Novel anti-CTLA4 antibody

Abstract

The present disclosure provides antibodies, particularly heavy chain antibodies, more particularly single domain antibodies, that specifically bind to cytotoxic T lymphocyte associated antigen-4 (CTLA4). Therapeutic uses of the antibodies are also disclosed.

Description

Novel anti-CTLA4 antibodies Technical Field

[0001] The present disclosure relates to antibodies and their uses. Specifically, the present disclosure relates to antibodies, particularly heavy chain antibodies, and more particularly single domain antibodies, that specifically bind to cytotoxic T lymphocyte-associated antigen-4 (CTLA4); and therapeutic uses of the antibodies. Background Art

[0002] As the first clinically targeted immune checkpoint receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA4, also known as CD152) is expressed in T cells. CTLA4 is constitutively expressed in regulatory T cells (Tregs) and is upregulated in other T cells upon activation ( et al., 2015, Jago et al., 2004). CTLA4 shares the same ligands as CD28, namely CD80 / B7-1 and CD86 / B7-2. Chikuma proposed that the mechanism of CTLA4 is to inhibit T cell activation by outcompeting CD28 for binding to CD80 / B7-1 and CD86 / B7-2 (Chikuma, 2017). CTLA4 is highly expressed in the tumor microenvironment (TME), particularly on Tregs, which are considered key molecules for regulating Treg function and modulating anti-tumor immunity (Montler et al., 2016, Sutmuller et al., 2001). Furthermore, CTLA4 is constitutively expressed in Foxp3+ Treg cells, inhibiting their anti-tumor activity (Montler et al., 2016, Sutmuller et al., 2001).

[0003] To date, several mechanisms of action of therapeutic CTLA4 antibodies have been proposed, including activation of effector T cells by blocking the B7-CTLA4 pathway and depletion of Tregs through antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) (Arce Vargas et al., 2018, Du et al., 2018). However, the clinical significance of these mechanisms remains controversial (Du et al., 2018).

[0004] Ipilimumab Ipilimumab is the most widely known anti-CTLA4 antibody, approved by the US FDA in 2011 for the treatment of advanced melanoma. Clinically, Ipilimumab has demonstrated effective cancer immunotherapy (CITE) efficacy both as a monotherapy (Hodi et al., 2010) and as part of a combination therapy with nivolumab (Larki et al., 2015). However, these CTLA4 therapies are associated with severe immunotherapy-related adverse events (irAEs) (Calabrese et al., 2018), particularly when combined with nivolumab. This is due to systemic activation of T cells by blocking the B7-CTLA4 pathway, which can reduce patient tolerance to the antibody (Hodi, 2010, Bertrand et al., 2015).

[0005] Even so, CTLA4 remains an important immunotherapy target because it can induce durable immune responses in cancer patients (Maio et al., 2015, Schadendorf et al., 2015). The main challenge in generating CTLA4 antibodies is to improve their safety and efficacy. Recently, Zhang et al. reported a novel CTLA4 antibody, HL32 (ONC-392), with a modified Fc, that uniquely targets CTLA4 to selectively eliminate tumor-infiltrating Tregs without affecting T cell activation in peripheral T cells (Zhang et al., 2019). Compared with other commercial or clinical-stage anti-CTLA4 antibodies, HL32 has more robust CITEs but significantly reduced irAEs in preclinical models (Zhang et al., 2019, Du et al., 2018) and in a Phase 1A / 1B clinical trial (PRESERVE-001, NCT04140526). They proposed that clinically effective anti-CTLA4 mAbs induce tumor rejection through a mechanism that is independent of checkpoint blockade but dependent on host Fc receptors (Du et al., 2018).

[0006] However, despite the above progress, there is still a need in the art for improved anti-CTLA-4 antibodies with increased response rates (RR) and reduced irAEs, as well as more effective immunotherapies.

[0007] Summary of the Invention

[0008] The inventors of this application have screened for improved anti-CTLA4 antibodies with different blocking abilities against CD80 / 86 and retained Fc function, thereby providing improved response rates and reduced irAEs. As demonstrated in the examples of this application, the antibodies inhibited the binding of CD80 / CD86 to CTLA4 to varying degrees while exhibiting a strong ADCC response.

[0009] Thus, in one aspect, the present disclosure provides an antibody that specifically binds CTLA4, wherein the antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3, respectively, comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to:

[0010] (1) SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3;

[0011] (2) SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15; or

[0012] (3) SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

[0013] In some embodiments, the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO:1, the CDR2 comprises or consists of the sequence shown in SEQ ID NO:2, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:3.

[0014] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:31.

[0015] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:4.

[0016] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5.

[0017] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:7.

[0018] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8.

[0019] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:10.

[0020] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11.

[0021] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:31.

[0022] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:32.

[0023] In some embodiments, the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 13, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 14, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO: 15.

[0024] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:16 or SEQ ID NO:19.

[0025] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:16.

[0026] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:17.

[0027] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:19.

[0028] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:20.

[0029] In some embodiments, the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO:22, the CDR2 comprises or consists of the sequence shown in SEQ ID NO:23, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:24.

[0030] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:25 or SEQ ID NO:28.

[0031] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:25.

[0032] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26.

[0033] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:28.

[0034] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:29.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of an intact antibody, a single-chain antibody (scFv), a heavy chain antibody, a single-domain antibody, a Fab fragment, a Fab' fragment, a F(ab')2, a Fv, a Fd fragment, and a bispecific antibody (BsAb).

[0036] In some embodiments, the antibody or antigen-binding fragment thereof is a heavy chain-only antibody.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of two heavy chains.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof is a single domain antibody.

[0039] In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof exhibits significant antibody-dependent cell-mediated cytotoxicity (ADCC).

[0041] In one aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0042] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding the antibody.

[0043] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:30 or SEQ ID NO:33.

[0044] In one aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule.

[0045] In one aspect, the present disclosure provides a host cell, which expresses the antibody, and / or contains the nucleic acid molecule or the expression vector.

[0046] In one aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, comprising administering the antibody or pharmaceutical composition in a therapeutically effective amount, wherein the disease or condition is cancer or an autoimmune disease.

[0047] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0048] In some embodiments, the antibody or pharmaceutical composition is administered in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0049] In one aspect, the present disclosure provides use of the antibody or the pharmaceutical composition in the preparation of a medicament for treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.

[0050] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0051] In some embodiments, the antibodies or pharmaceutical compositions are for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0052] In one aspect, the present disclosure provides the antibody or the pharmaceutical composition for use in treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.

[0053] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0054] In some embodiments, the antibodies or pharmaceutical compositions are for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0055] In one aspect, the present disclosure provides a kit comprising

[0056] a) the antibody or the pharmaceutical composition; and

[0057] b) Instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figures 1A and 1B show FACS analysis of the binding specificity of anti-CTLA4 antibodies (GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS019-4, GBD008-S004, GBD008-hS004-5, and GBD008-hS005-3-2) to cell surface human CTLA4 protein, where Ipi represents Ipilimumab.

[0059] Figures 2A-2F show FACS analysis of anti-CTLA4 antibodies blocking CD80 and CD86 binding to CTLA4, where Ipi represents Ipilimumab. Figure 2A shows that anti-CTLA4 antibodies (GBD008-hS005-3 and GBD008-hS005-5) block CD80 binding to CTLA4; Figure 2B shows that anti-CTLA4 antibodies (GBD008-S019 and GBD008-hS019-4) block CD80 binding to CTLA4; Figure 2C shows that anti-CTLA4 antibodies (GBD008-S004 and GBD008-hS004-5) block CD80 binding to CTLA4; Figure 2D shows that anti-CTLA4 antibodies (GBD008-hS Figure 2E shows that anti-CTLA4 antibodies (GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS019-4, GBD008-S004, and GBD008-hS004-5) blocked CD86 binding to CTLA4; Figure 2F shows that anti-CTLA4 antibody (GBD008-hS005-3-2) blocked CD86 binding to CTLA4.

[0060] Figures 3A and 3B show analysis of IL2 production using CTLA4 antibodies (GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS019-4, GBD008-S004, GBD008-hS004-5, and GBD008-hS005-3-2) by SEB assay.

[0061] Figures 4A-4F show the binding ability of anti-CTLA4 antibodies (GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS019-4, GBD008-S004, GBD008-hS004-5, and GBD008-hS005-3-2) to human CTLA4 (Figures 4A and 4B), cynomolgus macaque CTLA4 (Figures 4C and 4D), and mouse CTLA4 (Figures 4E and 4F) by ELISA.

[0062] FIG5 shows the results of ADCC, wherein Ipi represents Ipilimumab, the assay using PBMCs as effector cells and Raji cells expressing full-length human CTLA4 as target cells (E:T=20:1), induced with anti-CTLA4 antibody.

[0063] Figure 6 shows the in vivo anti-tumor efficacy of anti-CTLA4 antibodies in the MC38 colorectal tumor model. Figure 6A) Average tumor volume over time. Figure 6B) Average tumor volume and TGI (Tumor Growth Inhibition) at the end of the experiment.

[0064] Figures 7A-7C show the toxicity (arthritis) of anti-CTLA4 antibodies in hCTLA4 / hPD1 Balb / C mice. Figure 7A) Trends in mouse body weight changes. Figure 7B) Arthritis scores (paw and joint inflammation) in mice. Figure 7C) Incidence of arthritis in mice.

[0065] Detailed Description of the Invention

[0066] The present disclosure provides antibodies that specifically bind to CTLA4. Administering a therapeutically effective amount of an anti-CTLA4 antibody of the present disclosure to a subject in need thereof can be used to treat diseases or conditions, including cancer and autoimmune diseases. Binding of the antibody to T cells expressing CTLA4 induces strong antibody-dependent cell-mediated cytotoxicity against cells expressing tumor-associated antigens.

[0067] The antibodies disclosed herein offer various advantages, such as efficient binding to CTLA4, effective induction of strong antibody-dependent cell-mediated cytotoxicity, and / or significantly reduced risk of adverse events (eg, toxicity).

[0068] In certain aspects, anti-CTLA4 antibodies in certain formats (e.g., heavy chain-only antibodies compared to typical full-length antibodies, i.e., heavy chain antibodies or single domain antibodies) bind to CTLA4 more efficiently, leading to higher efficacy and improved immunotherapy.

[0069] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of a conflict, the definitions in this specification shall prevail.

[0070] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present invention. Although only certain exemplary materials and methods are described herein, many methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure.

[0071] Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" as used herein are intended to include the plural forms as well. In addition, the open-ended expressions "including" and "comprising" are interpreted as also including structural components or method steps that are not mentioned, but it should be noted that such open-ended expressions also cover situations where the composition consists only of the components and method steps (i.e., they cover situations where the closed-ended expression "consists of...").

[0072] In general, the term "about" is used herein to refer to a numerical value above and below the stated value with a modification of 5%.

[0073] As used in the entire text, range is used as a shorthand form for describing each numerical value and all numerical values ​​within the range. Any numerical value within the range, such as an integer value, a value that increases progressively by one tenth (when the end value of the range is one decimal place), or a value that increases progressively by one hundredth (when the end value of the range is two decimal places) can be selected as the endpoint of the range. For example, range 1-10 is used to describe all numerical values ​​within the range, such as 1, 2, 3, 4, 5, 6, 7, 8 ... 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10 (values ​​that increase progressively by one tenth), and includes all subranges, such as 1-1.5, 2.0-3.0, 4.0-5.0, 6.0-7.0, 8.0-9.0, etc.

[0074] The term "antibody" as used herein may include whole antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portion") or single chains. In one embodiment, an "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises a heavy chain variable region (herein referred to as a V H ) and heavy chain constant region. In some naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2 and CH3. In some naturally occurring antibodies, each light chain comprises a light chain variable region (herein referred to as V L ) and the light chain constant region. The light chain constant region comprises one domain, CL. V H and VL The V domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), alternating with regions of greater conserved expression, termed framework regions (FRs). H and V L It comprises three CDRs and four framework regions (FRs), arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains comprise a binding domain that interacts with an antigen. 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. Unless otherwise indicated, immunoglobulins may be from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, for example, human IgG1, exist as several allotypes that differ from each other at most in a few amino acids.

[0075] Unless otherwise indicated, "antibody" may include, for example, whole antibodies, single-chain antibodies (scFv), heavy-chain antibodies, single-domain antibodies, Fab fragments, Fab' fragments, F(ab')2, Fv, Fd fragments, bispecific antibodies (BsAb), etc.; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies, etc.

[0076] As used herein, an "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen to which the intact antibody binds. An "antigen-binding fragment" is any proteinaceous structure that can exhibit binding affinity for a specific antigen. Antigen-binding fragments include those provided by any known technology such as enzymatic cleavage, peptide synthesis, and recombinant technology. Some antigen-binding fragments consist of portions of intact antibodies that retain the antigen-binding specificity of the parent antibody molecule. For example, an antigen-binding fragment may comprise at least one variable region (heavy chain variable region or light chain variable region) or one or more CDRs of an antibody known to bind to a specific antigen. Examples of suitable antigen-binding fragments include, but are not limited to, diabodies and single-chain molecules, as well as Fab, F(ab')2, Fc, Fabc, and Fv molecules; single-chain (Sc) antibodies; single antibody light chains; single antibody heavy chains; chimeric fusions between antibody chains or CDRs and other proteins; protein scaffolds; heavy chain monomers or dimers; light chain monomers or dimers; dimers consisting of one heavy chain and one light chain; monovalent fragments consisting of VL, VH, CL, and CH1 domains; or monovalent antibodies as described in WO2007059782; a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; an Fd fragment consisting essentially of the VH and CH1 domains; an Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody; a dAb fragment consisting essentially of a VH domain, also known as a domain antibody; alpacas or nanobodies; isolated complementarity-determining regions (CDRs), etc. All antibody isotypes can be used to generate antigen-binding fragments. Additionally, antigen-binding fragments may include non-antibody proteinaceous frameworks that can be successfully incorporated into polypeptide segments in an orientation that confers affinity for a given antigen of interest (such as a protein scaffold). Antigen-binding fragments may be produced recombinantly or by enzymatic or chemical cleavage of intact antibodies.

[0077] As used herein, the term "heavy chain antibody" or "heavy chain-only antibody" refers to a single-chain antibody comprising only a heavy chain. A "heavy chain antibody" lacks not only a light chain but also the constant region CH1 region found in a conventional antibody heavy chain. Compared to a heavy chain antibody, a "single domain antibody" as used herein lacks an Fc region.

[0078] As used herein, the term sequence "identity" refers to the relationship between two or more polynucleotide sequences or two or more polypeptide sequences. When a position in a sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparison sequence, the sequence is called "identical" at that position. Percent sequence "identity" is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences to generate the number of "identical" positions. Then, the number of "identical" positions is divided by the total number of positions in the comparison window and multiplied by 100 to generate the percentage of sequence "identity". The percentage of "identity" is determined by comparing the two best aligned sequences in the comparison window. In order to optimally align sequences for comparison, the portion of the polynucleotide or polypeptide sequence in the comparison window can include additions or deletions called spaces, while the reference sequence remains constant. The best alignment is the alignment that produces the maximum possible number of "identical" positions between the reference sequence and the comparison sequence even with spaces. For example, the percent "identity" between two sequences can be determined using a version of the program "BLAST 2 Sequences" available from the National Center for Biotechnology Information (NCBI), which includes the programs BLASTN (for nucleotide sequence comparisons) and BLASTP (for polypeptide sequence comparisons), which are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993).

[0079] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody, such as a mouse antibody, are replaced by corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domain are replaced by amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions are unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted as long as they do not abolish the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigenic specificity similar to that of the original antibody.

[0080] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, e.g., an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody. A "hybrid" antibody refers to an antibody having heavy and light chains of different types, e.g., a mouse (parent) heavy chain and a humanized light chain, or vice versa.

[0081] As used herein, the term "monoclonal antibody" refers to an antibody that displays a single binding specificity and affinity for a particular epitope, or an antibody composition in which all antibodies display a single binding specificity and affinity for a particular epitope.

[0082] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region genes (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0083] As used herein, the terms "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated process in which nonspecific cytotoxic cells expressing FcγRs (e.g., monocytes, such as natural killer (NK) cells and macrophages) recognize bound antibodies (or other proteins capable of binding to FcγRs) on target cells and subsequently cause target cell lysis. In principle, any effector cell with an activating FcγR can be triggered to mediate ADCC. The primary cell mediating ADCC is NK cells, which express only FcγRIII, while monocytes can express FcγRI, FcγRII, and FcγRIII depending on their activation, localization, or differentiation state.

[0084] In one aspect, the present disclosure provides an antibody that specifically binds to CTLA4, wherein the antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 each comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to:

[0085] (1) SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3;

[0086] (2) SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15; or

[0087] (3) SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

[0088] In some embodiments, the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO:1, the CDR2 comprises or consists of the sequence shown in SEQ ID NO:2, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:3.

[0089] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:31.

[0090] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:4.

[0091] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5.

[0092] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:7.

[0093] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8.

[0094] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:10.

[0095] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11.

[0096] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:31.

[0097] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:32.

[0098] In some embodiments, the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 13, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 14, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO: 15.

[0099] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:16 or SEQ ID NO:19.

[0100] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:16.

[0101] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:17.

[0102] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:19.

[0103] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:20.

[0104] In some embodiments, the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO:22, the CDR2 comprises or consists of the sequence shown in SEQ ID NO:23, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:24.

[0105] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:25 or SEQ ID NO:28.

[0106] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:25.

[0107] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26.

[0108] In some embodiments, the heavy chain variable region comprises or consists of SEQ ID NO:28.

[0109] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:29.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of an intact antibody, a single-chain antibody (scFv), a heavy chain antibody, a single-domain antibody, a Fab fragment, a Fab' fragment, a F(ab')2, a Fv, a Fd fragment, and a bispecific antibody (BsAb).

[0111] In some embodiments, the antibodies of the present disclosure consist of only heavy chains. In some embodiments, the antibodies of the present disclosure comprise or consist of two heavy chains. In some embodiments, the antibodies of the present disclosure are single domain antibodies.

[0112] In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0113] In some embodiments, the antibody is a monoclonal antibody.

[0114] In some embodiments, the antibody is isolated.

[0115] In some embodiments, the antibody is an isolated monoclonal antibody.

[0116] Also provided are "conservative sequence modifications" to the antibody sequences provided herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate binding of the antibody encoded by the nucleotide sequence or comprising the amino acid sequence to the antigen. For example, modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with 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 amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0117] In some embodiments, the antibodies exhibit potent antibody-dependent cell-mediated cytotoxicity (ADCC).

[0118] In one aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody and a pharmaceutically acceptable carrier, which may be an inert or physiologically active carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any physiologically compatible solvent, dispersion medium, coating, antibacterial agent, antifungal agent, etc. Examples of suitable carriers include water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and any combination thereof.

[0119] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding the antibody.

[0120] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:30 or SEQ ID NO:33.

[0121] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 6. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 9. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 12. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 18. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 21. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 27.In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 30. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 33.

[0122] In one aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule.

[0123] In one aspect, the present disclosure provides a host cell, which expresses the antibody, and / or contains the nucleic acid molecule or the expression vector.

[0124] In one aspect, the present disclosure provides the antibody or the pharmaceutical composition for use in treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.

[0125] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0126] In some embodiments, the antibodies or pharmaceutical compositions are for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0127] In one aspect, the present disclosure provides use of the antibody or the pharmaceutical composition in the preparation of a medicament for treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.

[0128] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0129] In some embodiments, the antibodies or pharmaceutical compositions are for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0130] In one aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, comprising administering the antibody or pharmaceutical composition in a therapeutically effective amount, wherein the disease or condition is cancer or an autoimmune disease.

[0131] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0132] In some embodiments, the antibody or pharmaceutical composition is administered in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0133] "Administer" refers to the use of any of the various methods and delivery systems known to those skilled in the art to introduce a composition comprising a therapeutic agent into a subject. The route of administration of the pharmaceutical composition of the present disclosure includes intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration routes, such as by injection or infusion. The phrase "parenteral administration" used herein refers to the administration method generally injected except enteral and topical administration, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion and in vivo electroporation. In some embodiments, the composition is administered by non-parenteral route, in some embodiments, by oral administration. Other non-parenteral routes include topical, epidermal or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual or topical. Administration can also, for example, be performed once, repeatedly and / or over one or more extended time periods.

[0134] As used herein, the term "effective amount" refers to the amount of an active agent required to provide a therapeutic and / or prophylactic benefit to a subject.

[0135] The "subject in need thereof" refers to any mammal, such as but not limited to humans, horses, cows, cats, mice, rabbits, rats, goats, etc. Preferably, the mammal is a human.

[0136] In one aspect, the present disclosure provides a kit comprising

[0137] a) the antibody or the pharmaceutical composition; and

[0138] b) Instructions for use.

[0139] Table 1A. Sequence information

[0140] Table 1B. Sequence Description

[0141] The sequences of exemplary antibodies disclosed herein are shown in Table 2 below:

[0142] Table 2: Full-length sequences of exemplary antibodies

[0143] The invention is further described with reference to the following non-limiting examples. Example

[0144] The development, characterization and in vivo anti-tumor efficacy of the above eight antibodies: GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS019-4, GBD008-S004, GBD008-hS004-5 and GBD008-hS005-3-2 are exemplarily described below.

[0145] Example 1. Preparation of anti-CTLA4 antibodies

[0146] Camelids, such as camels or alpacas, are ideal animal models for producing antibodies containing only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. VHH antibodies naturally lack light chains but retain structural stability and effective antigen binding properties. Methods for obtaining antigen- or epitope-specific VHH domains have been previously described, for example, in WO2006 / 040153 and WO2006 / 122786; R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 Jun. 2009 and WO94 / 04678. The anti-CTLA4 VHH antibody sequence was obtained by immunizing alpacas with human CTLA4 / CD153 protein (Acro Biosystem, Catalog # CT4-H5229). The VHH sequence obtained from alpacas was optimized by changing the amino acids in the original VHH sequence and replacing them with the common amino acids that appear in the VH domain of human antibodies.

[0147] Example 2. FACS analysis of the binding specificity of anti-CTLA4 antibodies to cell surface human CTLA4 protein

[0148] CHOK1-CTLA4 cells (1x10 5 Cells were washed twice in FACS buffer (PBS + 2% BSA) per well and resuspended in 100 μl of FACS buffer containing serially diluted (1:5) anti-CTLA4 mAb and incubated at 4°C for 1 hour. The cells were then washed twice in FACS buffer and bound antibodies were detected by incubation with APC anti-human IgG Fc (Biolegend, Catalog #410712) for 1 hour at 4°C. Afterwards, the cells were washed twice in FACS buffer and then collected and analyzed using a BD LSRFortessa (BD Biosciences).

[0149] As shown in Figures 1A and B, the results showed that GBD008-S004, GBD008-hS005, GBD008-S019, and the humanized GBD008-hS005-3, GBD008-hS005-5, GBD008-hS019-4, GBD008-hS004-5, and GBD008-hS005-3-2 antibodies all exhibited specific binding to the CTLA4 protein. In particular, the mean fluorescence intensity (MFI) trends of GBD008-S019 and GBD008-hS019-4 were similar to those of ipilimumab, indicating that their binding specificity was comparable to that of ipilimumab. The mean fluorescence intensity (MFI) trends of GBD008-S005, GBD008-hS005-3, GBD008-hS005-3-2 and GBD008-hS005-5 were second best, and the MFI trends of GBD008-S004, GBD008-hS004-5 and CTLA4 were weaker.

[0150] Example 3. FACS analysis of anti-CTLA4 antibodies blocking CD80 and CD86 binding to CTLA4

[0151] CHOK1-CTLA4 cells (1x10 5 Cells were washed twice in FACS buffer (PBS + 2% BSA) per well and resuspended in 100 μl of FACS buffer containing serially diluted (1:5) anti-CTLA4 mAb and CD80 Biotinylated tag (SinoBiological, Catlog #10698-H49H-B) or CD86 Biotinylated tag (SinoBiological, Catlog #10699-H08H-B) and incubated at 4°C for 0.5 hours. The cells were then washed twice in FACS buffer and bound antibodies were detected by incubation with Streptavidi-Alexa Flour 488 (Invitrogen, Catlog #S32354) for 0.5 hours at 4°C. Afterwards, the cells were washed twice in FACS buffer and then collected and analyzed using a BD LSRFortessa (BD Biosciences).

[0152] As shown in Figures 2A-F, the results showed that the anti-CTLA4 antibodies GBD008-S004, GBD008-hS005, GBD008-S019, and the humanized GBD008-hS005-3, GBD008-hS005-5, GBD008-hS005-3-2, GBD008-hS019-4, and GBD008-hS004-5 antibodies all partially inhibited the binding of CD80 or CD86 to CTLA4. In particular, the mean fluorescence intensity (MFI) trends of GBD008-S019 and GBD008-hS019-4 were almost identical to those of ipilimumab, indicating that their blocking abilities were comparable to those of ipilimumab. The mean fluorescence intensity (MFI) trends of GBD008-S005, GBD008-hS005-3, GBD008-hS005-3-2, and GBD008-hS005-5 were second best, indicating that their blocking abilities were second best, and the mean fluorescence intensity (MFI) trends of GBD008-S004 and GBD008-hS004-5 with CTLA4 were weaker, indicating that their blocking abilities were weaker.

[0153] Example 4. Affinity of CTLA-4 Antibody Fc Fusion Protein for CTLA-4 (Octet Method)

[0154] The binding kinetics of CTLA-4 antibody Fc fusion protein and recombinant human CTLA-4 were analyzed qualitatively and quantitatively by Octet method (Octect RH-16, Sartorius). on ) and dissociation rate (k off ) was calculated using a simple one-to-one binding model (Octet Evaluation Software version 12.2). The equilibrium dissociation constant (k D ) is calculated as the ratio k off / k on The results are shown in Table 3. The results in Table 3 indicate that all CTLA4 antibody Fc fusion proteins bind to the CTLA4 antigen with strong affinity.

[0155] Table 3. Affinity results of CTLA4 antibody Fc fusion protein to CTLA4

[0156] Example 5. SEB assay for analyzing IL2 production by using CTLA4 antibody

[0157] Serially diluted antibodies and Staphylococcal enterotoxin B (SEB) (Sigma-Aldrich, Catlog # S4881) were added to effector human peripheral blood mononuclear cells (1×10 5 / well), these cells were obtained from individual donors ( Biotechnologies) and cultured in RPMI 1640 medium (Gibco, Catlog #A10491-01) containing 10% FBS (Gibco, Catlog #10099-141). The cells were cultured at 37°C for 4 days, after which the sample supernatants were collected and mixed with premixed IL2-HTRF antibody (PerkinElmer, Catlog #62HIL02PET). The sample plates were sealed and incubated at room temperature for 3 hours. Afterwards, the plates were removed from the seal and read on an EnVision 2105 multimode plate reader (PerkinElmer).

[0158] As shown in Figures 3A and B, the results showed that GBD008-S004, GBD008-hS005, GBD008-S019, and humanized GBD008-hS005-3, GBD008-hS005-3-2, GBD008-hS005-5, GBD008-hS019-4, and GBD008-hS004-5 antibodies all significantly promoted the production of IL2 in effector cells in a concentration-dependent manner, among which the IL2 production of GBD008-S019 and GBD008-hS019-4 was comparable to that of Ipilimumab. GBD008-S004, GBD008-hS005, and humanized GBD008-hS005-3, GBD008-hS005-3-2, and GBD008-hS005-5 produced the least IL2, while GBD008-hS004-5 and CTLA4 produced the least IL2. These results indicate that the antibodies disclosed herein have the potential to treat cancer or autoimmune diseases.

[0159] Example 6. Binding ability of anti-CTLA4 antibodies to cynomolgus monkey and mouse CTLA4 (by ELISA)

[0160] Plates were coated overnight at 4°C with 1 μg / ml human (Acro Biosystem, Catlog #CT4-H52H9), cynomolgus monkey (Acro Biosystem, Catlog #Ct4-C82E5), or mouse CTLA4 (Acro Biosystem, Catlog #CT4-M52H5). Blocking was then performed with blocking buffer at room temperature. A gradient dilution series of anti-CTLA4 antibodies was then added and allowed to react for 1 hour at room temperature. Goat anti-human IgG Fc (HRP) (Abcam, Catlog #ab97225) was added and allowed to react for 1 hour at room temperature. The colorimetric solution was added and then stopped, and the absorbance was read at a wavelength of 450 nm using an EnVision 2105 multimode plate reader (PerkinElmer). The results are shown in Figures 4A-C.

[0161] The absorbance data in Figures 4A-F show that GBD008-S004, GBD008-hS005, GBD008-S019, and humanized GBD008-hS005-3, GBD008-hS005-3-2, GBD008-hS005-5, GBD008-hS019-4, and GBD008-hS004-5 antibodies all bind to human and cynomolgus monkey CTLA4, but do not bind to mouse CTLA4.

[0162] Example 7. ADCC killing assay of anti-CTLA4 antibodies

[0163] Cytotoxic activity was assessed using FACS analysis. Effector cells Human peripheral blood mononuclear cells were obtained from individual human donors ( Biotechnologies) and cultured overnight with 10 ng / ml hIL-2 (PeproTech, 200-02). Target Raji cells expressing full-length human CTLA4 were labeled with 3 ml of detection buffer from the EuTDA kit mixed with 5 μl of DELFIA BATDA reagent (PerkinElmer, Catlog #AD16) at 37°C for 20 minutes, washed twice with RPMI 1640 medium (Gibco, Catlog #A10491-01) containing 10% FBS (Gibco, Catlog #10099-141), and seeded in 96-well round-bottom plates at a 20:1 effector cell to target cell ratio. Serially diluted anti-CTLA4 mAb was added to the designated rows of the assay plate. After incubation at 37°C for 2 hours, 20 μl of the supernatant was centrifuged and incubated with 200 μl of europium solution at room temperature for 10 minutes and read on an EnVision 2105 multimode plate reader (PerkinElmer). The results are shown in FIG5 .

[0164] The results demonstrate that the anti-CTLA4 antibodies of the present disclosure effectively induce target cell lysis and death.

[0165] Example 8. In vivo anti-tumor efficacy of anti-CTLA4 antibodies in the MC38 colorectal tumor model

[0166] MC38 (NTCC-MC38) C57BL / 6 human CTLA4 knock-in mice were subcutaneously inoculated with colon cancer cells (n=6 per group, female, 7-8 weeks old). 3 ), two doses of ipilimumab (0.3 mg / kg and 3 mg / kg) and two doses of the anti-CTLA4 antibody disclosed herein (0.15 mg / kg and 1.5 mg / kg) were administered by intraperitoneal injection. Treatment was continued twice a week for three weeks. Tumor growth was monitored every three days and reported as mean tumor volume. The results are shown in Figure 6.

[0167] As can be seen from Figure 6, the average tumor volume of mice treated with the anti-CTLA4 antibodies of the present disclosure was significantly smaller than that of the IgG1 control. In addition, at the end of the experiment, the average tumor volume of mice was reduced by 62% for the 0.15 mpk dose, and by 99% for the 1.5 mpk dose.

[0168] Example 9. Toxicity of anti-CTLA4 antibodies in the hCTLA4 / hPD1 Balb / C mouse model (arthritis)

[0169] Human CTLA4 / hPD1 knock-in Balb / C mice (n=8 per group, female, 4 weeks old) were intraperitoneally injected with IgG1 (30 mg / kg), a combination of ipilimumab and nivolumab (15 mg / kg + 15 mg / kg), and a combination of GBD008-hS005-3-2 and nivolumab (7.5 mg / kg + 15 mg / kg). Treatment was continued twice a week until arthritis (paw and joint inflammation) resolved. Mouse body weight and arthritis scores were monitored every three or four days. The results are shown in Figures 7A-C.

[0170] As can be seen from Figure 7B, the arthritis incidence in mice treated with the anti-CTLA4 antibody (GBD008-hS005-3-2) of the present disclosure in combination with Nivolumab was significantly less than that in the IgG1 control group and the group treated with the combination of Ipilimumab and Nivolumab. Furthermore, as can be seen from Figure 7C, the incidence of arthritis in mice treated with the combination of GBD008-hS005-3-2 and Nivolumab was also significantly less than that in the IgG1 control group and the group treated with the combination of Ipilimumab and Nivolumab.

[0171] The above examples demonstrate that the anti-CTLA4 antibodies disclosed herein inhibit the binding of CD80 / CD86 to CTLA4 to varying degrees while exhibiting a strong ADCC response. By varying degrees of CD80 / 86 blocking ability and retaining the antibody's Fc function, the anti-CTLA4 antibodies exhibit varying degrees of IL-2 response. Furthermore, the anti-CTLA4 antibodies disclosed herein exhibit excellent in vivo anti-tumor efficacy and minimal toxicity in mouse models.

[0172] Although various embodiments of the present invention have been described above, it should be understood that they are provided by way of example only and not limitation. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications will fall within the scope of the present invention as claimed. The scope of the present invention as claimed is defined by the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CTLA4, wherein the antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 each comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to: (1) SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; (2) SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15; or (3) SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:

24.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 1, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 2, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:

3.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO:

31. 4 . The antibody or antigen-binding fragment thereof according to claim 3 , wherein the heavy chain variable region comprises or consists of SEQ ID NO:

4.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

5. 6 . The antibody or antigen-binding fragment thereof according to claim 3 , wherein the heavy chain variable region comprises or consists of SEQ ID NO:

7.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

8.

8. The antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises or consists of SEQ ID NO:

10.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

11. 10 . The antibody or antigen-binding fragment thereof according to claim 3 , wherein the heavy chain variable region comprises or consists of SEQ ID NO:

31.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

32.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 13, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 14, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:

15.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 16 or SEQ ID NO:

19. 14 . The antibody or antigen-binding fragment thereof according to claim 13 , wherein the heavy chain variable region comprises or consists of SEQ ID NO:

16.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

17.

16. The antibody or antigen-binding fragment thereof according to claim 13, wherein the heavy chain variable region comprises or consists of SEQ ID NO:

19.

17. The antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

20.

18. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 22, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 23, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:

24.

19. The antibody or antigen-binding fragment thereof of claim 18, wherein the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 25 or SEQ ID NO:

28.

20. The antibody or antigen-binding fragment thereof according to claim 19, wherein the heavy chain variable region comprises or consists of SEQ ID NO:

25.

21. The antibody or antigen-binding fragment thereof of claim 20, wherein the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

26.

22. The antibody or antigen-binding fragment thereof of claim 19, wherein the heavy chain variable region comprises or consists of SEQ ID NO:

28.

23. The antibody or antigen-binding fragment thereof of claim 22, wherein the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

29.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a whole antibody, a single-chain antibody (scFv), a heavy chain antibody, a single domain antibody, a Fab fragment, a Fab' fragment, a F(ab')2, a Fv, a Fd fragment, and a bispecific antibody (BsAb).

25. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, wherein the antibody or antigen-binding fragment thereof is a heavy chain-only antibody.

26. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, wherein the antibody or antigen-binding fragment thereof comprises or consists of two heavy chains.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, wherein the antibody or antigen-binding fragment thereof is a single domain antibody.

28. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 27, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.

29. The antibody of any one of claims 1-28, wherein the antibody or antigen-binding fragment thereof exhibits significant antibody-dependent cell-mediated cytotoxicity (ADCC).

30. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

31. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-29.

32. The nucleic acid molecule of claim 31 , wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:21 , SEQ ID NO:27, SEQ ID NO:30 or SEQ ID NO:

33.

33. An expression vector comprising the nucleic acid molecule of claim 31 or 32.

34. A host cell expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, and / or comprising the nucleic acid molecule according to claim 31 or 32 or the expression vector according to claim 33.

35. A method of treating a disease or condition in a subject in need thereof, the method comprising administering the antibody or antigen-binding fragment thereof of any one of claims 1-29 or the pharmaceutical composition of claim 30 in a therapeutically effective amount, wherein the disease or condition is cancer or an autoimmune disease.

36. The method of claim 35, wherein the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

37. The method of claim 35 or 36, wherein the antibody or antigen-binding fragment thereof or pharmaceutical composition is administered in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines or other antibodies.

38. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 29 or the pharmaceutical composition of claim 30 in the preparation of a medicament for treating a disease or condition, wherein the disease or condition is cancer or an autoimmune disease.

39. The method of claim 38, wherein the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

40. The use of claim 38 or 39, wherein the antibody or antigen-binding fragment thereof or pharmaceutical composition is for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines or other antibodies.

41. A medicine kit comprising a) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 29 or the 30 pharmaceutical composition; and b) Instructions for use.

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