Anti-CTLA4 monoclonal antibodies or their antigen-binding fragments, pharmaceutical compositions and uses

Abstract

This invention relates to monoclonal antibodies against CTLA4 or their antigen-binding fragments, pharmaceutical compositions, and uses. The invention belongs to the fields of tumor therapy and molecular immunology, providing monoclonal antibodies against CTLA4 or their antigen-binding fragments, pharmaceutical compositions thereof, and their uses. The monoclonal antibodies of this invention can block the binding of CTLA4 to B7, relieve the immunosuppression caused by CTLA4, and activate T lymphocytes.

Description

[0001] This application is a divisional application of Chinese Patent Application 201580040171.X, filed on July 31, 2015, entitled "Anti-CTLA4 Monoclonal Antibody or Antigen-Binding Fragment thereof, Pharmaceutical Composition and Use". Technical Field

[0002] This invention belongs to the fields of tumor therapy and molecular immunology, and relates to anti-CTLA4 monoclonal antibodies or antigen-binding fragments thereof, pharmaceutical compositions thereof, sequences encoding them, and methods and uses of them for diagnosis, prevention, treatment and / or adjuvant therapy. Background Technology

[0003] Cytotoxic T lymphocyte associated antigen-4 (CTLA4) and CD28 are very similar in gene structure, chromosomal location, sequence homology, and gene expression. Both are receptors for the co-stimulatory molecule B7 and are mainly expressed on the surface of activated T cells. However, as co-stimulatory signals for lymphocyte activation, CTLA4 and CD28 have opposite functions. After binding to B7, CTLA4 can inhibit the activation of mouse and human T cells, playing a negative regulatory role in T cell activation.

[0004] CTLA4 mAbs or CTLA4 ligands can block the binding of CTLA4 to its natural ligand, thereby blocking the transmission of negative regulatory signals by CTLA4 to T cells and enhancing the responsiveness of T cells to various antigens. In this respect, in vivo and in vitro studies have yielded largely consistent results. Currently, CTLA4 mAbs (10D1, 11.2.2) are in clinical trials for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, and malignant melanoma (Grosso JF., Jure-Kunkel MN., CTLA-4blockade in tumor models: an overview of preclinical and translational research. Cancer Immun. 2013; 13: 5. Epub 2013 Jan 22; US 6984720 B1 and US 6682736B1). Among them, 10D1 and 11.2.2 are considered to be among the most effective CTLA4 monoclonal antibodies to date.

[0005] Interleukin-2 (IL-2), produced by T cells, is a growth factor that regulates T cell subsets and is an important factor in regulating immune responses. It can also promote the proliferation of activated B cells and participate in antibody responses, hematopoiesis, and tumor surveillance. Recombinant human IL-2 has been approved by the US FDA for the treatment of malignant tumors (including melanoma and renal tumors) and is currently undergoing clinical trials for the treatment of chronic viral infections (Chavez, AR, et al., Pharmacologic administration of interleukin-2. Ann NY Acad Sci, 2009. 1182: pp. 14-27).

[0006] CTLA4 and CTLA4 mAb, as important influencing factors of T cell function, can exert specific therapeutic effects on diseases and achieve high efficacy by intervening in the body's immune microenvironment, supplementing the shortcomings of traditional drugs and thus opening up new avenues for gene therapy. CTLA4 and CTLA4 mAb are applied in various stages of experimental and clinical trials: for example, effectively inhibiting airway hyperresponsiveness in asthma animal models of autoimmune diseases, preventing the development of rheumatic diseases, and inducing immune tolerance in allogeneic transplantation. However, although no adverse reactions have been found in short-term clinical trials of gene therapy, we should also be aware of the potential impact of its long-term use. For example, excessive blocking of CTLA4-B7 signaling by CTLA4 mAb can lead to the occurrence of autoimmune diseases. Because antibodies can specifically bind to their ligands and cause target cell lysis or block pathological processes, the development and utilization of antibodies, especially human antibody drugs, are of great significance for the clinical treatment of human malignant tumors and other immune diseases.

[0007] Currently, there is a need to develop new antibodies and humanized antibodies that block the binding of CTLA4 to B7. Summary of the Invention

[0008] Through in-depth research and creative work, the inventors expressed recombinant CTLA4 using a mammalian cell expression system as an antigen to immunize mice. Hybridoma cells were obtained by fusing mouse spleen cells with myeloma cells. Through screening a large number of samples, the inventors obtained a hybridoma cell line capable of secreting a specific monoclonal antibody that binds specifically to CTLA4, and this monoclonal antibody effectively blocks the binding of CTLA4 to B7. Furthermore, a humanized antibody was prepared. Thus, the following invention is provided:

[0009] One aspect of the present invention relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein,

[0010] The monoclonal antibody includes a complementarity-determining region (CDR) selected from the following:

[0011] It contains HCDR1 with the amino acid sequence SEQ ID NO: 27.

[0012] Contains HCDR2 with the amino acid sequence SEQ ID NO: 28, and

[0013] It contains HCDR3 with the amino acid sequence SEQ ID NO: 29;

[0014] and / or

[0015] Contains LCDR1 with the amino acid sequence SEQ ID NO: 30.

[0016] LCDR2 containing the amino acid sequence SEQ ID NO: 31, and

[0017] LCDR3 contains amino acid sequences selected from SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34.

[0018] According to any one of the monoclonal antibodies or antigen-binding fragments thereof of the present invention, wherein,

[0019] The amino acid sequence of the heavy chain variable region (VH) of the monoclonal antibody is selected from SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14 and SEQ ID NO: 18;

[0020] and / or

[0021] The amino acid sequence of the light chain variable region (VL) of the monoclonal antibody is selected from SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24.

[0022] According to any one of the monoclonal antibodies or antigen-binding fragments thereof of the present invention, wherein,

[0023] The monoclonal antibody includes:

[0024] (1) VH as shown in SEQ ID NO: 6 and VL as shown in SEQ ID NO: 8;

[0025] (2) VH as shown in SEQ ID NO: 10 and VL as shown in SEQ ID NO: 12;

[0026] (3) VH as shown in SEQ ID NO: 14 and VL as shown in SEQ ID NO: 16;

[0027] (4) VH as shown in SEQ ID NO: 18 and VL as shown in SEQ ID NO: 20;

[0028] (5) VH as shown in SEQ ID NO: 14 and VL as shown in SEQ ID NO: 22; or

[0029] (6) VH as shown in SEQ ID NO: 14 and VL as shown in SEQ ID NO: 24.

[0030] In this invention, the monoclonal antibodies in groups (1)-(6) are the amino acid sequences of the heavy chain variable region and the light chain variable region of 8D2 / 8D2(Re), 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 and 8D2H2L17, respectively.

[0031] Specifically, the methionine (Met) at position 18 in SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14 is independently replaced by an amino acid selected from the following:

[0032] Leucine (Leu), valine (Val), isoleucine (Ile), or alanine (Ala).

[0033] Antibody therapies, particularly monoclonal antibodies (MABs), have shown promising efficacy in treating a variety of diseases. Traditional experimental methods for obtaining these therapeutic antibodies involve immunizing animals with antigens to obtain antibodies targeting the antigens, or using affinity maturation to improve antibodies with low affinity for antigens. However, these methods require significant time and effort, and often fail to target specific epitopes on the antigen.

[0034] The variable regions of the light and heavy chains determine antigen binding; each chain's variable region contains three hypervariable regions called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3; these were named by Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda Md).

[0035] Using techniques well known to those skilled in the art, such as analyzing the amino acid sequence of the CDR region of the monoclonal antibody sequences in items (1)-(6) above through the VBASE2 database, the results are as follows:

[0036] Item (1):

[0037] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0038] HCDR1: GTFFSDNW (SEQ ID NO: 27)

[0039] HCDR2:IRNKPYNYET (SEQ ID NO: 28)

[0040] HCDR3:TAQFAY (SEQ ID NO: 29)

[0041] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0042] LCDR1:ENIYGG (SEQ ID NO:30)

[0043] LC DR2: GAT (SEQ ID NO: 31)

[0044] LCDR3: QNVLRSPFT (SEQ ID NO: 32)

[0045] Item (2):

[0046] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0047] HCDR1: GTFFSDNW (SEQ ID NO: 27)

[0048] HCDR2:IRNKPYNYET (SEQ ID NO: 28)

[0049] HCDR3:TAQFAY (SEQ ID NO: 29)

[0050] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0051] LCDR1:ENIYGG (SEQ ID NO:30)

[0052] LCDR2: GAT (SEQ ID NO: 31)

[0053] LCDR3: QNVLRSPFT (SEQ ID NO: 32)

[0054] Item (3):

[0055] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0056] HCDR1: GTFFSDNW (SEQ ID NO: 27)

[0057] HCDR2:IRNKPYNYET (SEQ ID NO: 28)

[0058] HCDR3:TAQFAY (SEQ ID NO: 29)

[0059] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0060] LCDR1:ENIYGG (SEQ ID NO:30)

[0061] LCDR2: GAT (SEQ ID NO: 31)

[0062] LCDR3: QNVLRSPFT (SEQ ID NO: 32)

[0063] Item (4):

[0064] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0065] HCDR1: GTFFSDNW (SEQ ID NO: 27)

[0066] HCDR2:IRNKPYNYET (SEQ ID NO: 28)

[0067] HCDR3:TAQFAY (SEQ ID NO: 29)

[0068] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0069] LCDR1:ENIYGG (SEQ ID NO:30)

[0070] LCDR2: GAT (SEQ ID NO: 31)

[0071] LCDR3: QNVLRSPFT (SEQ ID NO: 32)

[0072] Item (5):

[0073] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0074] HCDR1: GTFFSDNW (SEQ ID NO: 27)

[0075] HCDR2:IRNKPYNYET (SEQ ID NO: 28)

[0076] HCDR3:TAQFAY (SEQ ID NO: 29)

[0077] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0078] LCDR1:ENIYGG (SEQ ID NO:30)

[0079] LCDR2: GAT (SEQ ID NO: 31)

[0080] LCDR3: QNVLSRHPG (SEQ ID NO: 33)

[0081] Item (6):

[0082] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0083] HCDR1: GTFFSDNW (SEQ ID NO: 27)

[0084] HCDR2:IRNKPYNYET (SEQ ID NO: 28)

[0085] HCDR3:TAQFAY (SEQ ID NO: 29)

[0086] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0087] LCDR1:ENIYGG (SEQ ID NO:30)

[0088] LCDR2: GAT (SEQ ID NO: 31)

[0089] LCDR3: QNVLSSRPG (SEQ ID NO: 34)

[0090] According to any one of the present inventions, the monoclonal antibody or its antigen-binding fragment is selected from Fab, Fab′, F(ab′)2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody (e.g., scFv), humanized antibody, chimeric antibody or biantibody.

[0091] According to any one of the monoclonal antibodies or antigen-binding fragments thereof of the present invention, wherein the monoclonal antibody is at a concentration of less than about 10 -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10-10 M or smaller K D It binds to the CTLA4 protein.

[0092] According to any one of the monoclonal antibodies or antigen-binding fragments thereof of the present invention, wherein,

[0093] The monoclonal antibody includes a non-CDR region, and the non-CDR region is derived from a species other than rodents, such as from human antibodies.

[0094] The monoclonal antibody or its antigen-binding fragment of the present invention is an anti-CTLA4 monoclonal antibody or its antigen-binding fragment, which can specifically bind to CTLA4.

[0095] The monoclonal antibody or its antigen-binding fragment according to any one of the present invention is used for the prevention and / or treatment and / or adjuvant therapy and / or diagnosis of tumors; specifically, the tumors are selected from melanoma, kidney tumors, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer and liver cancer.

[0096] The monoclonal antibody according to any one of the present invention is used for:

[0097] Blocking the binding of CTLA4 to B7,

[0098] Adjusting (e.g., downregulating) CTLA4 activity or CTLA4 levels,

[0099] To relieve the immunosuppression caused by CTLA4, or

[0100] Drugs that activate T lymphocytes or increase IL-2 expression in T lymphocytes.

[0101] Another aspect of the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences capable of encoding variable regions of antibody heavy chains, wherein,

[0102] The antibody heavy chain variable region contains a CDR with the amino acid sequence SEQ ID NO: 27-29;

[0103] Specifically, the antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 18;

[0104] More specifically, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17.

[0105] This invention also provides isolated nucleic acid molecules encoding the monoclonal antibody or its antigen-binding fragment of the present invention. Such nucleic acid molecules can be isolated from hybridoma cells or obtained using genetic engineering recombination techniques or chemical synthesis methods.

[0106] Another aspect of the invention relates to isolated nucleic acid molecules comprising a nucleic acid sequence capable of encoding a variable region of an antibody light chain, wherein,

[0107] The variable region of the antibody light chain includes:

[0108] 1) The amino acid sequence is the CDR of SEQ ID NO: 30-32;

[0109] 2) The amino acid sequences are CDRs of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33; or

[0110] 3) The amino acid sequences are CDRs of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 34;

[0111] Specifically, the variable region of the antibody light chain has the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 24;

[0112] More specifically, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23.

[0113] Another aspect of the invention relates to a vector comprising the isolated nucleic acid molecule as described in any one of the present invention. The vector of the present invention can be a cloning vector or an expression vector. In a preferred embodiment, the vector of the present invention is, for example, a plasmid, a phage, a cosmid, etc.

[0114] Another aspect of the invention relates to a host cell comprising the isolated nucleic acid molecules described in any one of the inventions, or the vectors of the invention. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The cells of the invention can also be cell lines, such as 293T cells.

[0115] Another aspect of the invention relates to a method for preparing the monoclonal antibody or antigen-binding fragment thereof as described in any one of the invention, comprising culturing the host cells of the invention under suitable conditions, and recovering the monoclonal antibody or antigen-binding fragment thereof from the cell culture.

[0116] Another aspect of the present invention relates to conjugates comprising a monoclonal antibody or an antigen-binding fragment thereof and a conjugation portion, wherein the monoclonal antibody is any one of the monoclonal antibodies or an antigen-binding fragment thereof described in this invention, and the conjugation portion is a detectable label; specifically, the conjugation portion is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme (e.g., horseradish peroxidase).

[0117] Another aspect of the present invention relates to a kit comprising any monoclonal antibody or antigen-binding fragment thereof as described in any one of the present invention, or comprising a conjugate of the present invention;

[0118] Specifically, the kit further includes a second antibody that specifically recognizes the monoclonal antibody or its antigen-binding fragment; optionally, the second antibody further includes a detectable label, such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme (e.g., horseradish peroxidase).

[0119] Another aspect of the invention relates to the use of any monoclonal antibody or antigen-binding fragment thereof described in any one of the invention in the preparation of a kit for detecting the presence or level of CTLA4 in a sample.

[0120] Another aspect of the invention relates to a pharmaceutical composition comprising any monoclonal antibody or antigen-binding fragment thereof as described in any one of the invention, or a conjugate thereof; optionally, it further comprises a pharmaceutically acceptable carrier and / or excipient.

[0121] Another aspect of the present invention relates to the use of any monoclonal antibody or antigen-binding fragment thereof, or conjugate thereof, as described in any one of the present invention in the preparation of medicaments for the prevention and / or treatment and / or adjuvant treatment and / or diagnosis of tumors; specifically, the tumors are selected from melanoma, renal tumors, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, and liver cancer.

[0122] Another aspect of the present invention relates to the use of any monoclonal antibody or antigen-binding fragment thereof described in any one of the present invention, or conjugates thereof, in the preparation of the following pharmaceutical products:

[0123] The drug is used to detect the presence or level of CTLA4 in the sample.

[0124] Drugs that block the binding of CTLA4 to B7,

[0125] Drugs that regulate (e.g., downregulate) CTLA4 activity or CTLA4 levels,

[0126] Drugs that relieve the immunosuppression caused by CTLA4.

[0127] Drugs that activate T lymphocytes, or

[0128] Drugs that increase IL-2 expression in T lymphocytes.

[0129] Another aspect of the invention relates to an in vivo or in vitro method comprising the step of applying cells in an effective amount of any monoclonal antibody or antigen-binding fragment thereof or conjugate of the invention, said method being selected from the following:

[0130] Methods for detecting the presence or level of CTLA4 in a sample.

[0131] Methods to block the binding of CTLA4 and B7

[0132] Methods for regulating (downregulating) CTLA4 activity or CTLA4 levels

[0133] Methods to relieve the immunosuppression caused by CTLA4

[0134] Methods to activate T lymphocytes, or

[0135] Methods to increase IL-2 expression in T lymphocytes.

[0136] The method can be used for diagnostic or therapeutic purposes, or for non-diagnostic or therapeutic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0137] Another aspect of the invention relates to a method for preventing and / or treating and / or adjuvant treating and / or diagnosing tumors, comprising the step of administering to a subject an effective amount of any monoclonal antibody or antigen-binding fragment thereof as described in any one of the inventions, or a monoclonal antibody conjugate of the invention; specifically, the tumor is selected from melanoma, kidney tumors, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, and liver cancer.

[0138] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0139] As used herein, when referring to the amino acid sequence of the CTLA4 protein (Cytotoxic T-Lymphocyte Antigen 4), it includes the full-length CTLA4 protein, or the extracellular fragment CTLA4ECD (SEQ ID NO: 2) or a fragment containing CTLA4ECD; it also includes fusion proteins of CTLA4ECD, such as fragments fused with a fragment of the Fc protein (mFc) of mouse IgG (SEQ ID NO: 3). However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the CTLA4 protein without affecting its biological function. Therefore, in this invention, the term "CTLA4 protein" should include all such sequences, including the sequence shown in SEQ ID NO: 2 and its natural or artificial variants. Furthermore, when describing a sequence fragment of the CTLA4 protein, it includes not only the sequence fragment of SEQ ID NO: 2 but also the corresponding sequence fragments in its natural or artificial variants.

[0140] As used herein, unless otherwise specified, B7 refers to B7-1 and / or B7-2; the specific protein sequences are known sequences in the prior art, and can be found in existing literature or sequences published in GenBank. For example, B7-1 (CD80, NCBI Gene ID: 941); B7-2 (CD86, NCBI Gene ID: 942).

[0141] As used in this article, the term EC 50 The half-maximal effect concentration refers to the concentration that produces a 50% maximum effect.

[0142] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and heavy chains also contain "D" regions of approximately 3 or more amino acids. Each heavy chain is further divided by a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H 1. C H 2 and C H 3) Composition. Each light chain consists of a light chain variable region (VL ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L Each amino acid is assigned to a specific region or domain to form an antibody binding site. The allocation of amino acids to these regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The term “antibody” is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0143] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some cases, antigen-binding fragments include Fab, Fab′, F(ab′)2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0144] As used in this article, the term "Fd fragment" refers to a segment composed of V H and C H Antibody fragments composed of I domains; the term "Fv fragment" refers to a fragment consisting of the V domain of a single arm of the antibody. L and V H Antibody fragments composed of structural domains; the term "dAb fragment" refers to an antibody fragment composed of V... H Antibody fragments composed of structural domains (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" refers to a fragment composed of V... L V H C L and C H An antibody fragment consisting of a 1-domain structure; the term "F(ab′)2 fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region.

[0145] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), where V L and V H The domain enables linker pairing to form monovalent molecules as single polypeptide chains (see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules can have a general structure: NH2-V L -Connector-V H -COOH or NH2-V H -Connector-V L-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

[0146] In some cases, the antigen-binding fragment of an antibody is a biantibody, i.e., a bivalent antibody, where V... H and V L The domain is expressed on a single polypeptide chain, but the linker is too short to allow pairing between two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).

[0147] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the monoclonal antibodies 4B3, 13A10, 12B9, or 4H4 provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0148] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0149] As used herein, the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are usually obtained using hybridoma techniques first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see USP 4,816,567).

[0150] As used herein, monoclonal antibodies referred to by number are identical to those obtained from hybridomas with the same number. For example, monoclonal antibody 4B3 (or 13A10, 12B9, or 4H4) are identical to antibodies obtained from hybridoma cell lines 4B3 (or 13A10, 12B9, or 4H4) or their subclones or progeny cells.

[0151] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain is derived from a portion of an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)).

[0152] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, see, for example, Jones et al., Nature, 321: 522 525 (1986); Reichmann et al., Nature, 332: 323 329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593 596 (1992); and Clark, Immunol. Today 21: 397 402 (2000).

[0153] As used in this article, "neutralizing antibody" refers to an antibody or antibody fragment that can eliminate or significantly reduce the virulence (e.g., the ability to infect cells) of a target virus.

[0154] As used herein, the term “epitope” refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. “Epitope” is also referred to in the art as an “antigenic determinant.” Epitopes or antigenic determinants typically consist of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. For example, epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation, which can be “linear” or “conformal.” See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformational epitope, points of interaction are separated by protein amino acid residues.

[0155] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0156] As used herein, the term "Escherichia coli expression system" refers to an expression system consisting of Escherichia coli (strain) and a vector, wherein the Escherichia coli (strain) is derived from commercially available strains, such as, but not limited to: GI698, ER2566, BL21(DE3), B834(DE3), BLR(DE3).

[0157] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0158] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0159] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit is occupied (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48: 444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0160] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the essential properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0161] As used in this article, the term "immunogenicity" refers to the ability of an antigen to stimulate the body to form specific antibodies or sensitized lymphocytes. It refers both to the property of an antigen to stimulate specific immune cells, causing them to activate, proliferate, and differentiate, ultimately producing immune effector substances such as antibodies and sensitized lymphocytes, and to the specific immune response of the body's immune system to form antibodies or sensitized T lymphocytes after antigen stimulation. Immunogenicity is the most important property of an antigen; whether an antigen can successfully induce an immune response in the host depends on three factors: the nature of the antigen, the host's reactivity, and the mode of immunization.

[0162] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10- 10 M or lower affinity (K) D () binds to the antigen.

[0163] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies (e.g., the monoclonal antibodies 4B3, 13A10, 12B9, or 4H4 of this invention) have a dissociation equilibrium constant of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D The antigen (e.g., L1 protein) is bound to the antigen, for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR).

[0164] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0165] As used herein, the terms “hybridoma” and “hybridoma cell line” are used interchangeably, and when referring to the terms “hybridoma” and “hybridoma cell line”, they also include subclones and progeny cells of the hybridoma. For example, when referring to hybridoma cell line 4B3, it also refers to subclones and progeny cells of hybridoma cell line 4B3.

[0166] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0167] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when administered to the body along with or before an antigen, can enhance the body's immune response to the antigen or alter the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvant is more frequently used in clinical trials.

[0168] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., diseases associated with CTLA4-B7 binding or excessive CTLA4 activity, such as cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., diseases associated with CTLA4-B7 binding or excessive CTLA4 activity, such as cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0169] Beneficial effects of the invention

[0170] The monoclonal antibody 8D2 and its humanized antibody of the present invention can bind specifically to CTLA4. Specifically, the binding efficiency of antibody 8D2 and 8D2(Re) to murine CTLA4 antigen is stronger than that of control antibodies 10DI (Alan J. Korman, Edward L. Halk, et al., HUMAN CTLA-4 ANTIBODIES, United States Patent No. US 6984720 B1) and 11.2.1 (Douglas Charles Hanson, Mark Joseph Neveu, et al., Human monoclonal antibodies to CTLA-4, United States Patent No. US 682736 B1). The binding efficiency of humanized antibody 8D2H1L1 to murine CTLA4 antigen is stronger than that of control antibody 10D1 and comparable to that of 11.2.1. The binding efficiency of humanized antibody 8D2H2L2 to human CTLA4 antigen is comparable to that of 10D1. The binding efficiencies of humanized antibodies 8D2H2L2 and 8D2H3L3 to monkey CTLA4 antigen were comparable to those of 10D1. Antibodies 8D2H2L15 and 8D2H2L17 showed stronger binding efficiencies to human CTLA4 antigen than control antibodies 10DI and 11.2.1.

[0171] Antibodies 8D2, 8D2(Re), and humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17 can competitively bind to antigen CTLA4 with B7-2. Among them, 8D2, 8D2(Re), 8D2H1L1, and 8D2H2L2 exhibit stronger competitive binding to CTLA4 with B7-2 than 10D1; 8D2H1L1, 8D2H2L2, and 8D2H3L3; and 8D2H2L15 and 8D2H2L17 exhibit stronger competitive binding to CTLA4 with B7-1 and B7-2 than antibodies 10D1 and 11.2.1.

[0172] The monoclonal antibody 8D2 and its humanized antibody of the present invention can effectively block the binding of CLTA4 and B7, specifically relieve the immunosuppression caused by CTLA4, and activate T lymphocytes. Among them, 8D2H2L2 and 8D2H2L15 are more potent against T lymphocytes than the control antibodies 10D1 and 11.2.1. Attached Figure Description

[0173] Figure 1SDS-PAGE results of CTLA4ECD-mFc fusion protein. The samples and loading volumes in the four lanes from left to right are as follows: M: marker 10 μL; CTLA4ECD-mFc fusion protein 1 μg; CTLA4ECD-mFc fusion protein 2 μg; CTLA4ECD-mFc fusion protein 3 μg.

[0174] Figure 2 SDS-PAGE results of 8D2 antibody. The samples and their loading volumes in the four lanes from left to right are as follows: M: marker 10 μL; reduced protein electrophoresis loading buffer sample antibody 0.3 μg; non-reduced protein electrophoresis loading buffer 2 μL; non-reduced protein electrophoresis loading buffer sample antibody 0.3 μg.

[0175] Figure 3 SDS-PAGE results of 8D2 recombinant antibody (8D2(Re)). The samples and their loading volumes in the four lanes from left to right are as follows: M: marker 10 μL; reduced protein electrophoresis loading buffer sample antibody 1 μg; non-reduced protein electrophoresis loading buffer 2 μL; non-reduced protein electrophoresis loading buffer sample antibody 1 μg.

[0176] Figure 4 SDS-PAGE results of the humanized antibody 8D2H1L1 of 8D2. The samples and their loading volumes in the four lanes from left to right are as follows: M: marker 10 μL; reduced protein electrophoresis loading buffer sample antibody 1 μg; non-reduced protein electrophoresis loading buffer 2 μL; non-reduced protein electrophoresis loading buffer sample antibody 1 μg.

[0177] Figure 5 SDS-PAGE results of the humanized antibody 8D2H2L2 (8D2H2L2). The samples and loading volumes for the four lanes from left to right are as follows: M: marker 10 μL; reduced protein electrophoresis loading buffer, sample antibody 1 μg; non-reduced protein electrophoresis loading buffer 2 μL; non-reduced protein electrophoresis loading buffer, sample antibody 1 μg.

[0178] Figure 6 SDS-PAGE results of the humanized antibody 8D2H3L3 of 8D2. The samples and their loading volumes in the four lanes from left to right are as follows: M: marker 10 μL; reduced protein electrophoresis loading buffer sample antibody 1 μg; non-reduced protein electrophoresis loading buffer 2 μL; non-reduced protein electrophoresis loading buffer sample antibody 1 μg.

[0179] Figure 7SDS-PAGE results of the humanized antibody 8D2H2L15 for 8D2. Samples and loading volumes were as follows: M: marker 10 μL; 1: non-reducing protein electrophoresis loading buffer, sample antibody 1 μg; 2: reduced protein electrophoresis loading buffer, sample antibody 1 μg.

[0180] Figure 8 SDS-PAGE results of the humanized antibody 8D2H2L17 for 8D2. Samples and loading volumes were as follows: M: marker 10 μL; 1: non-reducing protein electrophoresis loading buffer, sample antibody 1 μg; 2: reduced protein electrophoresis loading buffer, sample antibody 1 μg.

[0181] Figure 9: Detection results of kinetic characteristic parameters of monoclonal antibody 8D2.

[0182] Figure 10 : Detection results of dynamic characteristic parameters of 8D2H1L1.

[0183] Figure 11 : Detection results of dynamic characteristic parameters of 8D2H2L2.

[0184] Figure 12 : Detection results of dynamic characteristic parameters of 8D2H3L3.

[0185] Figure 13 : Detection results of dynamic characteristic parameters of 8D2H2L15.

[0186] Figure 14 Results of dynamic characteristic parameter detection for 8D2H2L17.

[0187] Figure 15 Flow cytometry analysis of CTLA4 expression histogram (cell number - fluorescence (FITC)) in 293F cells without labeling, isotype control and 293F-CTLA4 cells.

[0188] Figure 16 Flow cytometry was used to detect the mean fluorescence intensity (MFI) of CTLA4 expression in unlabeled 293F cells, isotype control, and 293F-CTLA4 cells.

[0189] Figure 17 ECMO of monoclonal antibody 8D2 with labeled 293F-CTLA4 cells 50 result.

[0190] Figure 18 ECMO cells bound to 8D2(Re) antibody and labeled 293F-CTLA4 cells 50 result.

[0191] Figure 19ECG binding of 8D2H1L1 to labeled 293F-CTLA4 cells 50 result.

[0192] Figure 20 ECG binding of 8D2H2L2 to labeled 293F-CTLA4 cells 50 result.

[0193] Figure 21 ECG binding of 8D2H3L3 to labeled 293F-CTLA4 cells 50 result.

[0194] Figure 22 The ELISA method was used to detect the binding of recombinant antibodies 8D2, 8D2H1L1, and 8D2 to CTLA4.

[0195] Figure 23 The ELISA method was used to detect the binding of recombinant antibodies 8D2H2L2 and 8D2H3L3 to human CTLA4.

[0196] Figure 24 The ELISA method was used to detect the binding of recombinant antibodies 8D2H2L2 and 8D2H3L3 to monkey CTLA4.

[0197] Figure 25 The ELISA method was used to detect the binding of recombinant antibodies 8D2H2L15 and 8D2H2L17 to monkey CTLA4.

[0198] Figure 26 The recombinant antibodies 8D2, 8D2H1L1, and 8D2 competed with B7-1 for ELISA results.

[0199] Figure 27 The recombinant antibodies 8D2, 8D2H1L1, and 8D2 competed with B7-2 for ELISA results.

[0200] Figure 28 The ELISA results showed that antibodies 8D2H2L2 and 8D2H3L3 competed with B7-1.

[0201] Figure 29 The 8D2H2L2 and 8D2H3L3 antibodies competed with B7-2 for ELISA results.

[0202] Figure 30 The ELISA results showed that antibodies 8D2H2L15 and 8D2H2L17 competed with B7-1.

[0203] Figure 31 Antibodies 8D2H2L15 and 8D2H2L17 compete with B7-2 for ELISA results.

[0204] Figure 32 The effects of co-culturing peripheral blood mononuclear cells (PBMCs), Raji cells, and humanized antibodies 8D2H1L1, 8D2H2L2, and 8D2H3L3 for 72 hours on T lymphocyte IL-2 secretion levels were detected using ELISA. The results showed that the humanized antibody 8D2 increased T lymphocyte IL-2 secretion by blocking the CTLA4 receptor.

[0205] Figure 33 The effects of co-culturing peripheral blood mononuclear cells (PBMCs), Raji cells, and humanized antibodies 8D2H2L15 and 8D2H2L17 for 72 hours on T lymphocyte IL-2 secretion levels were detected by ELISA. The results showed that the humanized antibody 8D2 increased T lymphocyte IL-2 secretion by blocking the CTLA4 receptor.

[0206] Figure 34 Growth curve of subcutaneous transplanted tumor in the hu-SCID-raji model: 8D2H2L2. Detailed Implementation

[0207] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased on the market.

[0208] In the following embodiments of the present invention, the BALB / C mice used were purchased from the Guangdong Provincial Medical Laboratory Animal Center.

[0209] In the following embodiments of the present invention, the T cells used are from Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0210] The control antibody 10D1 was prepared according to US Patent 6984720 B1; 11.2.1 was prepared according to US 6682736 B1.

[0211] Example 1: Obtaining the CTLA4-8D2 hybridoma cell line LT001 and preparing the monoclonal antibody 8D2.

[0212] Recombinant CTLA4 was expressed using a mammalian cell expression system and used as an antigen to immunize mice. Hybridoma cells were obtained by fusing mouse spleen cells with myeloma cells. Through extensive sample screening, a hybridoma cell line (CTLA4-8D2 hybridoma cell line LT001) was obtained, which can secrete a monoclonal antibody 8D2 that specifically binds to CTLA4. The specific methods are as follows:

[0213] 1. Synthesis of the gene CTLA4ECD-mFc:

[0214] The amino acid sequence corresponding to the extracellular fragment CTLA4ECD (Cytotoxic T-Lymphocyte Antigen 4, NCBIGene ID: 1493, SEQ ID NO: 1) of the gene CTLA4 (SEQ ID NO: 2) was fused with the Fc protein fragment (mFc) of mouse IgG (SEQ ID NO: 3). Here, mFc refers to the Fc protein fragment of mouse IgG, and its amino acid sequence is shown in the underlined part of SEQ ID NO: 3.

[0215] To improve the expression efficiency of the target gene in the 293f cell expression system, GenScript was commissioned to optimize the nucleic acid sequence corresponding to the protein sequence SEQ ID NO: 3. The optimization mainly considered factors such as codon bias, GC content, mRNA secondary structure, and repetitive sequences. The final optimized sequence of the CTLA4ECD-mFc fusion protein gene is shown below (SEQ ID NO: 4), and GenScript was commissioned to synthesize it.

[0216] The sequence of the gene CTLA4ECD: (375 bp)

[0217]

[0218]

[0219] The protein sequence encoded by CTLA4ECD: (125aa)

[0220]

[0221] CTLA4ECD-mFc fusion protein sequence: (364aa)

[0222] The part with a wavy underline is CTLA4ECD, and the part with a solid underline is mFc.

[0223]

[0224] The gene coding sequence corresponding to the CTLA4ECD-mFc fusion protein: (1092 bp)

[0225] The part with a wavy underline is CTLA4ECD, and the part with a solid underline is mFc.

[0226]

[0227] 2. Obtaining the pUC57simple-CTLA4ECD-mFc plasmid:

[0228] The synthesized CTLA4ECD-mFc fusion gene (SEQ ID NO: 4) was cloned into the pUC57simple expression vector (provided by GenScript) by GenScript to obtain the pUC57simple-CTLA4ECD-mFc plasmid.

[0229] 3. Construction of the pcDNA3.1-CTLA4ECD-mFc recombinant plasmid:

[0230] The plasmid pUC57simple-CTLA4ECD-mFc was digested with enzymes (XbaI and BamHI), and the fusion gene fragment CTLA4ECD-mFc obtained by electrophoresis was ligated into the pcDNA3.1 expression vector (purchased from Invitrogen) to obtain pcDNA3.1-CTLA4ECD-mFc. This was then transfected into competent E. coli cells DH5α (purchased from TIANGEN), and transfection and culture were performed according to the manufacturer's instructions. Positive pcDNA3.1-CTLA4ECD-mFc clones were screened, and E. coli were amplified using standard methods. The pcDNA3.1-CTLA4ECD-mFc recombinant plasmid was then extracted using a kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., DP103-03) according to the kit's instructions.

[0231] 4. Transfect 293F cells (purchased from Invitrogen) with the recombinant plasmid pcDNA3.1-CTLA4ECD-mFc according to the method of the lipofectamin transfection kit (purchased from Invitrogen).

[0232] 5. Seven days after transfecting 293F cells with the recombinant plasmid pcDNA3.1-CTLA4ECD-mFc, the culture medium was purified into the CTLA4ECD-mFc fusion protein by high-speed centrifugation, vacuum filtration through a microporous membrane, and HiTrap protein A HP column. The purified sample was then added to reduced protein electrophoresis loading buffer for SDS-PAGE electrophoresis detection. Figure 1 As shown, the target protein is located at approximately 45 kDa.

[0233] 6. Establishment of the CTLA4-8D2 hybridoma cell line LT001

[0234] Using CTLA4ECD-mFc fusion protein as an antigen, spleen cells from immunized BALB / c mice (purchased from Guangdong Medical Laboratory Animal Center) were fused with mouse myeloma cells to form hybridoma cells, following established methods (e.g., Stewart, SJ, "Monoclonal Antibody Production", in Basic Methods in antibody Production and Characterization, Eds. G. Howard and D.R. Thiell, Boca Raton: CRC Press, 2000).

[0235] Using CTLA4 as the antigen to coat ELISA plates, hybridoma cells secreting novel antibodies that specifically bind to CTLA4 were screened using indirect ELISA. The hybridoma cells obtained from indirect ELISA screening were then further screened using competitive ELISA to identify hybridoma cell lines that secrete monoclonal antibodies that competitively bind to CTLA4 with ligands B7-1 (CD80, NCBI Gene ID: 941) and B7-2 (CD86, NCBI Gene ID: 942). Stable hybridoma cell lines were obtained through limiting dilution. These hybridoma cell lines were named CTLA4-8D2 hybridoma cell lines, and stable CTLA4-8D2 cell lines (also referred to as LT001 cells in this invention, and the monoclonal antibody secreted by them is named 8D2) were obtained through limiting dilution.

[0236] 7. Preparation of antibody 8D2

[0237] The CTLA4-8D2 (LT001) cell line of this invention was cultured with 10% low IgG fetal bovine serum, and the cell culture supernatant was collected after 7 days for purification to prepare antibody 8D2.

[0238] 8. SDS-PAGE electrophoresis detection of antibody 8D2:

[0239] The purified samples were added to reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer, respectively, and then boiled before detection. The results showed that the target protein in the reduced protein samples was located at approximately 50 kDa and 25 kDa, while the target protein in the non-reduced protein samples was located at approximately 150 kDa. Figure 2 ).

[0240] Example 2: Obtaining the light and heavy chain sequences of monoclonal antibody 8D2

[0241] mRNA was extracted from the CTLA4-8D2 hybridoma cell line (LT001 cells) prepared in Example 1 according to the method of the cultured cell bacterial total RNA extraction kit (Tiangen, catalog number DP430).

[0242] According to Invitrogen III. First-Strand Synthesis System for RT-PCR kit: cDNA was synthesized and PCR amplification was performed. The PCR amplification product was directly cloned using TA (transgen CT101) kit, following the instructions. The TA cloned product was then sequenced. The sequencing results are as follows:

[0243] DNA sequencing results of the heavy chain variable region: (345bp)

[0244]

[0245]

[0246] Its encoded protein sequence is: (115aa)

[0247]

[0248] DNA sequencing results of the light chain variable region: (318bp)

[0249]

[0250] Its encoded protein sequence is: (106aa)

[0251]

[0252] Example 3: Light chains of humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 and 8D2H2L17 and Design of heavy chain sequences

[0253] Based on the three-dimensional crystal structure of the CTLA4 protein (Nat. Struct. Biol. (1997) 4p. 527) and the sequence of antibody 8D2 obtained in Example 2, variable region sequences (antibody constant region sequences, from the NCBI database) of antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17 were designed using a computer-simulated antibody model, based on the antibody sequences and structural models. The variable region sequences are as follows:

[0254] 1. Light and heavy chain sequences of monoclonal antibody 8D2H1L1

[0255] DNA sequence of the heavy chain variable region: (345 bp)

[0256]

[0257] Its encoded protein sequence is: (115aa)

[0258]

[0259] DNA sequence of the light chain variable region: (321 bp)

[0260]

[0261]

[0262] Its encoded protein sequence is: (107aa)

[0263]

[0264] Light and heavy chain sequences of the 2.8D2 humanized monoclonal antibody 8D2H2L2

[0265] DNA sequence of the heavy chain variable region: (345 bp)

[0266]

[0267] Its encoded protein sequence is: (115aa)

[0268]

[0269] DNA sequence of the light chain variable region: (321 bp)

[0270]

[0271]

[0272] Its encoded protein sequence is: (107aa)

[0273]

[0274] Light and heavy chain sequences of the 3.8D2 humanized monoclonal antibody 8D2H3L3

[0275] DNA sequence of the heavy chain variable region: (345 bp)

[0276]

[0277] Its encoded protein sequence is: (115aa)

[0278]

[0279] DNA sequence of the light chain variable region: (321 bp)

[0280]

[0281] Its encoded protein sequence is: (107aa)

[0282]

[0283] 4. Light and heavy chain sequences of the humanized monoclonal antibody 8D2H2L15.

[0284] DNA sequence of the heavy chain variable region: (345 bp)

[0285]

[0286] Its encoded protein sequence is: (115aa)

[0287]

[0288] DNA sequence of the light chain variable region: (321 bp)

[0289]

[0290] Its encoded protein sequence is: (107aa)

[0291]

[0292] 5. Light and heavy chain sequences of the humanized monoclonal antibody 8D2H2L17.

[0293] DNA sequence of the heavy chain variable region: (345 bp)

[0294]

[0295]

[0296] Its encoded protein sequence is: (115aa)

[0297]

[0298] DNA sequence of the light chain variable region: (321 bp)

[0299]

[0300] Its encoded protein sequence is: (107aa)

[0301]

[0302] Example 4: 8D2 recombinant antibody 8D2(Re) and 8D2 humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, Preparation and SDS-PAGE electrophoresis detection of 8D2H2L15 and 8D2H2L17

[0303] 1. Preparation and SDS-PAGE electrophoresis detection of 8D2 recombinant antibody (8D2(Re)).

[0304] The heavy chain cDNA sequence of 8D2 (its variable region sequence is shown in SEQ ID NO: 5) and the light chain cDNA sequence (its variable region sequence is shown in SEQ ID NO: 7) were cloned into the pUC57simple vector (provided by GenScript) to obtain the pUC57simple-8D2H and pUC57simple-8D2L plasmids, respectively.

[0305] Plasmids pUC57simple-8D2H and pUC57simple-8D2L were digested with HindIII and EcoRI, respectively. The heavy and light chains recovered by electrophoresis were subcloned into the pcDNA3.1 vector, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was purified by high-speed centrifugation, vacuum filtration through a microporous membrane, and HiTrap protein A HP column. The purified samples were then added to reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer, respectively, and after boiling, SDS-PAGE electrophoresis was performed for detection. Figure 3 As shown, the target protein in the reduced protein sample is approximately at 50kD and 25kD, while the target protein in the non-reduced protein sample is approximately at 150kD.

[0306] 2. Preparation and SDS-PAGE electrophoresis detection of humanized 8D2 antibodies 8D2H1L1, 8D2H2L2 and 8D2H3L3

[0307] The heavy chain cDNAs of 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17 (with variable region sequences shown in SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 13, and SEQ ID NO: 13, respectively) and the light chain cDNAs (with variable region sequences shown in SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 13, respectively) were combined. The plasmids pUC57simple-8D2H1L1, pUC57simple-8D2H2L2, pUC57simple-8D2H3L3, pUC57simple-8D2H2L15, and pUC57simple-8D2H2L17 (as shown in NO: 23) were cloned into the pUC57simple vector (provided by GenScript) to obtain the plasmids pUC57simple-8D2H2L17. The plasmids were then subcloned into the pcDNA3.1 vector, using the same method as described above for 8D2(Re).

[0308] The recombinant plasmid was transfected into 293F cells, and the culture medium was purified and then analyzed (using the same method as described above for 8D2(Re)). The results are as follows: Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown, the target protein in the reduced protein sample is approximately at 50kD and 25kD, while the target protein in the non-reduced protein sample is approximately at 150kD.

[0309] The 8D2 recombinant antibody 8D2(Re) and the 8D2 humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 and 8D2H2L17 used in the following examples were all prepared according to the method of this example.

[0310] Example 5: Determination of antibody kinetic parameters

[0311] The kinetic parameters of the binding of antibody 8D2 and humanized 8D2H1L1, 8D2H2L2, and 8D2H3L3 to antigen CTLA4 (NCBI Gene ID: 1493, encoding nucleic acid sequence SEQ ID NO: 25, encoding amino acid sequence SEQ ID NO: 26) were determined using a Fortebio molecular interaction analyzer.

[0312] 1. The CTLA4-mFc protein was digested with TEV protease (the synthesis method of CTLA4-mFc is the same as that of CTLA4ECD-mFc described in Example 1), and the CTLA4 antigen was obtained by column purification.

[0313] The sequence of the CTLA4 gene: (636 bp)

[0314]

[0315]

[0316] The corresponding amino acid sequence is: (212aa)

[0317]

[0318] 2. Antibody 8D2 and its humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17 were immobilized on the surface of the AR2G sensor via amino-coupling. After blocking with ethanolamine and equilibration in PBST, they were bound to antigen CTLA4. CTLA4 was diluted two-fold with PBST to concentrations of 300, 150, 75, 37.5, 18.75, 9.38, 4.69, and 0 nM, and then dissociated in PBST. The detection method for humanized 8D2 H1L1, H2L2, H3L3, H2L15, and H2L17 was the same as for 8D2, with antigen concentrations of 180, 90, 45, 22.5, 11.25, 5.625, 2.813, and 0 nM.

[0319] The kinetic parameters of antibody 8D2 and its humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17 are shown in Table 1. The detection results of the kinetic characteristic parameters are as follows: Figure 9-14 As shown.

[0320] Table 1: Kinetic parameters of antibodies 8D2, 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17

[0321]

[0322]

[0323] K D K is the affinity constant; kon is the antigen-antibody binding rate; kdis is the antigen-antibody dissociation rate; K D =kdis / kon.

[0324] The results showed that all six antibodies had good affinity for the antigen, comparable to or even better than the control antibodies 10D1 and 11.2.1.

[0325] Example 6: Flow cytometry method for detecting the binding activity of antibody to CTLA4 antigen on the surface of hybridoma cell line

[0326] First, host cells 293F expressing the CTLA4 antigen were constructed. Then, the host cells were labeled with the monoclonal antibody 8D2 prepared in this invention (see Example 1), as well as the prepared 8D2(Re) and humanized 8D2 antibodies 8D2H1L1, 8D2H2L2, and 8D2H3L3 (see Example 4). Flow cytometry analysis was then used to verify that antibodies 8D2, 8D2(Re), and the humanized 8D2 antibodies 8D2H1L1, 8D2H2L2, and 8D2H3L3 specifically bind to antigens with their native conformation on the cell surface.

[0327] The specific steps are as follows:

[0328] 1. Construction of host cell 293F expressing CTLA4 antigen

[0329] Following the method described in the Lipofectamin transfection kit (purchased from Invitrogen), 293F cells were transfected with the vector pLenti6.3-CTLA4 (pLenti6.3 was purchased from Invitrogen). After screening, a stable clonal population of 293F-CTLA4 cells expressing CTLA4 was obtained.

[0330] 2. Antibody labeling and flow cytometry detection

[0331] The host cells 293F expressing the CTLA4 antigen were obtained using the above steps via conventional trypsin digestion, with each collection tube containing 2 × 10⁻⁶ cells. 5 8D2 antibody dilutions at concentrations of 20 nM, 10 nM, 5 nM, 1 nM, 0.1 nM, 0.01 nM, and 0 nM were prepared using PBS (1% BSA). These solutions were incubated on ice with 293F cells expressing CTLA4 for 2 hours. Then, 100 μL of FITC-Goat-Anti-Mouse IgG (1:500) was added to each tube, and the cells were incubated on ice for 1 hour. Finally, 300 μL of PBS was added, and fluorescence signals were detected using the FITC channel on a flow cytometer. Detection of other antibodies followed the same procedure as for the 8D2 antibody.

[0332] 3. Experimental Results

[0333] The results of CTLA4 expression validation in 293F-CTLA4 cells are as follows: Figure 15 and Figure 16 As shown in the figure. The results of detecting the binding of 8D2, 8D2(Re) antibodies and three humanized antibodies to 293F cells are as follows. Figure 17-21 As shown in the figure, the 8D2 antibody and its humanized antibody can effectively bind to the target CTLA protein on the surface of host cell 293F, and the binding efficiency is dose-dependent. The fluorescence intensity of each dose is shown in Table 2.

[0334] By performing quantitative fluorescence analysis on the bound 8D2 and its humanized antibody, the EC50 of the 8D2 and its humanized antibody was simulated using curve analysis. 50 As shown in Table 3.

[0335] Table 2: Fluorescence intensity analysis of 8D2, 8D2(Re), and humanized antibodies 8D2H1L1, 8D2H2L2, and 8D2H3L3 after binding to the CTLA4 host cell 293F surface antigen CTLA4 by flow cytometry.

[0336]

[0337] Table 3: Flow cytometry analysis curves show the binding efficiency (ECG) of simulated 8D2, 8D2(Re), and 8D2 humanized antibodies 8D2H1L1, 8D2H2L2, and 8D2H3L3 to the CTLA4 host cell 293F surface antigen CTLA4. 50

[0338] 8D2 8D2(Re) 8D2H1L1 8D2H2L2 8D2H3L3 <![CDATA[EC 50 (nM)]]> 3.84 1.38 5.06 4.37 4.54

[0339] The results showed that antibodies 8D2, 8D2(Re), and humanized antibodies 8D2H1L1, 8D2H2L2, and 8D2H3L3 all had a strong binding ability to the CTLA4 host cell 293F surface antigen CTLA4.

[0340] Example 7: ELISA method for detecting the binding activity of antibody to antigen CTLA4

[0341] The ELISA plate was coated with CTLA4 and incubated overnight at 4°C. After blocking with 1% BSA at 37°C for 2 hours, CTLA4 antibodies 8D2, 8D2(Re), and humanized 8D2 antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17, as well as control antibodies 10D1 (Alan J. Korman, Edward L. Halk, et al., HUMAN CTLA-4 ANTIBODIES, United States Patent No. US 6984720 B1) and 11.2.1 (Douglas Charles Hanson, Mark Joseph Neveu, et al., Human monoclonal antibodies to CTLA-4, United States Patent No. US 682736B1) were added and reacted for 30 minutes. After incubation with enzyme-labeled secondary antibody for 30 minutes, the absorbance at 450 nm was measured on an ELISA reader.

[0342] The results of detecting the binding of 8D2 antibody and its humanized antibody to antigen CTLA4 are as follows: Figure 22-25 As shown in the figure, 8D2, 8D2(Re) antibodies, and humanized 8D2 antibodies can effectively bind to CTLA4 protein, and their binding efficiency is dose-dependent. The fluorescence intensity of each dose is shown in Tables 4-8. Quantitative fluorescence analysis of the bound 8D2, 8D2(Re), and humanized antibodies was performed, and the curves simulated the binding efficiency EC50 of 8D2, 8D2(Re), and humanized antibodies. 50 (Table 9)

[0343] Table 4: Binding of 8D2 and 8D2 recombinant antibodies to mouse CTLA4 (ELISA)

[0344]

[0345]

[0346] Table 5: Binding of 8D2, 8D2H1L1, and 8D2 recombinant antibodies to human CTLA4 (ELISA)

[0347]

[0348] Table 6: Binding of 8D2H2L2 and 8D2H3L3 antibodies to human CTLA4 (ELISA)

[0349]

[0350]

[0351] Table 7: Binding of 8D2H2L2 and 8D2H3L3 antibodies to monkey CTLA4 (ELISA)

[0352]

[0353] Table 8: Binding of antibodies 8D2H2L15 and 8D2H2L17 to human CTLA4 (ELISA)

[0354]

[0355]

[0356] Table 9: ELISA analysis curves show the binding efficiencies (EC5) of 8D2, 8D2(Re), and humanized 8D2 antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17 to the CTLA4 antigen. 50

[0357]

[0358]

[0359] Note: 8D2H2L2 was tested in three parallel trials.

[0360] The above results indicate that antibodies 8D2 and 8D2(Re) exhibit stronger binding efficiency to murine CTLA4 antigen than control antibodies 10D1 and 11.2.1. The humanized antibody 8D2H1L1 shows stronger binding efficiency to murine CTLA4 antigen than control antibody 10D1, and comparable to 11.2.1.

[0361] The binding efficiency of humanized antibody 8D2H2L2 to human CTLA4 antigen was comparable to that of 10D1. The binding efficiencies of humanized antibodies 8D2H2L2 and 8D2H3L3 to monkey CTLA4 antigen were comparable to those of 10D1. Antibodies 8D2H2L15 and 8D2H2L17 showed significantly stronger binding efficiencies to human CTLA4 antigen than control antibodies 10D1 and 11.2.1.

[0362] Example 8: Detection of the binding activity of antibody and B7-1 / 2 to antigen CTLA4 in a competitive ELISA method

[0363] 1. ELISA detection of the binding activity of antibody against B7-1 competitively binding antigen CTLA4.

[0364] The microplate was coated with B7-1 and incubated overnight at 4°C, then blocked with 1% BSA at 37°C for 2 hours. Anti-CTLA4 monoclonal antibodies 8D2, 8D2(Re), and humanized 8D2 antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17, along with control antibodies 10D1 and 11.2.1, were added and incubated for 10 minutes. Then, CTLA4-mFc was added and incubated at 37°C for 40 minutes, followed by incubation with enzyme-labeled secondary antibody at 37°C for 30 minutes. The absorbance at 450 nm was measured using a microplate reader.

[0365] 2. ELISA detects the binding activity of antibodies against B7-2 in competing with antigen CTLA4.

[0366] CTLA4-mFc-coated ELISA plates were incubated overnight at 4°C, blocked with 1% BSA at 37°C for 2 hours, and then incubated with anti-CTLA4 monoclonal antibodies 8D2, 8D2(Re), and humanized 8D2 antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17, as well as control antibodies 10D1 and 11.2.1 for 10 minutes. After incubation with B7-2-his at 37°C for 40 minutes, ELISA-labeled secondary antibody was added and incubated at 37°C for 30 minutes. The absorbance at 450 nm was measured using an ELISA reader. The binding results of 8D2, 8D2(Re) antibodies and their humanized antibodies to the CTLA4 antigen are shown below. Figure 26-31As shown in the figure, 8D2, 8D2(Re) antibodies, and humanized 8D2 antibodies can effectively bind to CTLA protein, and their binding efficiency is dose-dependent. The fluorescence intensity of each dose is shown in Tables 10-16. Quantitative fluorescence analysis of the bound 8D2, 8D2(Re), and humanized antibodies was performed, and the curves simulated the binding efficiency EC50 of 8D2, 8D2(Re), and humanized antibodies. 50 (Table 17).

[0367] Table 10: ELISA Competition between 8D2, 8D2(Re), and B7-1

[0368]

[0369] Table 11: ELISA Competition between 8D2, 8D2H1L1, 8D2(Re) and B7-1

[0370]

[0371]

[0372] Table 12: Competition between 8D2, 8D2H1L1, and 8D2 recombinant antibodies and B7-2 in ELISA

[0373]

[0374] Table 13: Competition between 8D2H2L2 and 8D2H3L3 antibodies and B7-1 in ELISA

[0375]

[0376]

[0377] Table 14: Competitive binding of 8D2H2L2 and 8D2H3L3 antibodies to B7-2 in CTLA4 ELISA

[0378]

[0379] Table 15: Competitive binding of 8D2H2L15 and 8D2H2L17 antibodies to B7-1 in CTLA4 ELISA

[0380]

[0381] Table 16: Competitive binding of 8D2H2L15 and 8D2H2L17 antibodies to B7-2 in CTLA4 ELISA

[0382]

[0383]

[0384] Table 17: Competitive ELISA analysis curves show the binding efficiencies (ECs) of 8D2, 8D2(Re), and humanized 8D2 antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17 with B7 in competitive binding to antigen CTLA4. 50

[0385]

[0386] Note: 8D2H2L2 was tested in three parallel trials.

[0387] The above results indicate that antibodies 8D2, 8D2(Re), and humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17 can all competitively bind to antigen CTLA4 with B7. Among them, 8D2, 8D2(Re), 8D2H1L1, and 8D2H2L2 exhibit stronger competitive binding to CTLA4 with B7-2 than 10D1; 8D2H2L17 shows significantly stronger competitive binding to CTLA4 with both B7-1 and B7-2 than antibodies 10D1 and 11.2.1.

[0388] Example 9: Monoclonal antibody 8D2 and humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 and Cell biological activity analysis of 8D2H2L17

[0389] To investigate the effects of monoclonal antibody 8D2 and humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17, and control antibody 10D1, on IL-2 expression in peripheral blood mononuclear cells (PBMCs), peripheral blood was collected from healthy individuals using heparinized blood collection tubes. After dilution with PBS and centrifugation (2550 rpm, 20 min) with separation buffer, a cell suspension, i.e., PBMCs, was obtained. SEB (1 μg / mL) / PHA (30 μl / mL) was added to the cell suspension, and the cells were cultured in a saturated humidity incubator at 37°C and 5% CO2 for 48 hours. Raji lymphocytes and antibodies were then added for co-incubation. After washing PBMCs twice with PBS, 10,000 cells / well were added to each well of a 96-well plate. Then, antibodies of appropriate concentration gradients were added, and the plates were incubated for 20 minutes. Following this, 10,000 cells / well of Raji cells treated with MMC for 1 hour were added, and the plates were co-cultured for 72 hours. After 72 hours of co-incubation, the cell culture supernatant was collected, and IL-2 expression in the supernatant was detected using ELISA (refer to the kit instructions: DAKEW, DKW12-1020-096).

[0390] The experimental results were statistically analyzed, such as Figures 32-33As shown. Compared with the T cells and with Raji cells groups, the humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17 of the monoclonal antibody 8D2 effectively blocked the binding of CTLA4 to B7 and increased the expression of IL-2 in T lymphocytes. Figures 32-33 In particular, the inventors surprisingly discovered that 8D2H2L2, 8D2H2L15, and 8D2H2L17 were significantly superior to the control antibodies 10D1 and 11.2.1. The IL-2 levels achieved by these antibodies at a concentration of 10 nM were comparable to or even better than those achieved by 10D1 or 11.2.1 at a concentration of 100 nM. It is evident that the antibodies of the present invention can increase IL-2 levels at a lower concentration, for example, approximately 10 nM.

[0391] Example 10: In vivo antitumor activity of monoclonal antibody 8D2H2L2

[0392] The in vivo antitumor activity of 8D2H2L2 was evaluated using a hu-SCID-raji animal model. Human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll reagent and activated with SEB 1 μg / ml for 3 days. Then, 1.25 million activated PBMCs were mixed with 5 million Burkitt lymphoma cells (raji) and 8D2H2L2 (20 mg / kg) and subcutaneously inoculated onto the dorsal side of SCID-Beige mice. An isotype control group was also included, with 5 animals in each group. The mice were then administered intravenously once weekly at a dose of 20 mg / kg for 3 consecutive weeks. Tumor volume was measured twice weekly until the end of the experiment or when the tumor volume reached 1000 mmHg. 3 .

[0393] like Figure 34 As shown, 8D2H2L2 significantly inhibited tumor growth in the hu-SC1D-raji model. This result indicates that this antibody can be used clinically to treat lymphoma.

[0394] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. A monoclonal antibody comprising a heavy chain variable region of SEQ ID NO: 14 and a light chain variable region of SEQ ID NO: 16, wherein the methionine at position 18 of SEQ ID NO: 14 is replaced by leucine, wherein the monoclonal antibody has an IgG isotype selected from IgG1, IgG2, IgG3 or IgG4.

2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody has an IgG isotype of IgG1.

3. The monoclonal antibody according to any one of claims 1 to 2, wherein, The monoclonal antibody is less than 10 -5 M of K D The K protein binds to the CTLA4 protein. D It was measured in a BIACORE instrument using surface plasmon resonance.

4. An isolated nucleic acid molecule encoding a monoclonal antibody according to any one of claims 1 to 3.

5. A carrier comprising the isolated nucleic acid molecule of claim 4.

6. A host cell comprising the vector of claim 5.

7. A method for preparing the monoclonal antibody according to any one of claims 1 to 3, comprising culturing the host cell of claim 6 under suitable conditions, and recovering the monoclonal antibody or an antigen-binding fragment thereof from the cell culture.

8. A pharmaceutical composition comprising the monoclonal antibody of any one of claims 1 to 3 and a pharmaceutically acceptable carrier and / or excipient.

9. Use of the monoclonal antibody according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of cancers selected from melanoma, renal tumors, lymphoma, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, and liver cancer.

10. Use of the monoclonal antibody according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of human patients with cancers selected from melanoma, renal tumors, lymphoma, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, and liver cancer.

11. The use of claim 9 or 10, wherein the cancer is colorectal cancer.

12. The use of claim 9 or 10, wherein the cancer is lymphoma.

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