Arg Ile Asp Pro Asn Arg Gly Thr Ile Tyr Tyr Asn Glu Lys Phe Asn 1 5 10 15 Asn <210> 38 <211> 10 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 of mouse D1H3 <400> 38 Gly Gly Ser Asn Phe Tyr Ala Met Asp Tyr 1 5 10 <210> 39 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 of mouse D1H3 <400> 39 Arg Thr Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 40 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 of mouse D1H3 <400> 40 Asn Ala Lys Thr Leu Ala Glu 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 of mouse D1H3 <400> 41 Gln Asn His Asp Gly Ile Pro Phe Thr 1 5 <210> 42 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 of mouse D1H4 <400> 42 Asp Tyr Tyr Met Asn 1 5 <210> 43 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 of mouse D1H4 <400> 43 Ala Ile Asn Pro Asp His Gly Gly Ser Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 44 <211> 10 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 of mouse D1H4 <400> 44 Asp Gly Ser Ile His Tyr Val Met Asp Asp 1 5 10 <210> 45 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 of mouse D1H4 <400> 45 Lys Ser Ser Gln Ser Leu Leu His Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 46 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 of mouse D1H4 <400> 46 Gly Ala Ser Thr Arg Glu Ser 1 5 <210> 47 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 of mouse D1H4 <400> 47 Gln Asn Asp Tyr Gly Tyr Pro Tyr Thr 1 5 <210> 48 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 of mouse D2A4 <400> 48 Ser Tyr Trp Val His 1 5 <210> 49 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 of mouse D2A4 <400> 49 Arg Ile Asp Pro Asn Arg Gly Gly Thr Tyr Tyr Asn Glu Asn Phe Lys 1 5 10 15 Thr <210> 50 <211> 10 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 of mouse D2A4 <400> 50 Gly Gly Leu Asn Tyr Tyr Ala Leu Asp Tyr 1 5 10 <210> 51 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VL CDR1 of mouse D2A4 <400> 51 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 52 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR2 of mouse D2A4 <400> 52 Asn Ala Asn Thr Leu Thr Glu 1 5 <210> 53 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 of mouse D2A4 <400> 53 Gln His His Tyr Gly Ile Pro Phe Thr 1 5 <210> 54 <211> 115 <212> PRT <213> Artificial sequence <220> <223> VH of mouse and chimeric C1D1 <400> 54 Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Pro Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Asn 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85 90 95 Tyr Cys Thr Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110 Val Ser Ser 115 <210> 55 <211> 115 <212> PRT <213> Artificial sequence <220> <223> VH of huC1D1-V1 - huC1D1-V12 <220> <221> Other features <222> (49)..(49) <223> Xaa can be Ala or Gly <220> <221> Other features <222> (81)..(81) <223> Xaa can be Ala or Val <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Asn 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Xaa Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Xaa Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Gly Gly Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 56 <211> 108 <212> PRT <213> Artificial sequence <220> <223> VL of mouse and chimeric C1D1 <400> 56 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Gly Ala Ser Glu Ile Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Met Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Gln Tyr Ser Leu Lys Ile Ser Ser Leu His Pro 65 70 75 80 Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Lys Ile Leu Ser Pro Pro Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 57 <211> 108 <212> PRT <213> Artificial sequence <220> <223> VL of huC1D1-V1 - huC1D1-V12 <220> <221> Other features <222> (43)..(43) <223> Xaa can be Ser or Val <220> <221> Other features <222> (58)..(58) <223> Xaa can be Met or Val <220> <221> Other features <222> (69)..(69) <223> Xaa can be Arg or Thr <220> <221> Other features <222> (71)..(71) <223> Xaa can be Tyr or Phe <400> 57 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Ile Ile Tyr Gly Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Xaa Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Xaa Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Xaa Asp Xaa Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Ile Leu Ser Pro Pro Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 58 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Mouse and chimeric D1D5 VH <400> 58 Glu Val Lys 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 Tyr 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ala Phe Ile Ser Asn Leu Ala Tyr Ser Val Tyr Tyr Ala Asp Thr Glu 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Glu Asp Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 59 <211> 118 <212> PRT <213> Artificial sequence <220> <223> VH of huD1D5-V1 - huD1D5-V12 <220> <221> Other features <222> (45)..(45) <223> Xaa can be Pro or Leu <220> <221> Other features <222> (49)..(49) <223> Xaa can be Ala or Ser <220> <221> Other features <222> (74)..(74) <223> Xaa can be Asp or Asn <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Xaa Glu Trp Val 35 40 45 Xaa Phe Ile Ser Asn Leu Ala Tyr Ser Val Tyr Tyr Ala Asp Thr Glu 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Xaa Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 60 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL of murine and chimeric D1D5 <400> 60 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Ile Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Arg Met Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 61 <211> 107 <212> PRT <213> Artificial sequence <220> <223> VL of huD1D5-V1 - huD1D5-V12 <220> <221> Other features <222> (43)..(43) <223> Xaa can be Thr or Val <220> <221> Other features <222> (44)..(44) <223> Xaa can be Val or Pro <220> <221> Other features <222> (87)..(87) <223> Xaa can be Phe or Tyr <400> 61 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Xaa Xaa Lys Leu Leu Ile 35 40 45 Tyr Tyr Ile Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Xaa Cys Gln Gln Gly Arg Met Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 62 <211> 115 <212> PRT <213> Artificial sequence <220> <223> VH of murine C1G4 <400> 62 Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Pro Ile Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Asn 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Thr Lys Asp Met Gly Ile Tyr 85 90 95 Tyr Cys Thr Gly Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 <210> 63 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Mouse C1G4 VL <400> 63 Asp Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Gly 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Met Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Gln Tyr Ser Leu Lys Ile Ser Arg Leu His Pro 65 70 75 80 Asp Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Asn Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 64 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Mouse D1A7 VH <400> 64 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Met Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Ser Ser 20 25 30 Lys Leu Gly Val Gly Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asn Asp Asp Asn Tyr Tyr Val Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Asn Val Asp Ala Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Val Gln Val Pro Tyr Tyr Thr Thr Thr Gln Pro Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 65 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Mouse D1A7 VL <400> 65 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 20 25 30 Leu Ala Trp Tyr Leu Gln Asn Gln Gly Arg Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Thr Glu Thr Leu Ala Asp Gly Val Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Phe Leu Lys Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Leu Trp Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 66 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH of murine D1B6 <400> 66 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Asn Thr Phe Thr Thr Tyr 20 25 30 Val Leu Asn Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Asn Pro Tyr Asn Asp Gly Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Glu Gly Gly Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 67 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL of Mouse-derived D1B6 <400> 67 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Gly Thr Ser 20 25 30 Arg Asn Thr Tyr Ile His Trp Tyr Gln Gln Lys Leu Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asp Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 68 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> VH of Mouse-derived D1B8 <400> 68 Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu 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 Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asn Asn Asn Thr Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ile Asp Thr Ser Ser Ser Thr Ala Phe 65 70 75 80 Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser <210> 69 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL of murine D1B8 <400> 69 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Ile Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Thr Gly Tyr 20 25 30 Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Gln Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 70 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH of Mouse-derived D1G6 <400> 70 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Asn Thr Phe Thr Thr Tyr 20 25 30 Val Leu Asn Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Asn Pro Tyr Asn Asp Ala Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Glu Gly Gly Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 71 <211> 111 <212> PRT <213> Synthetic Sequence <220> <223> VL of murine D1G6 <400> 71 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Gly Thr Ser 20 25 30 Ser Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asp Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 72 <211> 119 <212> PRT <213> Synthetic Sequence <220> <223> VH of murine D1H3 <400> 72 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Asn Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Leu His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Arg Gly Thr Ile Tyr Tyr Asn Glu Lys Phe 50 55 60 Asn Asn Lys Ala Thr Val Thr Val Asp Lys Pro Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Arg Leu Thr Leu Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ser Asn Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 73 <211> 107 <212> PRT <213> Artificial sequence <220> <223> VL of murine D1H3 <400> 73 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Glu Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln Asn His Asp Gly Ile Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 74 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH of murine D1H4 <400> 74 Glu Val Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asn Pro Asp His Gly Gly Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ser Ile His Tyr Val Met Asp Asp Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 75 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> VL of murine D1H4 <400> 75 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Thr Val Ser Ala Gly 1 5 10 15 Asp Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Trp Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 76 <211> 119 <212> PRT <213> Artificial sequence <220> <223> VH of murine D2A4 <400> 76 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ser 1 5 10 15 Ser Val Asn Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Val His Trp Val Lys Gln Gly Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Arg Gly Gly Thr Tyr Tyr Asn Glu Asn Phe 50 55 60 Lys Thr Lys Ala Ala Leu Thr Val Asp Ser Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Asn Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 77 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL of murine-derived D2A4 <400> 77 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Phe Ala Ser Leu Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Asn Thr Leu Thr Glu Gly Val Pro Ser Ser Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Thr Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His His Tyr Gly Ile Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 78 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Constant Region <400> 78 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> 79 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Constant Region <400> 79 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> 80 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Mouse and chimeric C1D1 VH <400> 80 gaggtgaagc tggatgagac tggaggaggc ttggtgcaac ctgggaggcc cctgaaactc 60 tcctgtgttg cctctggatt cacttttagt gacaactgga tgaactgggt ccgccagtct 120 ccagagaaag gactggagtg ggtagcacaa attagaaaca aaccttataa ttatgaaaca 180 tattattcag attctgtgaa aggcagattc accatctcaa gagatgattc caaaagtagt 240 gtctacctgc aaatgaacaa cttaagagtt gaagacatgg gtatctatta ctgtacaggc 300 gggatggact actggggtca aggaacctca gtcaccgtct cctca 345 <210> 81 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Mouse and chimeric C1D1 VH <400> 81 60. gaggtgaagc tggacgagac cggcggcggc ctggtgcaac caggaagacc cctgaagctg tcctgcgtgg ccagcggctt cacattcagc gacaattgga tgaactgggt gaggcagagc cctgagaagg gcctggagtg ggtggcccag atcagaaata agccttacaa ctacgagacc taktacagcg actccgtgaa gggcaggttc acaatcagca gagacgacag caagtccagc gtgtacctgc agatgaacaa tctgagagtg gaggacatgg gcatctacta ctgtaccggc ggcatggatt actggggcca gggcacaagc gtgaccgtgt ccagc 345 <210> 82 <211> 345 <212> DNA <213> The snowstorm <220> <223> huC1D1-V4, huC1D1-V8 and huC1D1-V12 VH <400> 82 60. gaggtgcagc tggtggagtc cggcggcgga ctggtgcagc ctggaagaag cctgagactg tcctgtaccg ccagcggctt caccttctcc gacaactgga tgaattgggt gaggcaggcc cctggcaagg gcctggagtg ggtgggacag atcagaaata agccctacaa ttacgagaca tactactccg attccgtgaa gggcagattc accatctcca gggatgatag caagagcatc gcctacctgc agatgaattc cctgaagacc gaggacaccg ccgtgtacta ctgtaccggc 300 ggcatggact actggggcca gggcaccaca gtgacagtgt ccagc 345 <210> 83 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Mouse and chimeric C1D1 VL <400> 83 gacattcaga tgactcagtc tccagcttca ctgtctgcat ctgtgggaga aactgtcacc 60 atcacatgtg gagcaagtga gattatttac ggtgctttaa attggtatca gcagaaacag 120 ggaaaatctc ctcagctcct gatctatggt gcaaccaact tggcagatgg catgtcatcg 180 aggttcagtg gcagtggatc tggtagacaa tattctctca agatcagtag cctgcatcct 240 gacgatgctg caacatatta ctgtcaaaaa atattaagtc ctcctccgtg gacgttcggt 300 ggaggcacca agctggagat caaa 324 <210> 84 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Mouse and chimeric C1D1 VL <400> 84 gacatccaga tgacacagag ccccgccagc ctgtccgcca gcgttggaga gaccgtgaca 60 atcacatgtg gcgcctccga gatcatctac ggcgccctga attggtatca acagaagcag 120 ggcaagagcc cccagctgct gatctacggc gctaccaatc tggccgacgg catgagctcc 180 aggttctccg gcagcggcag cggcaggcag tacagcctga agatctccag cctgcacccc 240 gacgacgccg ccacatacta ctgccagaag atcctgtccc cccctccttg gacattcggc 300 ggcggcacca agctggagat caag 324 <210> 85 <211> 324 <212> DNA <213> Artificial sequence <220> <223> VL of huC1D1-V5 - huC1D1-V8 <400> 85 gatatccaga tgacacagtc cccctcctcc ctgagcgcct ccgtgggaga cagagtgacc 60 atcacctgtg gcgcctccga gatcatctac ggcgccctga attggtatca acagaagccc 120 ggcaagagcc ccaagctgct gatctacggc gctacaaacc tggccgatgg cgtgccttcc 180 aggtttagcg gctccggctc cggcaccgac ttcaccctga ccatctcctc cctgcagccc 240 gaggatgtgg ccacatacta ctgtcagaag atcctgagcc cccccccttg gaccttcggc 300 ggaggaacaa aggtggagat caag 324 <210> 86 <211> 354 <212> DNA <213> Artificial sequence <220> <223> VH of mouse and chimeric D1D5 <400> 86 gaggtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactacggaa tggcgtgggt tcgacaggct 120 ccagggaagg ggcctgagtg ggtagcattc attagtaatt tggcatatag tgtctactat 180 gcagacactg agacgggccg attcaccatc tctagagagg atgccaagaa caccctgtac 240 ttggaaatga gcagtctgag gtctgaggac acagccatgt attactgtgc aagaagtgga 300 ctaccctatg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354 <210> 87 <211> 354 <212> DNA <人工智能序列 <220> <223> VH of mouse-derived and chimeric D1D5 <400> 87 gaggtgaagc tggtggagtc cggcggcggc ctggtgaagc caggaggaag cctgaagctg 60 tcctgcgccg cctccggctt cacattctcc gactacggca tggcctgggt gaggcaggcc 120 cctggaaagg gccctgagtg ggtggccttc atctccaatc tggcctacag cgtgtactac 180 gccgataccg agacaggcag gttcaccatc tccagagagg acgccaagaa tacactgtac 240 ctggagatga gcagcctgag atccgaggac acagccatgt actactgcgc caggagcggc 300 ctgccttacg ccatggatta ctggggccag ggcacaagcg tgaccgtgag ctcc 354 <210> 88 <211> 354 <212> DNA <213> Artificial sequence <220> <223> VH of huD1D5-V1, huD1D5-V5 and huD1D5-V9 <400> 88 gaggtgcagc tggtggagtc cggcggcgga ctggtgaagc ctggcggatc cctgaggctg 60 tcctgtgccg cctccggctt caccttctcc gactacggca tggcctgggt gaggcaggcc 120 cctggaaagg gccccgagtg ggtggctttc atctccaatc tggcctacag cgtgtactac 180 gccgatacag agacaggcag gttcacaatc agcagggatg acgccaagaa cagcctgtac 240 ctgcagatga actccctgag ggccgaggat accgccgtgt actactgtgc caggtccggc 300 ctgccctacg ccatggatta ctggggccag ggcacaacag tgacagtgag cagc 354 <210> 89 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Mouse-derived and chimeric D1D5 VL <400> 89 gatatccaga tgacacagac tacatcctcc ctgtctgcct cgctgggaga cagagtcacc 60 atcagttgca gggccagtca ggacattagc aattatttaa actggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctactac atatcaagat tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tgggacagat ttttctctca ccattagcaa cctggaacaa 240 gaagatattg ccacttacttttgccaacag ggtcgtatgc ttccgtggac gttcggtgga 300 ggcaccaggc tggaaatcaa a 321 <210> 90 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Mouse-derived and chimeric D1D5 VL <400> 90 gatatccaga tgacccagac aacaagcagc ctgagcgcct ccctgggcga tagagtgaca 60 atctcctgca gggccagcca ggatatcagc aactacctga attggtatca acagaagcct 120 gatggcaccg tgaagctgct gatctactac atctccagac tgcacagcgg cgtgcccagc 180 agattctccg gcagcggcag cggcaccgac ttctccctga ccatctccaa tctggagcag 240 gaggatatcg ccacatactt ctgccagcag ggcagaatgc tgccttggac attcggcggc 300 ggcaccagac tggagatcaa g 321 <210> 91 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL of huD1D5-V9 - huD1D5-V12 <400> 91 gacatccaga tgacacagtc ccctagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacatgta gagccagcca ggacatctcc aattacctga attggtatca acagaagccc 120 ggcaaggtgc ctaagctgct gatctactac atcagcaggc tgcactccgg cgtgccctcc 180 agattcagcg gcagcggctc cggcaccgat tttaccctga caatctccag cctgcagcct 240 gaggacgtgg ccacatacta ctgccagcag ggcaggatgc tgccttggac attcggcggc 300<x ggcacaaagg tggagatcaa g 321 <210> 92 <211> 984 <212> DNA <213> Artificial sequence <220> e<223> Heavy chain constant region <400> 92 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 ccagggaggac cctgaggtgc agttcaattg gtacgtggat 480 ggcgtggagg tgcacaatgc caacaaag cccagagagg agcagtttaa ttccacatac 540 agggtggtgt ccgtgctgac cgtgctgcac caggattggc tgaacggcaa ggatcaag 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> 93 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Light chain constant region <400> 93 cgtacggtgg cggcgccatc 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 ttga 324

Claims

1. An isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to cytotoxic T lymphocyte-associated antigen 4 (CTLA4), comprising: i) a heavy chain variable region comprising a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, and ii) a light chain variable region comprising a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, The amino acid sequences of the VH CDR1 region, VH CDR2 region, VH CDR3 region, VL CDR1 region, VL CDR2 region and VL CDR3 region are shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively.

2. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1 , wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 54 or 55, wherein amino acid residues 49 and 81 of SEQ ID NO: 55 are A and V; A and A; G and V; or G and A, respectively.

3. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1 , wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NOs: 56 or 57, wherein amino acid residues 43, 58, 69, and 71 of SEQ ID NO: 57 are S, M, R, and Y; S, V, T, and F; or V, V, T, and F, respectively.

4. The isolated monoclonal antibody or antigen-binding portion thereof of 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: 54 and 56; or SEQ ID NOs: 55 and 57, respectively. wherein the 49th and 81st amino acid residues of SEQ ID NO:55 are A and V, respectively, and the 43rd, 58th, 69th, and 71st amino acid residues of SEQ ID NO:57 are S, M, R, and Y, respectively; wherein the 49th and 81st amino acid residues of SEQ ID NO:55 are A and A, respectively, and the 43rd, 58th, 69th, and 71st amino acid residues of SEQ ID NO:57 are S, M, R, and Y, respectively; wherein the 49th and 81st amino acid residues of SEQ ID NO:55 are G and V, respectively, and the 43rd, 58th, 69th, and 71st amino acid residues of SEQ ID NO:57 are S, M, R, and Y, respectively; wherein the 49th and 81st amino acid residues of SEQ ID NO:55 are G and A, respectively, and the 43rd, 58th, 69th, and 71st amino acid residues of SEQ ID NO:57 are S, M, R, and Y, respectively; wherein the 49th and 81st amino acid residues of SEQ ID NO:55 are A and V, respectively, and the 43rd, 58th, 69th, and 71st amino acid residues of SEQ ID NO:57 are S, V, T, and F, respectively; wherein the 49th and 81st amino acid residues of SEQ ID NO:55 are A and A, respectively, and the 43rd, 58th, 69th, and 71st amino acid residues of SEQ ID NO:57 are S, V, T, and F, respectively; wherein the 49th and 81st amino acid residues of SEQ ID NO:55 are G and V, respectively, and the 43rd, 58th, 69th, and 71st amino acid residues of SEQ ID NO:57 are S, V, T, and F, respectively; wherein the 49th and 81st amino acid residues of SEQ ID NO:55 are G and A, respectively, and the 43rd, 58th, 69th, and 71st amino acid residues of SEQ ID NO:57 are S, V, T, and F, respectively; wherein the 49th and 81st amino acid residues of SEQ ID NO:55 are A and V, respectively, and the 43rd, 58th, 69th, and 71st amino acid residues of SEQ ID NO:57 are V, V, T, and F, respectively; wherein the 49th and 81st amino acid residues of SEQ ID NO:55 are A and A, respectively, and the 43rd, 58th, 69th, and 71st amino acid residues of SEQ ID NO:57 are V, V, T, and F, respectively; wherein amino acid residues 49 and 81 of SEQ ID NO:55 are G and V, respectively, and amino acid residues 43, 58, 69, and 71 of SEQ ID NO:57 are V, V, T, and F, respectively; or wherein the 49th and 81st amino acid residues of SEQ ID NO: 55 are G and A, respectively, and the 43rd, 58th, 69th and 71st amino acid residues of SEQ ID NO: 57 are V, V, T and F, respectively.

5. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO: 78 and is connected to the heavy chain variable region, and the light chain constant region has an amino acid sequence as shown in SEQ ID NO: 79 and is connected to the light chain variable region.

6. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, which (a) binds to human CTLA4; (b) binds to monkey CTLA4; (c) blocks CTLA4-CD80 / CD86 interaction; and / or (d) promotes T cell responses.

7. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1 which is mouse, chimeric or humanized.

8. The isolated monoclonal antibody or antigen-binding portion thereof of claim 1, which is of IgG1, IgG2 or IgG4 subtype.

9. A nucleotide encoding the isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 8. An expression vector comprising the nucleotide sequence of claim 9 . A host cell comprising the nucleotide sequence of claim 9 . A host cell comprising the expression vector according to claim 10 .

13. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 8, the expression vector according to claim 10, or the host cell according to claim 11 or 12, and a pharmaceutically acceptable carrier. 14 . A pharmaceutical composition comprising the nucleotide according to claim 9 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 13, further comprising an anti-tumor agent.

16. The pharmaceutical composition of claim 14, further comprising an anti-tumor agent.

17. Use of the pharmaceutical composition according to claim 15 or 16 in the preparation of a medicament for inhibiting tumor growth, wherein the tumor is melanoma, colorectal cancer, hepatocellular carcinoma, pleural mesothelioma, non-small cell lung cancer, or renal cell carcinoma.

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