CN108948194B - Novel CTLA-4 monoclonal antibody - Google Patents

Abstract

The present invention provides a CTLA-4 monoclonal antibody, especially a high-affinity CTLA-4 humanized monoclonal antibody. The present invention also provides functional monoclonal antibodies that cross-react with human, cynomolgus and mouse CTLA-4. The present invention further provides the amino acid sequence of the antibody of the present invention, a cloning or expression vector, a host cell and a method for expressing or isolating the antibody, and identifying the epitope of the antibody. Therapeutic compositions comprising antibodies of the invention are also provided. The invention also provides methods of treating cancer and other diseases with anti-CTLA-4 antibodies.

Description

A new CTLA-4 monoclonal antibody

technical field

The present invention mainly relates to anti-CTLA-4 antibodies and compositions thereof, and immunotherapy for cancer, infection or other human diseases using anti-CTLA-4 antibodies.

Background technique

Cancer immunotherapy has become a hot research field in the treatment of cancer. Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is one of the validated targets of immune checkpoints. Following T cell activation, CTLA-4 is rapidly expressed on T cells, usually within 1 h of antigen engagement with the TCR. CTLA-4 can inhibit T cell signaling by competing with CD28. CD28 mediates one of the well-characterized T cell co-stimulatory signals: binding of CD28 to ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells leads to T cell proliferation, induction of interleukin-2 and Anti-apoptotic factor. Since CTLA-4 has a higher affinity for CD80 and CD86 than CD28, CTLA-4 can compete with CD28 on CD80 and CD86 for binding, resulting in the inhibition of T cell activation. In addition to induced expression on activated T cells, CTLA-4 is constitutively expressed on the surface of regulatory T cells (Treg), suggesting that CTLA-4 may be required for contact-mediated suppression and is associated with immunosuppressive Cytokines such as transforming growth factor beta and interleukin-10 are associated with Treg production.

CTLA-4 blockade has been demonstrated in many preclinical and clinical studies to induce tumor regression. Two antibodies against CTLA-4 are in clinical development. Ipilimumab (MDX-010, BMS-734016), a fully human anti-CTLA-4 monoclonal antibody of the IgG1-κ type, is an immunomodulator approved for monotherapy in the treatment of advanced melanoma. The proposed mechanism of action of Ipilimumab is to interfere with the interaction between CTLA-4 expressed on activated T cell subsets and CD80/CD86 molecules on professional antigen-presenting cells. The inhibitory regulation of T cell activation promoted by the interaction between CTLA-4 and CD80/CD86 is blocked, which leads to enhanced T cell function. The resulting T cell activation, proliferation, and lymphocyte infiltration into the tumor lead to tumor cell death. The commercial formulation is a 5 mg/mL concentrate for infusion. Ipilimumab is also being used in clinical studies in other tumor types, including prostate and lung cancer. Tremelimumab, another anti-CTLA-4 antibody, is also being studied as a therapy in melanoma and malignant mesothelioma.

Contents of the invention

The invention provides isolated antibodies, particularly monoclonal antibodies or humanized monoclonal antibodies.

In one aspect, the present invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds to human, monkey, or mouse CTLA-4.

An antibody or antigen-binding fragment as described above inhibits binding of CTLA-4 to CD80 or CD86.

In the above-mentioned antibody or antigen-binding fragment, the binding antigen epitope of the antibody or antigen-binding fragment includes N145 or N145 glycosylation modification of CTLA-4.

In one aspect, the present invention provides an antibody or antigen-binding fragment thereof, wherein said antibody or said antigen-binding fragment binds to human or monkey CTLA-4, wherein the binding epitope of said antibody or antigen-binding fragment comprises CTLA P138 of -4.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof

a) bind to human CTLA-4 with a _KD of 4.77E-10M or less; and

b) Binding to mouse CTLA-4, K _D of 1.39E-09M or less.

The aforementioned antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment has at least one of the following properties:

a) binds to human CTLA-4 with a K _D of 4.77E-10M to 2.08E-10M, and binds to mouse CTLA-4 with a K _D of 1.39E-09M to 9.06E-10;

b) increasing interleukin-2 secretion from stimulated PBMCs;

c) Substantially does not bind to proteins such as coagulation factor VIII, FGFR, PD-1, CD22, VEGF, CD3, HER3, OX40, and 4-1BB.

The present invention provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, sequences in the group consisting of 11, 12, 13, and 14 have at least 70%, 80%, 90% or 95% homology,

Wherein the antibody or antigen-binding fragment specifically binds CTLA-4.

The present invention provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and sequences in the group consisting of 14,

Wherein the antibody or antigen-binding fragment specifically binds CTLA-4.

The present invention provides an antibody or an antigen-binding fragment thereof, comprising:

a) a heavy chain variable region having an amino acid sequence at least 70%, 80%, 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7 % or 95% homology; and

b) a light chain variable region having an amino acid sequence at least 70%, 80%, 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14 % or 95% homology,

Wherein the antibody or antigen-binding fragment specifically binds CTLA-4.

The present invention provides an antibody or an antigen-binding fragment thereof, comprising:

a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, and 7; and

b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14,

Wherein the antibody or antigen-binding fragment specifically binds CTLA-4.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises:

a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; and

b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8,

wherein the antibody or antigen-binding fragment specifically binds CTLA-4;

or said antibody or antigen-binding fragment thereof, comprising:

a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; and

b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9,

wherein the antibody or antigen-binding fragment specifically binds CTLA-4;

or said antibody or antigen-binding fragment thereof, comprising:

a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 3; and

b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10,

wherein the antibody or antigen-binding fragment specifically binds CTLA-4;

or said antibody or antigen-binding fragment thereof, comprising:

a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 4; and

b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11,

wherein the antibody or antigen-binding fragment specifically binds CTLA-4;

or said antibody or antigen-binding fragment thereof, comprising:

a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 5; and

b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12,

wherein the antibody or antigen-binding fragment specifically binds CTLA-4;

or said antibody or antigen-binding fragment thereof, comprising:

a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 6; and

b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13,

wherein the antibody or antigen-binding fragment specifically binds CTLA-4;

or said antibody or antigen-binding fragment thereof, comprising:

a) a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 7; and

b) a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14,

Wherein the antibody or antigen-binding fragment specifically binds CTLA-4.

See Table 1 and sequence listing information for details of the sequence:

Table 1. Deduced antibody amino acid sequences

In another aspect, the present invention provides an antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) whose amino acid sequence is selected from the group consisting of SEQ ID NOs: 15-41,

Wherein the antibody or antigen-binding fragment specifically binds CTLA-4.

In another aspect, the present invention provides an antibody or antigen-binding fragment thereof, comprising:

a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences; and

a light chain variable region comprising CDR1, CDR2 and CDR3 sequences,

Wherein the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 16, 17, and 18 and conservative modifications thereof,

Wherein the antibody or antigen-binding fragment specifically binds CTLA-4.

Preferably, the CDR3 sequence of the antibody light chain variable region in the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, and 22 and conservative modifications thereof .

Preferably, the CDR2 sequence of the heavy chain variable region in the antibody or its antigen-binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, 27, and 28 and its conservative sexual modification.

Preferably, the light chain variable region CDR2 sequence of the antibody or its antigen-binding fragment comprises amino acid sequences selected from the group consisting of SEQ ID NO: 29, 30, 31, and 32 and conservative modifications thereof.

Preferably, the CDR1 sequence of the heavy chain variable region in the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 33, 34, 35, and 36 and conservative modifications thereof.

Preferably, the CDR1 sequence of the light chain variable region in the antibody or its antigen-binding sheet comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37, 38, 39, 40, and 41 and conservative modifications thereof .

In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to CTLA-4, and comprises:

a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences; and

A light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein

a) a heavy chain variable region CDR1 whose sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 33, 34, 35, and 36,

heavy chain variable region CDR2, the sequence comprising the amino acid sequence shown in the group consisting of SEQ ID NO: 23, 24, 25, 26, 27, and 28,

a heavy chain variable region CDR3 whose sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 16, 17, and 18,

b) and light chain variable region CDR1, the sequence comprising the amino acid sequence shown in the group consisting of SEQ ID NO: 37, 38, 39, 40, and 41,

A light chain variable region CDR2 whose sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 30, 31, and 32,

a light chain variable region CDR3 whose sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 20, 21, and 22,

Wherein the antibody or antigen-binding fragment thereof specifically binds CTLA-4.

In some embodiments, the antibody or antigen-binding fragment comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 15,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 23,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 33,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

e) light chain variable region CDR2, the sequence comprising the amino acid sequence selected from SEQ ID NO: 29,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 37,

Wherein the antibody or antigen-binding fragment thereof specifically binds CTLA-4.

In some embodiments, the antibody or antigen-binding fragment comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 16,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 24,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 34,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 20,

e) light chain variable region CDR2, the sequence comprising an amino acid sequence selected from SEQ ID NO: 30,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 38,

Wherein the antibody or antigen-binding fragment thereof specifically binds CTLA-4.

In some embodiments, the antibody or antigen-binding fragment comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 17,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 25,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 35,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 19,

e) light chain variable region CDR2, the sequence comprising an amino acid sequence selected from SEQ ID NO: 31,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 39,

Wherein the antibody or antigen-binding fragment thereof specifically binds CTLA-4.

In some embodiments, the antibody or antigen-binding fragment comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 18,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 26,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 36,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 22,

e) light chain variable region CDR2, the sequence comprising an amino acid sequence selected from SEQ ID NO: 32,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 40,

Wherein the antibody or antigen-binding fragment thereof specifically binds CTLA-4.

In some embodiments, the antibody or antigen-binding fragment comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 16,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 27,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 34,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 20,

e) light chain variable region CDR2, the sequence comprising an amino acid sequence selected from SEQ ID NO: 30,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 38,

Wherein the antibody or antigen-binding fragment thereof specifically binds CTLA-4.

In some embodiments, the antibody or antigen-binding fragment comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 17,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 25,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 35,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 21,

e) light chain variable region CDR2, the sequence comprising an amino acid sequence selected from SEQ ID NO: 31,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 39,

Wherein the antibody or antigen-binding fragment thereof specifically binds CTLA-4.

In some embodiments, the antibody or antigen-binding fragment comprises:

a) CDR1 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 18,

b) CDR2 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 28,

c) CDR3 of the heavy chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 36,

d) CDR1 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 22,

e) light chain variable region CDR2, the sequence comprising an amino acid sequence selected from SEQ ID NO: 32,

f) CDR3 of the light chain variable region, the sequence comprising the amino acid sequence selected from SEQ ID NO: 41,

Wherein the antibody or antigen-binding fragment thereof specifically binds CTLA-4.

See Table 2 and sequence listing information for specific CDR sequences:

Table 2. CDR sequences of antibodies

Antibodies of the invention may be chimeric antibodies.

Antibodies of the present invention may be humanized antibodies.

Antibodies of the invention may be fully human antibodies.

Antibodies of the invention may be rat antibodies.

The antibody or antigen-binding fragment of the present invention has at least one of the following properties:

a) bind to human CTLA-4 with a _KD of 2.08E-09M or less, and/or bind to mouse CTLA-4 with a _KD of 1.39E-09M or less;

b) Increased interleukin-2 secretion from stimulated PBMCs.

In yet another aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in the present invention.

The present invention provides a cloning or expression vector comprising the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention.

The present invention provides a host cell comprising one or more of the above-mentioned cloning or expression vectors.

In another aspect, the invention provides a method for producing any of the antibodies of the invention, comprising culturing the host cell described in the invention, and isolating the antibody.

The antibody was prepared by immunizing SD rats with the extracellular domain of human CTLA-4 and the extracellular domain of mouse CTLA-4.

The invention provides a transgenic animal, such as a rat, comprising human immunoglobulin heavy chain and light chain transgenes, wherein the rat expresses any antibody described in the invention.

The present invention provides a hybridoma obtained from the above rat, characterized in that the hybridoma produces the antibody.

In another aspect, the present invention also provides a pharmaceutical composition, which comprises any antibody or antigen-binding fragment thereof described in the present invention, and more than one pharmaceutically acceptable excipient, diluent or carrier.

The invention also provides an immunoconjugate comprising any antibody or antigen-binding fragment thereof of the invention linked to a therapeutic agent.

The present invention also provides a pharmaceutical composition, which comprises the above immunoconjugate and more than one pharmaceutically acceptable excipient, diluent or carrier.

The present invention also provides a method for preparing an anti-CTLA-4 antibody or an antigen-binding fragment thereof, comprising:

(a) provide:

(i) an antibody sequence of a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NO: 33-36, a CDR2 sequence selected from the group consisting of SEQ ID NO: 23-28 and a CDR3 sequence selected from the group consisting of SEQ ID NO: 15-18; and/or

(ii) an antibody sequence of a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 37-41, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 29-32 and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 19-22; and

(b) Expressing the altered antibody sequence as a protein.

The invention also provides a method of modulating an immune response in a subject, comprising administering to the subject any antibody or antigen-binding fragment thereof described in the invention.

The present invention also provides the use of any antibody or antigen-binding fragment thereof as described in the present invention in the preparation of a medicament for treating or preventing immune disorders or cancer.

The present invention also provides a method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of any antibody or antigen-binding fragment thereof described in the present invention, so as to inhibit the growth of tumor cells.

In the present invention, the above-mentioned tumor cells are selected from melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular malignant melanoma Cancers in the group consisting of melanoma, uterine cancer, ovarian cancer, and rectal cancer.

In the present invention, the above-mentioned antibody is a chimeric antibody, a humanized antibody, a human antibody, or a rat antibody.

Beneficial Effects of the Invention

The inventors utilized proprietary hybridoma technology to generate humanized antibodies against CTLA-4, wherein the antibodies inhibit the binding of CTLA-4 to its ligands CD80 and CD86. The antibody disclosed in the invention has high binding affinity, specifically binds human and monkey CTLA-4 proteins; and effectively regulates immune response and increases interleukin-2 production.

One of the antibodies not only binds human and monkey CTLA-4, but also murine CTLA-4, which could greatly facilitate preclinical validation of its efficacy in mouse tumor models.

Description of drawings

Figure 1 shows a graph of chimeric antibody binding to human CTLA-4 in ELISA.

Figure 2 shows a graph of chimeric antibody binding to cynomolgus CTLA-4 in ELISA.

Figure 3 shows a graph of chimeric antibody binding to mouse CTLA-4 in ELISA.

Figure 4 shows a graph of binding of chimeric antibodies to human CTLA-4 on cells by FACS.

Fig. 5 shows the results of binding to human CTLA-4 by the SPR chimeric antibody.

Figure 6 shows the results of chimeric antibodies blocking the binding of CTLA-4 to ligands.

Figure 7 shows graphs of chimeric antibodies inhibiting the binding of CD80 or CD86 expressing cells to CTLA-4.

Figure 8 shows the results of chimeric antibodies enhancing cytokine release from SEB-stimulated PBMCs.

Figure 9 shows graphs of humanized antibody binding to human, cynomolgus and mouse CT LA-4 in ELISA.

Figure 10a shows a graph (FACS) of humanized antibody binding to CTLA-4 on cells.

Figure 10b shows an affinity map of humanized antibodies detected by FACS.

Figure 11 shows that humanized antibodies block CTLA-4 binding to ligands by ELISA.

Figure 12 shows that humanized antibodies block CTLA-4 binding to its ligands by FACS.

Figure 13 shows that humanized antibodies enhance cytokine release in SEB assay.

Figure 14 shows the SEC curves of W3162-1.146.19-Z12 or W3162-1.154.8-Z35 under different conditions.

Figure 15 shows the in vivo anti-tumor efficacy of antibody W3162-146.19-z12.

Figure 16 shows that the W3162 antibody specifically binds to CTLA-4.

Fig. 17 shows the binding activity of anti-CTLA4 antibodies to human CTLA-4/CTLA-4 mutants. (A) Ipilimumab, (B) W3162-1.146.19-z12 and (C) W3162-1.154.8-z35 antibodies were captured with 2 μg/mL goat anti-human IgG antibody, and diluted hCTLA4-His (WT) or Mutant proteins (N113Q and N145Q) were incubated together, and then HRP-labeled anti-6XHis tag antibody was added for detection.

Figure 18 shows the binding residues or epitopes mapped to human CTLA-4: (A) CD80 (PDB: 1I8L), (B) CD86 (PDB: 1I85), (C) tremelimumab (PDB: 5GGV), ( D) Ipilimumab, (E) W3162-1.146.19-z12 and (F) W3162-1.154.8-z35. IAH1 was used in CTLA-4 structures D–F to show the structure of glycosylation.

Detailed ways

The present invention will be further described below through specific embodiments and experimental data. Although specific terms are used hereinafter for purposes of clarity, these terms are not meant to define or limit the scope of the invention.

As used herein, the terms "cytotoxic T-lymphocyte-associated antigen 4", "protein CTLA-4", "CTLA-4", "CTLA4", "CD152" are used interchangeably and include human CTLA-4 Variants, subtypes, species homologues or other species of CTLA-4 and analogs having at least one common epitope of CTLA-4.

As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof. "Antibody" refers to a protein comprising at least two heavy (H) and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen.

The term "antibody", as used in this application, refers to an immunoglobulin or fragment or derivative thereof, and includes any polypeptide comprising an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, heterozygous, mutant , grafted antibodies. The term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function, ie, are capable of specific binding to CTLA-4. Typically, such fragments will include antigen binding fragments.

The terms "antigen-binding fragment", "antigen-binding domain" and "binding fragment" refer to an antibody molecule comprising the amino acids responsible for the specific binding between antibody and antigen. For example, where the antigen is large, the antigen-binding fragment may only bind a portion of the antigen. The portion of the antigen molecule responsible for the specific interaction with the antigen-binding fragment is called an "epitope" or "antigenic determinant".

An antigen-binding fragment typically includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily have to include both. For example, a so-called Fd antibody fragment consists of only the VH domain but still retains some of the antigen-binding functions of the intact antibody.

The above term "epitope" is defined as an antigenic determinant which specifically binds/recognizes the binding fragment. Binding fragments can specifically bind/interact with conformational or continuous epitopes unique to the target structure, such as human CTLA-4 and murine CTLA-4. A conformational or discontinuous epitope is characterized by a polypeptide antigen as two or more discrete amino acid residues separated in the primary sequence, but aggregated together on the surface of the molecule when the polypeptide is folded into a native protein/antigen. Two or more discrete amino acid residues that make up an epitope are present on separate parts of one or more polypeptide chains. When the polypeptide chain folds into a three-dimensional structure, these residues gather on the surface of the molecule to form an epitope. In contrast, a continuous or linear epitope consisting of two or more discrete amino acid residues, which occurs in a single linear segment of a polypeptide chain.

The term "CTLA-4-binding epitope" refers to an antibody specifically binding to a specific epitope of CTLA-4, which can be defined by a linear amino acid sequence or a partial three-dimensional structure of CTLA-4. By binding is meant that the antibody has a significantly greater affinity for portions of CTLA-4 than it does for other related polypeptides. The term "substantially greater affinity" refers to a measurable increase in the affinity for a portion of CTLA-4 compared to the affinity for other related polypeptides. Preferably, the affinity for a particular portion of CTLA-4 is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 103-fold, ^{104 -} fold, ¹⁰⁵ -fold, ¹⁰⁶ -fold or greater compared to other proteins . Preferably, binding affinity is determined by enzyme-linked immunosorbent assay (ELISA), or by fluorescence-activated cell sorting (FACS) analysis or surface plasmon resonance (SPR). More preferably, binding specificity is determined by fluorescence activated cell sorting (FACS) analysis.

The term "cross-reactivity" as described herein refers to the binding of antigenic fragments of the same target molecule of human, monkey, and/or murine origin (mouse or rat). Thus, "cross-reactivity" should be understood as interspecies reactivity with the same molecule X expressed in a different species. The cross-reactivity specificity of monoclonal antibodies recognizing human CTLA-4, monkey, and/or murine CTLA-4 (mouse or rat) can be determined by FACS analysis.

As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably.

The terms "treatment" and "method of treatment" refer to both therapeutic treatment and prophylactic/preventive measures. Those in need of treatment include individuals already with the particular medical condition as well as those who may eventually acquire the condition.

The term "conservative modification", ie, modifications to the nucleotide and amino acid sequences that do not significantly affect or alter the binding properties of the antibody encoded by the nucleotide sequence or that comprise the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced into the sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include those 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), nonpolar side chains (e.g., alanine, valine, leucine , isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The experimental methods in the following examples are conventional methods unless otherwise specified.

Example:

Embodiment 1 Experimental material preparation

1. Expression and purification of soluble CTLA-4

Cloning human and mouse CTLA-4 extracellular domain (ECD) genes with hexahistidine (6×His) or Fc-tag into expression vectors, and then transfecting Expi293 cells using Expi293 Expression System Kit . Cells were cultured in Expi293 expression medium on a shaker platform rotating at 135 rpm in a 37 °C incubator in a humidified environment with 8% _CO2 . The collected supernatant was used for protein purification. Hexahistidine-tagged proteins were purified using a Ni-NTA column and Fc-tagged proteins were purified using a Protein A column.

2. Cell Line Establishment

The gene for full-length human CTLA-4 was cloned into an expression vector for stable cell line generation. Using Plasfect reagent, 30 μg of DNA was transfected into 293F cells with a density of 1×10 ⁶ /mL and a volume of 30 mL. Place the transfected cells in an incubator at 37 °C, 8% _CO2 , and a shaking speed of 100 rpm. 24-48 hours after transfection, stable clones were selected using blasticidin at a final concentration of 4 μg/mL to 6 μg/mL. Selected clones were tested by FACS using anti-CTLA-4 antibody.

In order to obtain cells expressing cynomolgus CTLA-4, the full-length cynomolgus CTLA-4 gene was cloned into an expression vector used for cell pool production. Using Plasfect reagent (Life Technology), 30 μg of DNA was transfected into 293F cells at a density of 1×10 ⁶ /mL and a volume of 30 mL. Place the transfected cells in an incubator at 37 °C, 8% _CO2 , and a shaking speed of 100 rpm. Twenty-four hours after transfection, the cell pool was selected using blasticidin at a final concentration of 4 μg/mL. Selected cell pools were tested by FACS using an anti-CTLA-4 antibody.

Example 2 Production of Antibody Hybridomas

1. Immunity

Human CTLA-4 and mouse CTLA-4 were used for immunization of SD rats. Specifically, 3 SD rats were immunized with an adjuvant containing 30 μg/rat of human and mouse CTLA-4ECD proteins. Adjuvants include Titer-Max, Adju-Phos and CpG-ODN. Rats were injected weekly via the footpad and subcutaneously. Antibody titers in serum were determined by ELISA every other month. When antibody titers were sufficiently high, a final boost was performed in rats with the highest titers using human and mouse CTLA-4 ECD proteins in Dulbecco's phosphate-buffered saline (DPBS) without adjuvant. Several days later, the spleen and lymph nodes were removed from the rats, and the lymphocytes were isolated for fusion.

2. Cell Fusion

Cell fusion was performed as follows: myeloma cells SP2/0 cells were revived one week before fusion and passaged at 1:2 every day until the day before fusion to maintain logarithmic growth. B lymphocytes and myeloma cells from the lymph nodes of immunized rats were treated with trypsin, and FBS was added to stop the reaction. B lymphocytes are fused with myeloma cells at a ratio of 1:1. The cell mixture was then washed and resuspended at 2 x ¹⁰⁶ cells/mL in electrolysis solution containing 0.3 M sucrose, 0.1 mM magnesium acetate and 0.1 mM calcium acetate. Electrofusion was performed using a Btx Electro Cell Manipulator (Ecm 2001) following the manufacturer's standard protocol. The cell suspension from the fusion chamber was then immediately transferred to a sterile bottle containing fresh medium and incubated in a 37 °C incubator for 2 h. The cell suspension was then mixed and transferred to 60 (1× ¹⁰⁴ cells/well) wells in a 96-well plate. The 96-well plate was monitored periodically at 37 °C and 5% _CO2 . When the clones are large enough (after 7-10 days), remove 180 μL/well of supernatant and add 200 μL of fresh medium to each well. After 72 hours, transfer 100 μL of the supernatant from the tissue culture plate to a 96-well assay plate for screening.

3. Screening of Hybridomas

A large number of hybridoma clones were screened for binding to human, murine and monkey CTLA-4 proteins as well as engineered human CTLA-4 expressing cells. Once specific binding to CTLA-4 and blocking activity were verified by the first and second screens, positive hybridoma lines were subcloned into 96-well plates by limiting dilution. Plates were incubated at 37°C, 5% CO ₂ until positive clones competed with the ligands CD80 and CD86 for binding to CTLA-4 for further screening. Culture supernatants of selected positive clones were collected for antibody purification and further characterization. Select lead candidates for VH and VL sequencing.

4. Determination of hybridoma VH and VL sequences

VH and VL genes of antibodies from selected hybridoma clones were isolated by RT-PCR or 5'RACE. Specifically, total RNA was isolated from hybridoma cells using RNeasy Plus Mini Kit (Qiagen). First strand cDNA was reverse transcribed using oligo dT. The VH and VL genes of the antibody were amplified from cDNA using 3'-constant region degenerate primers and 5'-degenerate primer sets. 5' degenerate primers were designed based on the upstream signal sequence coding region of the Ig variable sequence. Then the PCR product was ligated into the pMD18-T vector, and 10 μL of the ligated product was transformed into Top10 competent cells. Transformed cells were plated on 2xYT plates with carbenicillin and incubated overnight at 37 °C. 15 positive colonies were randomly selected for DNA sequencing by Biosune. Alternatively, use 5' RACE to identify the VH and VL sequences of selected hybridoma clones. First, RNA was first reverse-transcribed into cDNA using 5'-RACE kit (Takara-28001488), and then PCR was performed using 3'-degenerate primers and 3'-linker primers (ExTaq: Takara-RR001B). The PCR fragment was inserted into pMD18-T vector (Takara-D101C) and sent for sequencing (Biosune, Shanghai).

Example 3 : Generation and Characterization of Chimeric Antibodies

1. Generation of Chimeric Antibody

The deduced amino acid sequences of VH and VL are listed in Table 3. Underlined sequences are CDRs defined by the Kabat system. The variable regions of these rat antibodies were fused to the constant regions of human antibodies, and chimeric antibodies were expressed from Expi293 cells and purified using protein A chromatography.

Table 3 Amino acid sequences of the light chain and heavy chain of the rat anti-CTLA-4 antibody

2. Characterization of Chimeric Antibody

2.1 Antibodies binding to human, monkey and mouse CTLA-4 (ELISA, FACS and SPR)

Chimeric antibodies with rat variable regions and human constant regions were expressed in mammalian cells and purified using protein A affinity chromatography.

Antibodies were tested for binding to CTLA-4 in an ELISA. As shown in Figures 1, 2 and 3, four antibodies that bind to human and monkey CTLA-4 have comparable EC ₅₀ to Ipilimumab (WBP316-BMK1), but only one antibody, W3162-1.146.19, also binds to murine CTLA- 4 binding with an EC ₅₀ of 0.01 nM. To confirm that the antibodies were able to bind CTLA-4 on the cell surface, a CTLA-4 expressing cell line was used in the FACS assay. These antibodies also bound CTLA-4 on the cell surface (Figure 4) with _EC50s ranging from 1.14 nM to 9.42 nM. W3162-1.146.19 binds to CTLA-4 on the cell surface with an _EC50 of 3.25 nM, and W3162-1.154.8 binds to CTLA-4 on the cell surface with an _EC50 of 1.26 nM.

The binding kinetics of the four antibodies were measured using SPR. Antibodies were captured on immobilized goat anti-human Fc, followed by sequential injections of different concentrations of human CTLA-4 ECD. Subtract the sensorgrams for the reference and buffer channels from the test sensorgram. Data were used for 1:1 binding analysis. As shown in Figure 5 and Table 4, all four antibodies had higher affinity for the human CTLA-4 ECD domain than Ipilimumab (WBP316-BMK1), with _KD ranging from 2.08E-09nM to 6.80E-11nM.

Table 4. Kinetics of Antibody Binding to Human CTLA-4ECD

2.2 Ligand competition with chimeric antibody

CTLA-4 was found to bind CD80 and CD86 with 20 to 50 times greater affinity than CD28 (Krummel, 1996). Therefore, it was tested whether anti-CTLA-4 antibodies compete with the binding of CD80 and CD86 on CTLA-4. Both ELISA and FACS were used as competition assays. In an ELISA-based competition assay, human CTLA-4 is coated on a plate and the antibody mixed with a biotinylated ligand is added to the plate. Bound ligand was detected by HRP-conjugated streptavidin. As shown in Figures 6a and 6b, all four antibodies competed with the ligands CD80 (B7-1, L1) and CD86 (B7-2, L2) in CTLA-4 binding, except for W3162-1.101.2, in which Three antibodies had _EC50s comparable to Ipilimumab (WBP316-BMK1). In the FACS assay, a mixture of antibody and biotinylated human CTLA-4 was added to CD80 or CD86 expressing cells, and bound human CTLA-4 was detected by PE-conjugated streptavidin. As shown in Figures 7a (upper panel) and 7b (lower panel), all four of these antibodies effectively blocked CTLA-4 binding to ligand-expressing cells. Three antibodies except W3162-1.154.8 could completely block CTLA-4 binding on CD80 cells, while Ipilimumab WBP316-BMK could only partially block the binding even at the highest concentration of 200 nM (Fig. 7a). In a FACS assay for blocking CTLA-4 binding on CD86 cells (Fig. 7b), all four antibodies could completely block CTLA-4 binding on CD86 cells, while Ipilimumab could only partially block it even at the highest concentration of 200 nM. block this binding. The kinetics of W3162-1.101.2 behave differently: at low concentrations, it is less effective than Ipilimumab, while at high concentrations, Ipilimumab is more effective. The other three antibodies were more effective at blocking CTLA-4 than ipilimumab at all concentrations tested.

2.3 Function of chimeric antibody in SEB test

The function of anti-CTLA-4 antibodies with different concentrations of 1.34 nM, 3.35 nM, 8.71 nM, 21.4 nM, 53.6 nM, 134 nM was tested in a modified T cell stimulation assay (SEB assay). Staphylococcal enterotoxin B (SEB) was used as a stimulator of human T cell activation, in which CTLA-4 was reported as an important player. T cell activation was measured by secretion of IL-2. As shown in Figure 8, all four antibodies promoted IL-2 secretion in a dose-dependent manner comparable to or superior to Ipilimumab.

Embodiment 4 : Humanized antibody characterization

1. Humanization

The "Best Fit" method was used to humanize antibody light and heavy chains.

Three anti-CTLA-4 antibodies (except W3162-1.101.2 because of its relatively low binding activity in ELISA and FACS) were selected for humanization using the CDR grafting technique. The CDRs (underlined in Table 5) and FRs of the variable regions of the antibodies were defined using the Kabat system. Based on sequence homology and structural similarity, the genes of the rat region FR1-3 were replaced by the humanized region FR1-3, while the region FR4 of the rat gene was replaced by the humanized ones derived from the JH and JK genes with the most similar structure CLFR4 region substitution. Alter variable region post-translational modification (PTM) hotspots to reduce PTM risk. After verifying the template sequence and codon optimization, the heavy and light chain variable regions were synthesized, cloned into expression vectors, and then used for the expression of humanized antibodies. Humanized antibodies were purified using protein A chromatography and kinetic binding of human, monkey and mouse CTLA-4 was determined using SPR.

Table 5. Amino acid sequences of light chain and heavy chain of humanized anti-CTLA-4 antibody

Table 6. Gene sequences of light chain and heavy chain of humanized anti-CTLA-4 antibody

2. Characterization of Humanized Antibody

2.1 Antibodies that bind human, monkey and mouse CTLA-4

2.1.1 CTLA-4-binding ELISA

Humanized antibodies are expressed in mammalian cells and purified using protein A affinity chromatography. Ipilimumab is from a commercial source. WuXi Biologics produces isotype control antibodies, human CTLA-4ECD and mouse CTLA-4.ECD-hFc with different tags (hFc or 6xHis). Mouse CTLA-4.ECD-6xHis and cynomolgus CTLA-4ECD-6xHis were purchased from SinoBiological. HRP-conjugated goat anti-human IgG Fc was purchased from Bethyl (catalogue number: A80-304P).

ELISA was used to test the binding of anti-human CTLA-4 antibodies to human, murine and cynomolgus CTLA-4 proteins. Coat 96 cells with human CTLA-4.ECD-6xHis (1.0 μg/mL), cynomolgus monkey CTLA-4.ECD-6xHis (0.5 μg/mL) or mouse CTLA-4.ECD-6xHis (0.5 μg/mL) Orifice plate, react at 4°C for 16-20 hours. After blocking with 2% BSA in DBPS for 1 hour, test antibodies, as well as positive and negative control antibodies, were added to the plate and incubated for 1 hour at room temperature. Antibody bound to the plate was detected by incubation with HRP-conjugated goat anti-human IgG antibody (diluted 1:5000) for 1 hour. Color was developed with 100 μL TMB substrate for 8 minutes and then stopped with 100 μL 2N HCl. Absorbance at 450 nM was measured using a microplate spectrophotometer.

As shown in Figure 9, the EC ₅₀ of the two antibodies W3162-1.146.19-z12-IgGk and W3162-1.154.8-z35-IgGk binding to human CTLA-4 were 0.03nM and 0.04nM, slightly higher than the EC 0.01 nM Ipilimumab (WBP316-BMK1) at _{50 Å} (FIG. 9A). Both antibodies also bound to monkey CTLA-4 with an _EC50 of 0.05 nM (Figure 9B), but only W3162-1.146.19-z12-IgG bound to murine CTLA-4 with an _EC50 of 0.19 nM. Neither W3162-1.154.8-z35-IgGk nor Ipilimumab bound murine CTLA-4 (Fig. 9C).

2.1.2 CTLA-4 combined with FACS

Human CTLA4 expressing 293F cell line was developed by WuXi Biologics. PE-conjugated goat anti-human IgG Fc fragment was purchased from Jackson (cat. no. 109-115-098). Add 1 x ¹⁰⁵ cells per well to a 96-well plate, centrifuge at 4 °C for 5 min, and remove the supernatant. Serial dilutions of the test antibody, positive and negative controls were added to the resuspended cells and incubated for 1 hour at 4°C. Cells were washed twice with 200 [mu]L DPBS containing 1% BSA. PE-conjugated goat anti-human IgG (1:100) diluted in DPBS containing 1% BSA was added to the cells and incubated at 4°C for 1 hour. Perform two additional washing steps with 200 μL of DPBS containing 1% BSA, followed by centrifugation at 1500 rpm for 4 min at 4 °C. Finally, cells were resuspended in 100 μL DPBS containing 1% BSA, and fluorescence values were measured by flow cytometry and analyzed by FlowJo.

These antibodies were also able to bind human CTLA-4 on the cell surface in a FACS assay. As shown in Figure 11 (Figure 10a and Figure 10b), the _EC50 of W3162-1.146.19-z12-IgG, W3162-1.154.8-z35-IgGk and Ipilimumab were slightly different, being 1.58nM, 0.66nM and 0.83 nM.

2.2 Antibody binding kinetics

2.2.1 Measuring the binding kinetics of these antibodies using SPR

The experiment is based on SPR technique to measure the rate constant (ka) and dissociation constant (kd) of CTLA-4ECD antibody. The affinity constant (K _D ) was thus determined.

Biacore T200S series sensor chip CM5, amine coupling kit and 10 × HBS-EP were purchased from GE Healthcare. Goat anti-human IgG Fc antibody was purchased from Jackson ImmunoResearch Lab (cat. no. 109-005-098). In the fixation step, the activation buffer was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection, and the CM5 sensor chip was activated by the activation buffer for 420 seconds, and then injected in 10 mM NaAc (pH4.5) at a flow rate of 5 μL/min 30 μg/mL goat anti-human IgG Fcγ antibody was injected into the Fc1-Fc4 channel for 200 seconds, the chip was deactivated by 1M ethanolamine-HCl (GE), and then the antibody was captured on the chip. 4 μg/mL antibody in running buffer (HBS-EP+) was injected separately into the Fc3 channel for 30 s at a flow rate of 10 μL/min. The analyte CTLA-4 (WBP316.hCTLA-4.ECD-6xHis) and blank running buffer at 30 μL The flow rate per minute sequentially subjected the Fc1-Fc4 channels to a 120 s association phase followed by a 2400 s dissociation phase. After each dissociation phase, regeneration buffer (10 mM glycine pH 1.5) was injected for 30 s at a rate of 10 μL/min.

The binding kinetics of these antibodies were measured using SPR. Antibodies were captured on immobilized anti-human Fc and sequentially injected with different concentrations of CTLA-4-ECD. Subtract the sensorgrams for the reference and buffer channels from the test sensorgram. The data were used for a 1:1 binding assay of human, monkey and mouse CTLA-4.ECD-6xHis. As shown in Table 7, humanized antibodies W3162-1.146.19-Z12, W145 and W3162-1.154.8-Z35 bound to the human CTLA-4-ECD domain with affinities of 0.477nM, 1.84nM and 0.0968nM, respectively. Humanized antibodies have similar affinities compared to rat antibodies. W3162-1.146.19-Z12 and W3162-1.154.8-Z35 had significantly higher affinity than Ipilimumab (K _D =3.68 nM). Antibody W3162-1.146.19-Z12 can also bind murine CTLA-4, and its affinities before and after humanization are shown in Table 9. After humanization, its affinity of 1.39nM is slightly lower than that of the parental antibody of 0.906nM.

The affinities of W3162-1.146.19-Z12, W3162-1.145.10-z7 and W3162-1.154.8-Z35 to cynomolgus CTLA-4-ECD were 1.92nM, 0.598nM and 0.131nM, respectively (Table 8).

Table 7. Kinetics of Antibody Binding to Human CTLA-4ECD

Table 8. Kinetics of Antibody Binding to Monkey CTLA-4ECD

Table 9. Kinetics of Antibody Binding to Murine CTLA-4ECD

2.2.2 FACS detection of affinity

FITC-conjugated goat anti-human IgG Fc was purchased from Jackson Immunological Research Laboratories (cat. no. 109-095-098) and a BD CantoII was used for the assay. HEK293 cells expressing human CTLA-4 were transferred into 96-well U-bottom plates (BD) at a density of 5 × ¹⁰⁴ cells/well. Test antibodies were serially diluted 1:2 times in 1% BSA in PBS and incubated with cells for 1 hour at 4°C. After centrifugation at 1500 rpm for 4 minutes, the supernatant was discarded. Add the secondary antibody, FITC-conjugated goat anti-human IgG Fc (3.2 FITC per IgG, Jackson Immunological Research Laboratories), resuspend the cells to a final concentration of 14 μg/mL, and incubate at 4°C in the dark for 30 min . Cells were then washed once, resuspended in 1% BSA in PBS, and analyzed by flow cytometry (BD). Fluorescence intensity was converted to bound molecules/cell based on quantitative beads (QuantumTM MESF Kits, Bangs Laboratories). _KD was calculated using Graphpad Prism5.

The binding affinity of the humanized antibody to cell surface CTLA-4 was determined by flow cytometry and modified using the Benedict method [Benedict 1997 JIM]. After measuring antibody fluorescence bound to CTLA-4 expressing CHO cells, bound and free antibodies were analyzed and fitted to the equation shown in Figure 5, as shown in Figure 5. Based on the data and formulas, the calculated affinity constants _KD are shown in Table 10. The humanized antibodies W3162-1.146.19-Z12 and W3162-1.154.8-Z35 have high affinity, the affinity is 5.05 and 0.35nM, respectively, while the affinity of Ipilimumab is 0.97nM.

Table 10. Affinity Test (FACS)

2.3 Competition with ligands

To test whether the humanized antibodies retained their ability to block CTLA-4 binding to CD80 and CD86, both ELISA and FACS were used in competition assays. Two CTLA-4 ligands, CD80 and CD86, were purchased from Sino Biological (Catalogue No. 10698-H08H and 10699-H08H). Biotinylated anti-His tag antibody was purchased from Genscript (Catalog #A00613). HRP-conjugated streptavidin was purchased from Invitrogen (cat# SNN1004).

2.3.1 ELISA-based competition assay

ELISA is used to detect whether the antibody can inhibit the binding of human CTLA-4 to its ligands human CD80 and CD86. Plates were coated with human CTLA-4.ECD.hFc (0.5 μg/mL) for 16-20 hours at 4°C. After blocking with 2% BSA in DBPS for 1 hour, the test antibodies and positive and negative control antibodies were premixed with 0.25 μg/mL of CD80-6xHis or CD86-6xHis, then added to the plate and incubated for 1 hour at room temperature. After washing 3 times with PBS containing 0.05% Tween 20, the biotinylated anti-His tag antibody was diluted 1:2000 and added. Plates were incubated for 1 hour at room temperature. Bound ligand was detected by HRP-conjugated streptavidin (1 :20000). Color was developed with 100 μL TMB substrate for 8 minutes and then stopped with 100 μL 2N HCl. Absorbance at 450 nM was measured using a microplate spectrophotometer.

As shown in Figure 11, W3162-1.146.19-z12-IgGg and W3162-1.154.8-z35-IgGk have similar effects to Ipilimumab in blocking ligand binding to coated CTLA-4, for CD80, IC ₅₀ was 0.87nM, 0.63nM and 0.40nM; for CD86, _IC50 was 0.71nM, 0.50nM and 0.42nM.

2.3.2 FACS determination

To test whether the antibody could block the binding of CTLA-4 to cell surface CD80 and CD86, we used FACS to test this competition. CHO cell lines expressing CD80 and CD86 were developed by WuXi Biologics. Biotinylated CTLA-4.ECD.hFc is manufactured by WuXi Biologics. PE-conjugated streptavidin was purchased from eBioscience (cat# 12-4317).

CD80- or CD86-expressing cells were added to each well of a 96-well plate at 1 × 10 ⁵ /well, and centrifuged at 1500 rpm for 4 min at 4 °C, and then the supernatant was removed. Serial dilutions of test antibodies, positive and negative controls were mixed with biotinylated human CTLA4.ECD.hFc. Due to the different density of ligands on the cell surface, use 0.02 μg/mL of hCTLA-4.ECD.hFc-biotin for human CD80 cells and 0.08 μg/mL of hCTLA-4.ECD.hFc for human CD86 cells - Biotin. The antibody and CTLA-4 mixture was then added to the cells and incubated at 4°C for 1 hour. Cells were washed twice with 200 μL of FACS buffer (DPBS containing 1% BSA). Streptavidin PE diluted 1:333 in FACS buffer was added to the cells and incubated at 4°C for 1 hr. An additional washing step was performed twice with 200 μL of FACS buffer, followed by centrifugation at 1500 rpm for 4 min at 4 °C. Finally, the cells were resuspended in 100 μL of FACS buffer, the fluorescence value was detected by flow cytometry, and analyzed by FlowJo.

The results are shown in FIG. 12 . Two humanized antibodies blocked CTLA-4/ligand binding more effectively than Ipilimumab. At the highest concentration used, Ipilimumab blocked only 32% of CTLA-4 binding to CD80 and 40% of CTLA-4 binding to CD86. In contrast, antibody W3162-1.146.19-Z12 blocked 71% of CTLA-4 binding to CD80 and 73% of CTLA-4 binding to CD86, while antibody W3162-1.154.8-Z35 blocked 89% of CTLA-4 bound to CD80 and 98% of CTLA-4 bound to CD86. For CD80, the IC ₅₀ of Ipilimumab, W3162-1.146.19-Z12 and W3162-1.154.8-Z35 were 3.23nM, 6.60nM and 0.07nM, respectively. For CD86, the IC ₅₀ of Ipilimumab, W3162-1.146.19-Z12 and W3162-1.154.8-Z35 were 2.52nM, 5.15nM and 0.28nM, respectively.

2.4 Cytokine release from SEB-stimulated PBMCs

It was tested whether anti-CTLA4 antibody could enhance cytokine release from human PBMC after SEB (from Second Military Medical University) stimulation. Peripheral blood from healthy donors was obtained and cells were isolated by density gradient centrifugation with Ficoll (GE Healthcare, 17-1440-02). After removing the buoyant layer, the platelets were washed multiple times with medium. Add 1 x ¹⁰⁵ human PBMC cells to each well of a 96-well plate. Serial dilutions of the test antibody, positive and negative controls were mixed with SEB (10 ng/mL), then added to the pelleted cells and incubated at 37°C for 3 days. Supernatants were collected to measure human IL-2 concentrations.

For detection of human IL-2, microplates were pre-coated with 1.0 μg/mL human IL-2 antibody (R&D System MAB602) for 16-20 hours at 4°C. After blocking with 2% BSA (BovoGen) in DBPS for 1 hour, the supernatant containing IL-2 was added to the plate and incubated at room temperature for 2 hours. After washing 3 times with PBST (containing 0.05% Tween 20), diluted biotinylated human IL-2 antibody (R&D Systems, BAF202) was added at a concentration of 0.5 μg/mL. Plates were incubated for 1 hour at room temperature. Bound biotinylated antibody was detected by HRP-conjugated streptavidin (Invitrogen, SNN1004) diluted 1:20000. After 1 h of incubation, the color was developed by 100 μL of TMB substrate and then stopped with 100 μL of 2M HCl. Absorbance at 450 nm and 540 nm was measured using a microplate spectrophotometer.

In a cell-based assay, it was tested whether humanized antibodies (8.60 nM, 21.4 nM, 53.6 nM, 134 nM, 335 nM) could enhance superantigen SEB-stimulated human PBMC. Three days after stimulation, IL-2 from PBMCs was measured using ELISA. Both humanized antibodies (W3162-1.146.19-Z12, W3162-1.154.8-Z35) and Ipilimumab dose-dependently enhanced IL-2 release from PBMCs compared to isotype control antibodies (Figure 13) .

2.5 thermal stability

Test the stability of the lead antibody at different temperatures. 100 μL of each antibody sample was pipetted into each tube and the samples were incubated at 4°C or 37°C for 20 hours, or at 45°C or 50°C for 2 hours. The samples were then centrifuged at 12,000 rpm for 10 minutes. These samples were observed for possible precipitation and analyzed by SEC-HPLC for purity and elution time.

The SEC curves of W3162-1.146.19-Z12 under different conditions are shown in Fig. 14a–d. Neither the dilution time nor the main peak percentage (92.39% to 92.48%) changed significantly at high temperature compared to low temperature (92.24%). The SEC curves of W3162-1.154.8-Z35 under different high temperature conditions are shown in Fig. 14e–h. Neither the dilution time nor the percentage of the main peak (97.14% to 97.17%) changed significantly compared to the low temperature (96.84%). This set of data indicates that the antibody is stable under the high temperature conditions tested.

2.6 Non-specific binding

FACS and ELISA were used to test whether the antibody binds to other targets. In the FACS assay, different cell lines (Ramos, Raji, MDA-MB-453, BT474, Jurkat, Hut78, A431, A204, CaLu-6, A375, HepG2, BxPC-3, HT29, FaDu, 293F, CHO- K1) was adjusted to 1×10 ⁵ cells in each well. Test antibodies and isotype control antibodies were diluted to 10 μg/mL in PBS containing 1% BSA and incubated with cells for 1 hour at 4°C. Cells were washed twice with 180 μL of PBS containing 1% BSA. PE-conjugated goat anti-human IgG Fc fragment (Jackson, Cat. No. 109-115-098) was diluted to a final concentration of 5 μg/mL in PBS containing 1% BSA, then added to resuspend the cells, and incubated at 4 °C Incubate for 30 minutes in the dark. Perform two additional washing steps with 180 μL of PBS containing 1% BSA, followed by centrifugation at 1500 rpm for 4 min at 4 °C. Finally, cells were resuspended in 100 μL of PBS containing 1% BSA, and fluorescence values were measured by flow cytometry (BD CantoII) and analyzed by FlowJo.

In the ELISA assay, the test antibody, isotype control antibody and factors including factor VIII, FGFR-ECD, PD-1, CTLA-4.ECD, VEGF, HER3ECD, OX40.ECD, 1BB.ECD, CD22.ECD, CD3e.ECD 10 different target antigens. A 96-well plate was coated with each antigen (2 μg/mL) overnight at 4°C. After blocking with 2% BSA in PBS for 1 hour, wash 3 times with 300 μL of PBST. Test antibodies as well as isotype control antibodies were diluted to 10 μg/mL in PBS containing 2% BSA, then added to the plate and incubated for 2 hours at room temperature. After washing 3 times with 300 μL of PBST, HRP-coupled goat anti-human IgG antibody (diluted 1:5000 in 2% BSA) was added to the plate and incubated for 1 hour at room temperature. Finally, the plate was washed 6 times with 300 μL of PBST. Color was developed with 100 μL of TMB substrate for 12 minutes and then stopped with 100 μL of 2M HCl. Absorbance at 450 nM was measured using a microplate spectrophotometer.

In addition to CTLA-4, other unrelated proteins were also used to test whether antibodies W3162-1.146.19-Z12 and W3162-1.154.8-Z35 could bind these antigens. As shown in Figure 16, in the antigen group, only CTLA-4 was detected by both antibodies. Other antigens gave no signal in this ELISA assay. In contrast, the anti-OX40 antibody did bind OX40, indicating that this antigen was coated on the plate.

The specificity of both antibodies was also tested on a panel of different cell lines in a FACS assay. Antibodies did not produce any detectable signal for these cell lines (data not shown).

2.7 In vivo drug efficacy experiment

Since antibody W3162-1.146.19-Z12 cross-reacts with both human and murine CTLA-4, the antitumor efficacy of this antibody was tested in a syngeneic mouse model. Anti-CTLA-4 antibody W3162-1.146.19-Z12 was tested in a xenograft mouse model using the mouse cancer cell line CT26. An anti-mouse CTLA-4 antibody purchased from BioXCell was used as a positive control (BioXCell-BE0131). Tumor cells were maintained as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 5% CO ₂ at 37°C. After detaching cells by trypsin-EDTA treatment, tumor cells were routinely subcultured twice a week. Cells growing in exponential phase were harvested and seeded counted. Female Balb/C mice were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd., and mice aged 6-8 weeks and weighing about 18-22 grams were studied. Inoculate 1 x ¹⁰⁵ tumor cells subcutaneously in the right axilla of each mouse in 0.1 mL of PBS mixed with 50 μL of Matrigel. Animals were randomized when the average tumor volume reached 60-80 ^mm3 . Anti-CTLA-4 antibody and isotype control were used for treatment: intravenous injection twice a week. Tumor size was measured twice a week using vernier calipers, and tumor volume was calculated by the formula a×b ² ×π/6, where a is the length and b is the width (a>b).

W3162-1.146.19-Z12 (1 mg/kg, 3 mg/kg, 10 mg/kg) and control antibody (10 mg/kg) were injected twice weekly for ^two weeks when the average tumor volume reached approximately 70 mm. Animals were monitored for tumor growth and body weight over time. As shown in Figure 15, W3162-1.146.19-Z12 significantly inhibited tumor growth in a dose-dependent manner. At a dose of 1 mg/kg, W3162-1.146.19-Z12 inhibited tumor growth compared to the control group. At a dose of 3 mg/kg, W3162-1.146.19-Z12 suppressed tumor volume to 160 mm ³ at day 19, while 10 mg/kg W3162-1.146.19-Z12 induced tumor regression at the end of the study period.

2.8 Epitope positioning (mapping)

多位点定向诱变试剂盒(安捷伦科技公司，帕洛阿尔托，加州)将一组突变引物用于第一步PCR。在突变体链合成反应后，使用Dpn I核酸内切酶消化亲本模板。在第二步PCR中，在HEK293F细胞(LifeTechnologies，盖瑟斯堡，马里兰州)中扩增并瞬时表达由CMV启动子、CTLA4的突变ECD、His-标签和单纯疱疹病毒胸苷激酶(TK)多腺苷酸化组成的线性DNA表达盒，盖瑟斯堡，马里兰州)。此外，构建了三个质粒载体，以测试聚糖表位：pcDNA3.3-hCTLA4_ECD.His(N113Q)、pcDNA3.3-hCTLA4_ECD.His(N145Q)和pcDNA3.3-hCTLA4_ECD.His(N113Q，N145Q)。这三种突变蛋白在HEK293F细胞(Life Technologies，盖瑟斯堡，马里兰州)中瞬时表达。Alanine scanning was used to identify the CTLA-4 epitope of the antibody. In this experiment, the alanine residue on hCTLA4 was mutated to a glycine residue and all other residues were mutated to alanine. For each residue of the extracellular domain (ECD) of human CTLA4, spot amino acid substitutions were made using two sequential PCR steps. Use the pcDNA3.3-hCTLA4_ECD.His plasmid encoding the ECD of human CTLA4 and the C-terminal His tag as a template, and use

The Multiple Site-Directed Mutagenesis Kit (Agilent Technologies, Palo Alto, CA) used a set of mutation primers for the first PCR step. Following the mutant strand synthesis reaction, the parental template was digested with Dpn I endonuclease. In a second PCR step, amplified and transiently expressed in HEK293F cells (LifeTechnologies, Gaithersburg, MD) composed of the CMV promoter, mutant ECD of CTLA4, His-tag, and herpes simplex virus thymidine kinase (TK) polyadenylated linear DNA expression cassette, Gaithersburg, MD). In addition, three plasmid vectors were constructed to test glycan epitopes: pcDNA3.3-hCTLA4_ECD.His(N113Q), pcDNA3.3-hCTLA4_ECD.His(N145Q) and pcDNA3.3-hCTLA4_ECD.His(N113Q, N145Q) . These three muteins were transiently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD).

To test how mutations affect antibody binding, ELISA was performed. Ipilimumab, W3162-1.146.19-z12 and W3162-1.154.8-z35 (2 μg/mL) were captured by pre-coating with 2 μg/mL goat anti-human IgG Fc (Bethyl Laboratories, Montgomery, TX) . After interaction with the supernatant containing quantified CTLA4 mutein, HRP-conjugated anti-His antibody (1:5000, Rockland Immunochemicals, Pottstown, PA) was added as a detection antibody. TMB was used as a substrate for HRP. Absorbance was normalized to the mean of control mutants. The final identified epitope residues were determined after setting an additional cut-off point for fold change in binding (<0.55).

The binding activities of antibodies W3162-1.146.19-z12, W3162-1.154.8-z35 and Ipilimumab (W316-BMK1) to human CTLA4 were performed and all three antibodies were found to bind to human CTLA4 ( FIG. 17 ).

Test point mutations that affect antibody binding to CTLA-4 are shown in Table 11. Based on the human CTLA4 crystal structure (PDB code 1AH1), some amino acid residues (e.g., Met38, Val40, Tyr60, Val71, Val73, Arg75, Val84, Cys85, Cys129, Ile149) are unlikely to directly contact any antibody. The observed reduction in binding is most likely due to instability or even collapse of the CTLA4 structure after alanine substitution. The final identified epitope residues are listed in Table 12 and labeled on Figure 18.

As shown in Figure 18D and E, the epitopes of Ipilimumab and W3162-1.146.19-z12 overlap each other except for a few residues such as N145 and P138. In contrast, W3162-1.154.8-z35 bound to a smaller region of CTLA-4 than the other two antibodies (Fig. 18F). All three antibody-binding epitopes involve the ligand-binding domain of CTLA-4 (Figure 18A and B), and involve the MYPPPY motif.

The overlapping epitopes of Ipilimumab and W3162-1.146.19-z12 cannot explain the unique cross-species binding ability of antibody W3162-1.146.19-z12. Since the N145 mutation on CTLA-4 only affects CTLA-4-binding W3162-1.146.19-z12 and not the other two antibodies, we considered the N-glycosylation site as a potential epitope and made further Research. The effect of mutations at two glycosylation sites of CTLA4 on antibody binding activity is shown in FIG. 17 . Binding of Ipilimumab or W3162-1.154.8-z35 to mutant CTLA-4 was not significantly altered (Figure 17A and C). In contrast, binding of W3162-1.146.19-z12 to mutated CTLA-4 N145Q was significantly reduced, whereas binding of this antibody to CTLA-4 with N113Q was unchanged. This set of data suggested that the glycan on CTLA-4N145 (Fig. 18E) could be the epitope of W3162-1.146.19-z12. Whereas the N145 residue is conserved in CTLA-4 from cynomolgus monkeys and mice.

The present invention has been described above by means of examples. However, it should be understood by those skilled in the art that the present invention is not limited to these examples. The present invention may be embodied in other specific forms without departing from its forms or its essential characteristics. The scope of the invention is therefore indicated by the appended claims, rather than the foregoing description, and all changes which come within the scope of the appended claims and equivalents thereof are embraced.

Table 11. Effect of CTLA4 point mutation on antibody binding

^a The fold change in binding is the relative value of multiple alanine silent substitutions

Table 12. Discovery of three antibody epitopes

sequence listing

<110> Shanghai WuXi Biotechnology Co., Ltd.

<120> A novel CTLA-4 monoclonal antibody

<130> PCTF1701

<160> 47

<170> PatentIn version 3.3

<210> 1

<211> 114

<212> PRT

<213> Artificial Sequence

<400> 1

Glu Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Lys

1 5 10 15

Ser Leu Lys Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Arg Ser Ser

20 25 30

Ala Met His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Asp Trp Val

35 40 45

Ala Phe Ile Ser Ser Gly Gly Asp Thr Ala Tyr Ala Asp Ala Val Lys

50 55 60

Gly Arg Phe Ile Val Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu

65 70 75 80

Gln Leu Asn Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Val

85 90 95

Arg Met Glu Arg Ile Pro Thr Trp Gly Gln Gly Val Met Val Thr Val

100 105 110

Ser Ser

<210> 2

<211> 115

<212> PRT

<213> Artificial Sequence

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Asp Leu Thr Phe Ser Asn Tyr

20 25 30

Asp Met Ala Trp Val Arg Gln Thr Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Pro Asn Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Leu Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 3

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 3

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Tyr His Thr Ile Thr Ser Gly

20 25 30

Tyr Asp Trp Thr Trp Ile Arg Lys Phe Pro Gly Asn Gln Met Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu His Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Met Met Val Pro His Tyr Tyr Val Met Asp Ala Trp Gly Gln

100 105 110

Gly Ala Ser Val Thr Val Ser Ser

115 120

<210> 4

<211> 119

<212> PRT

<213> Artificial Sequence

<400> 4

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Ala Gly Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Val Asp Pro Glu Asn Gly Arg Ala Asp Tyr Ala Glu Lys Phe

50 55 60

Lys Lys Lys Ala Thr Leu Thr Ala Asp Thr Thr Ser Asn Thr Ala Tyr

65 70 75 80

Ile His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Arg Ala Met Asp Asn Tyr Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 5

<211> 115

<212> PRT

<213> Artificial Sequence

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Leu Thr Phe Ser Asn Tyr

20 25 30

Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Pro Ser Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 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 His Leu Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 6

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 6

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Tyr His Thr Ile Thr Ser Gly

20 25 30

Tyr Asp Trp Thr Trp Ile Arg Lys Pro Pro Gly Lys Gly Met Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Met Met Val Pro His Tyr Tyr Val Met Asp Ala Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 7

<211> 119

<212> PRT

<213> Artificial Sequence

<400> 7

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Val Asp Pro Glu Gln Gly Arg Ala Asp Tyr Ala Glu Lys Phe

50 55 60

Lys Lys Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Met Asp Asn Tyr Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 8

<211> 110

<212> PRT

<213> Artificial Sequence

<400> 8

Asp Ile Val Leu Thr Gln Ser Pro Val Leu Ala Val Ser Leu Gly Gln

1 5 10 15

Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Ser Ser

20 25 30

Ile Asn Leu Ile His Trp Tyr Gln Gln Arg Pro Gly Gln Gln Pro Lys

35 40 45

Leu Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asp Pro

65 70 75 80

Val Gln Ala Asp Asp Val Ala Asp Tyr Tyr Cys Gln Gln Ser Arg Glu

85 90 95

Ser Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 9

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 9

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Gln Ala Ser Gln Asp Ile Gly Ser Asn

20 25 30

Leu Ile Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Met Ile

35 40 45

Tyr Tyr Ala Thr His Leu Ala Asp Gly Val Pro Ser 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 Val Ala Asp Tyr His Cys Leu Gln Tyr Lys Gln Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 10

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 10

Asp Val Val Leu Thr Gln Thr Pro Pro Thr Ser Ser Ala Thr Ile Gly

1 5 10 15

Gln Ser Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gln Arg Pro Ser Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Val Ser Lys Leu Gly Ser Gly Val Pro

50 55 60

Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Gly Val Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Val Gln Gly

85 90 95

Thr His Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 11

<211> 106

<212> PRT

<213> Artificial Sequence

<400> 11

Glu Ile Met Leu Thr Gln Ser Pro Thr Ile Met Ala Ala Ser Leu Gly

1 5 10 15

Glu Lys Ile Thr Ile Thr Cys Ser Ala Asn Ser Ser Leu Ser Tyr Met

20 25 30

Tyr Trp Phe Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp Val His

35 40 45

Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Tyr Leu Thr Ile Asn Thr Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Phe Cys His His Trp Ser Asn Thr Gln Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 12

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 12

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 Gln Ala Ser Gln Asp Ile Gly Ser Asn

20 25 30

Leu Ile Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Met Ile

35 40 45

Tyr Tyr Ala Thr His Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Lys Gln Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 13

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Val Ser Lys Leu Gly Ser Gly Val Pro

50 55 60

Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Gly

85 90 95

Thr His Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 14

<211> 106

<212> PRT

<213> Artificial Sequence

<400> 14

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ala Leu Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Trp Val His

35 40 45

Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His His Trp Ser Asn Thr Gln Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 15

Met Glu Arg Ile Pro Thr

1 5

<210> 16

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 16

His Leu Trp Phe Ala Tyr

1 5

<210> 17

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 17

Met Met Val Pro His Tyr Tyr Val Met Asp Ala

1 5 10

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 18

Arg Ala Met Asp Asn Tyr Gly Phe Ala Tyr

1 5 10

<210> 19

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 19

Gln Gln Ser Arg Glu Ser Pro Leu Thr

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 20

Leu Gln Tyr Lys Gln Tyr Pro Arg Thr

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 21

Val Gln Gly Thr His Asp Pro Trp Thr

1 5

<210> 22

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 22

His His Trp Ser Asn Thr Gln Trp Thr

1 5

<210> 23

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 23

Phe Ile Ser Ser Gly Gly Asp Thr Ala Tyr Ala Asp Ala Val Lys Gly

1 5 10 15

<210> 24

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 24

Ser Ile Ser Pro Asn Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys

1 5 10 15

Gly

<210> 25

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 25

Tyr Ile Ser Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 26

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 26

Arg Val Asp Pro Glu Asn Gly Arg Ala Asp Tyr Ala Glu Lys Phe Lys

1 5 10 15

Lys

<210> 27

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 27

Ser Ile Ser Pro Ser Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys

1 5 10 15

Gly

<210> 28

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 28

Arg Val Asp Pro Glu Gln Gly Arg Ala Asp Tyr Ala Glu Lys Phe Lys

1 5 10 15

Lys

<210> 29

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 29

Arg Thr Ser Asn Leu Ala Ser

1 5

<210> 30

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 30

Tyr Ala Thr His Leu Ala Asp

1 5

<210> 31

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 31

Leu Val Ser Lys Leu Gly Ser

1 5

<210> 32

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 32

Gly Thr Ser Asn Leu Ala Ser

1 5

<210> 33

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 33

Ser Ser Ala Met His

1 5

<210> 34

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 34

Asn Tyr Asp Met Ala

1 5

<210> 35

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 35

Ser Gly Tyr Asp Trp Thr

1 5

<210> 36

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 36

Asn Tyr Phe Met Asn

1 5

<210> 37

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 37

Arg Ala Ser Gln Ser Val Ser Ile Ser Ser Ser Ile Asn Leu Ile His

1 5 10 15

<210> 38

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 38

Gln Ala Ser Gln Asp Ile Gly Ser Asn Leu Ile

1 5 10

<210> 39

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 39

Arg Ser Ser Gln Ser Leu Leu Asn Ser Asp Gly Asn Thr Tyr Leu Tyr

1 5 10 15

<210> 40

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 40

Ser Ala Asn Ser Ser Leu Ser Tyr Met Tyr

1 5 10

<210> 41

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 41

Ser Ala Asn Ser Ala Leu Ser Tyr Met Tyr

1 5 10

<210> 42

<211> 345

<212>DNA

<213> Artificial Sequence

<400> 42

gaggtgcagc tggtggagag cggcggagga ctggtgcaac ctggcggaag cctgagactg 60

agctgcgccg ccagcgacct gaccttcagc aactacgaca tggcctgggt gagacaggcc 120

cctggcaagg gactggagtg ggtggccagc atcagcccca gcggcggcaa cacctactac 180

agggacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa cagcctgtac 240

ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caggcacctg 300

tggttcgcct actggggcca gggcacactg gtgaccgtga gcagc 345

<210> 43

<211> 360

<212>DNA

<213> Artificial Sequence

<400> 43

caggtgcagc tgcaggagag cggacccgga ctggtgaagc cctccgagac cctgagcctg 60

acctgcagcg tgacctacca caccatcacc agcggctacg actggacctg gatcagaaag 120

ccccccggca aaggcatgga gtggatcggc tacatcagct acagcggcaa caccaactac 180

aacccccagcc tgaagagcag ggtgaccatc agcagggaca ccagcaagaa ccagttcttc 240

ctgaagctga gcagcgtgac agccgccgat accgccgtgt actactgcgc cagcatgatg 300

gtgccccact actacgtgat ggacgcctgg ggacagggca ccctggtgac agtgagcagc 360

<210> 44

<211> 357

<212>DNA

<213> Artificial Sequence

<400> 44

caggtgcagc tggtgcagag cggagccgag gtgaagaagc ccggcagcag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc aactacttca tgaactgggt gaggcaggcc 120

cctggacaag gcctggagtg gatgggcaga gtggatcccg agcagggcag ggccgactac 180

gccgagaagt tcaagaagag ggtgaccatc accgccgaca agagcaccag caccgcctac 240

atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcgc caggagagcc 300

atggacaact acggcttcgc ctactggggc cagggaaccc tggtgaccgt gagcagc 357

<210> 45

<211> 321

<212>DNA

<213> Artificial Sequence

<400> 45

gacatccaga tgacccagag ccctagcagc ctgagcgcca gcgtgggcga tagggtgacc 60

atcacctgcc aggccagcca ggacatcggc agcaacctga tctggttcca gcagaagccc 120

ggcaaggccc ccaagcctat gatctactac gccaccccacc tggccgatgg cgtgcctagc 180

agattcagcg gcagcagaag cggcaccgac tacaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgcctgcag tacaagcagt accccagaac cttcggcggc 300

ggcaccaagg tggagatcaa g 321

<210> 46

<211> 336

<212>DNA

<213> Artificial Sequence

<400> 46

gacatcgtga tgacccagac ccccctgagc ctgagcgtga cacctggaca gcccgccagc 60

atcagctgca ggtccagcca gagcctgctg aacagcgacg gcaacaccta cctgtactgg 120

tacctgcaga agcctggcca gagcccccag ctgctgatct acctggtgtc caagctgggc 180

agcggcgtgc ctaacaggtt tagcggcagc ggcagcggca ccgatttcac cctgaagatc 240

agcagggtgg aggccgagga tgtgggcgtg tactactgcg tgcagggcac ccacgatcct 300

tggaccttcg gcggcggaac caaggtggag atcaag 336

<210> 47

<211> 318

<212>DNA

<213> Artificial Sequence

<400> 47

gagatcgtgc tgacccagag ccccgacttc cagagcgtga cccccaagga gaaggtgacc 60

atcacctgca gcgccaacag cgccctgagc tacatgtact ggtaccagca gaagcccgac 120

cagagcccca agctgtgggt gcacggcacc agcaatctgg ccagcggcgt gcctagcaga 180

tttagcggca gcggcagcgg caccgatttc accctgacca tcaacagcct ggaggccgag 240

gacgccgcta cctactactg ccaccactgg agcaacaccc agtggacctt cggcggcggc 300

accaaggtgg agatcaag 318

Claims (18)

1. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds CTLA-4 and comprises a heavy chain variable region and a light chain variable region:

a. the heavy chain variable region comprises a CDR1 sequence shown in SEQ ID NO: 36, a CDR2 sequence shown in SEQ ID NO: 28 and a CDR3 sequence shown in SEQ ID NO: 18, and

b. the light chain variable region comprises a CDR1 sequence shown as SEQ ID NO: 41, a CDR2 sequence shown as SEQ ID NO: 32 and a CDR3 sequence shown as SEQ ID NO: 22.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO 7.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID No. 14.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO 14 and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO 7.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment inhibits binding of CTLA-4 to CD80 or CD86.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding epitope of the antibody or antigen-binding fragment comprises P138 of CTLA-4.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment binds to human and monkey CTLA-4.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment

a) Binding to human CTLA-4,K _D Is 4.77E-10M or less; and is

b) Binding to mouse CTLA-4,K _D Is 1.39E-09M or less.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment is a chimeric, humanized, or fully human antibody.

10. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 9.

11. A cloning or expression vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising one or more cloning or expression vectors of claim 11.

13. A method for producing the antibody of any one of claims 1 to 9, comprising culturing the host cell of claim 12, and isolating the antibody.

14. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

15. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 9 linked to a therapeutic agent.

16. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment or prevention of cancer.

17. The use of claim 16, wherein the cancer is melanoma, renal cancer, prostate cancer, lung cancer, head and neck cancer, rectal cancer, colon cancer, pancreatic cancer, or breast cancer.

18. The use of claim 17, wherein the melanoma is cutaneous or intraocular malignant melanoma.

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