Novel anti-CTLA4 antibody

Abstract

This disclosure provides antibodies, particularly heavy chain antibodies, and more particularly single-domain antibodies, that specifically bind cytotoxic T lymphocyte-associated antigen-4 (CTLA 4). Therapeutic uses of such antibodies are also disclosed.

Description

[Technical Field]

[0001] This disclosure relates to antibodies and their uses. Specifically, this disclosure relates to antibodies that specifically bind cytotoxic T lymphocyte-associated antigen-4 (CTLA 4), particularly heavy chain antibodies, and more particularly single-domain antibodies, and the therapeutic use of said antibodies. [Background technology]

[0002] Cytotoxic T lymphocyte-associated antigen-4 (CTLA4, also known as CD152) is expressed on T cells as the first clinically recognized targeted immune checkpoint receptor. CTLA4 is constitutively expressed in regulatory T cells (Tregs) and is upregulated in other T cells after activation (Sledzinska et al., 2015, Jago et al., 2004). CTLA4 shares the same ligands as CD28, namely CD80 / B7-1 and CD86 / B7-2. Chikuma has proposed that the mechanism of CTLA4 suppresses T cell activation by winning the binding competition with CD28 for CD80 / B7-1 and CD86 / B7-2 (Chikuma, 2017). CTLA4 is highly expressed in the tumor microenvironment (TME), and is considered to be an important molecule for regulating Treg function and modulating antitumor immunity, particularly in Tregs (Montler et al., 2016, Sutmuller et al., 2001). Furthermore, CTLA4 is constitutively expressed in Foxp 3+ Treg cells and suppresses antitumor activity (Montler et al., 2016, Sutmuller et al., 2001).

[0003] To date, several mechanisms of action for therapeutic CTLA 4 antibodies have been proposed, including activation of effector T cells by blocking the B7-CTLA 4 pathway, consumption of Tregs via antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP) (Arce Vargas et al., 2018, Du et al., 2018). However, the clinical significance of these mechanisms remains a subject of debate (Du et al., 2018).

[0004] Ipilimumab (Yervoy®) is the most well-known anti-CTLA4 antibody and was approved by the US FDA in 2011 for the treatment of advanced melanoma. Clinically, ipilimumab has shown effective cancer immunotherapy efficacy (CITE) as monotherapy (Hodi et al., 2010) and as part of combination therapy with nivolumab (Larki et al., 2015). However, the above CTLA4 therapy has shown serious immunotherapy-related adverse events (irAEs) (Calabrese et al., 2018), and in particular, when combined with nivolumab treatment, it led to decreased antibody resistance in patients by systemic activation of T cells through blockade of the B7-CTLA4 pathway (Hodi, 2010, Bertrand et al., 2015).

[0005] Nevertheless, CTLA4 is an important immunotherapy target because it can induce a sustained immune response in cancer patients (Maio et al., 2015, Schadendorf et al., 2015). The main challenge in producing CTLA4 antibodies is to enhance their safety and efficacy. Recently, Zhang et al. reported HL 32 (ONC-392), a novel CTLA4 antibody with modified Fc that uniquely targets CTLA4 to selectively remove tumor-infiltrating Tregs without affecting T cell activation in peripheral T cells (Zhang et al., 2019). Compared to other commercially available or clinical-stage anti-CTLA4 antibodies, HL32 showed milder CITEs in preclinical models (Zhang et al., 2019, Du et al., 2018) and Phase 1 A / 1 B clinical trials (PRESERVE-001, NCT 04140526), ​​but significantly reduced irAEs. They proposed that clinically effective anti-CTLA4 mAbs induce tumor rejection through a mechanism independent of checkpoint blockade but dependent on host Fc receptors (Du et al., 2018).

[0006] However, despite the aforementioned advancements, there is a need in this field for improved anti-CTLA-4 antibodies with enhanced response rates (RR) and reduced irAEs, as well as more effective immunotherapies. [Overview of the project] [Problems that the invention aims to solve]

[0007] The inventors of the present invention screened for antibodies that exhibit different blocking abilities against CD80 / 86 and retain Fc function, and provided an improved anti-CTLA4 antibody that exhibits improved reaction rate and reduced irAE. As confirmed by the examples of the present invention, the antibodies inhibited the binding of CD80 / CD86 to CTLA4 to different degrees while simultaneously exhibiting a strong ADCC reaction. [Means for solving the problem]

[0008] Accordingly, in one embodiment, the present disclosure provides an antibody that specifically binds to CTLA4, wherein the antibody includes a heavy chain variable region, and the heavy chain variable region comprises CDR1, CDR2, and CDR3, each containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with the following sequences. (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.

[0009] In some embodiments, the heavy chain variable region includes CDR1 containing or consisting of the sequence shown in SEQ ID NO:1, CDR2 containing or consisting of the sequence shown in SEQ ID NO:2, and CDR3 containing or consisting of the sequence shown in SEQ ID NO:3.

[0010] In some embodiments, the heavy chain variable region includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:31.

[0011] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:4.

[0012] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:5.

[0013] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:7.

[0014] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:8.

[0015] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:10.

[0016] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:11.

[0017] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:31.

[0018] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:32.

[0019] In some embodiments, the heavy chain variable region includes CDR1 containing or consisting of the sequence shown in SEQ ID NO:13, CDR2 containing or consisting of the sequence shown in SEQ ID NO:14, and CDR3 containing or consisting of the sequence shown in SEQ ID NO:15.

[0020] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16 or SEQ ID NO: 19.

[0021] In some embodiments, the heavy chain variable region comprises SEQ ID NO: 16 or consists of SEQ ID NO: 16.

[0022] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17.

[0023] In some embodiments, the heavy chain variable region comprises SEQ ID NO: 19 or consists of SEQ ID NO: 19.

[0024] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 20.

[0025] In some embodiments, the heavy chain variable region comprises a CDR1 comprising or consisting of the sequence shown in SEQ ID NO: 22, a CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 23, and a CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 24.

[0026] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 25 or SEQ ID NO: 28.

[0027] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:25.

[0028] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:26.

[0029] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:28.

[0030] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:29.

[0031] In some embodiments, the antibody or its antigen-binding fragment is selected from the group consisting of complete antibodies, single-chain antibodies (scFv), heavy-chain antibodies, single-domain antibodies, Fab fragments, Fab' fragments, F(ab')2, Fv, Fd fragments, and bispecific antibodies (BsAb).

[0032] In some embodiments, the antibody or its antigen-binding fragment is an antibody consisting only of the heavy chain.

[0033] In some embodiments, the antibody or its antigen-binding fragment contains or consists of two heavy chains.

[0034] In some embodiments, the antibody or its antigen-binding fragment is a single-domain antibody.

[0035] In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0036] In some embodiments, the antibody or its antigen-binding fragment exhibits significant antibody-dependent cell-mediated cytotoxicity (ADCC).

[0037] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier.

[0038] In one embodiment, the present disclosure provides an isolated nucleic acid molecule encoding the antibody.

[0039] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with 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.

[0040] In one embodiment, the present disclosure provides an expression vector comprising the nucleic acid molecule.

[0041] In one embodiment, the present disclosure provides a host cell that expresses the antibody and / or contains the nucleic acid molecule or the expression vector.

[0042] In one embodiment, the present disclosure provides a method for treating a disease or condition which is cancer or an autoimmune disease in a subject of interest, comprising administering the antibody or pharmaceutical composition in a therapeutically effective amount.

[0043] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, squamous cell carcinoma, melanoma, myeloma, stomach 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.

[0044] In some embodiments, the antibody or pharmaceutical composition is administered in combination with one or more other chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0045] In one embodiment, the present disclosure provides the use of the antibody or pharmaceutical composition in the manufacture of a pharmaceutical product for treating a disease or condition which is cancer or an autoimmune disease.

[0046] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, squamous cell carcinoma, melanoma, myeloma, stomach 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.

[0047] In some embodiments, the antibody or pharmaceutical composition is used in combination with one or more other chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0048] In one embodiment, the present disclosure provides an antibody or pharmaceutical composition for treating a disease or condition which is cancer or an autoimmune disease.

[0049] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, squamous cell carcinoma, melanoma, myeloma, stomach 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.

[0050] In some embodiments, the antibody or pharmaceutical composition is used in combination with one or more other chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0051] In one aspect, this disclosure is, a) The antibody or the pharmaceutical composition and b) Usage specifications and It provides a medicine container that includes [the necessary components]. [Brief explanation of the drawing]

[0052] [Figure 1] Figures 1A and 1B show FACS analysis of the binding specificity of anti-CTLA 4 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 protein on the cell surface, where Ipi represents ipilimumab. [Figure 2] Figures 2A-2F show FACS analysis of anti-CTLA4 antibodies blocking the binding of CD80 and CD86 to CTLA4, where Ipi represents ipilimumab. Figure 2A shows that anti-CTLA4 antibodies (GBD008-hS005-3 and GBD008-hS005-5) block the binding of CD80 to CTLA4. Figure 2B shows that anti-CTLA4 antibodies (GBD008-S019 and GBD008-hS019-4) block the binding of CD80 to CTLA4. Figure 2C shows that anti-CTLA4 antibodies (GBD008-S004 and GBD008-hS004-5) block the binding of CD80 to CTLA4. Figure 2D shows that anti-CTLA4 antibody (GBD008-hS005-3-2) blocks the binding of CD80 to CTLA4. Figure 2E shows that the anti-CTLA4 antibodies (GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS019-4, GBD008-S004, and GBD008-hS004-5) block the binding of CD86 to CTLA4. Figure 2F shows that the anti-CTLA4 antibody (GBD008-hS005-3-2) blocks the binding of CD86 to CTLA4. [Figure 3]Figures 3A and 3B show the analysis of IL2 generated 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 the SEB assay method. [Figure 4] Figures 4A-4F show the binding affinity (by ELISA) 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 monkey CTLA4 (Figures 4C and 4D), and mouse CTLA4 (Figures 4E and 4F). [Figure 5] Figure 5 shows the ADCC results, where Ipi represents ipilimumab. The measurement method uses PBMCs as effector cells and Raji cells expressing the full length of human CTLA4 (E:T=20:1) as target cells, and is induced by an anti-CTLA4 antibody. [Figure 6] Figure 6 shows the in vivo antitumor effect of anti-CTLA4 antibody in the MC38 colorectal tumor model. Figure 6A shows the mean values ​​of the change in tumor volume over time. Figure 6B shows the mean values ​​of tumor volume and TGI (Tumor Growth Inhibition) at the end of the experiment. [Figure 7] Figures 7A-7C show the toxicity (arthritis) of anti-CTLA4 antibodies in hCTLA4 / hPD1 Balb / C mice. Figure 7A shows the trend of weight change in mice. Figure 7B shows the arthritis (inflammation of the paws and joints) score in mice. Figure 7C shows the incidence of arthritis in mice. [Modes for carrying out the invention]

[0053] This disclosure provides an antibody that specifically binds to CTLA4. The anti-CTLA4 antibody of this disclosure can be used to treat diseases or conditions, including cancer and autoimmune diseases, by administering a therapeutically effective dose to the subject in need. The binding of the antibody to T cells expressing CTLA4 induces strong antibody-dependent cell-mediated cytotoxicity against cells expressing tumor-associated antigens.

[0054] The antibodies described herein offer various advantages, such as effectively binding to CTLA4, effectively inducing strong antibody-dependent cell-mediated cytotoxicity, and / or significantly reducing the risk of adverse events (e.g., toxicity).

[0055] In some embodiments, anti-CTLA4 antibodies bind more effectively to CTLA4 in several forms (e.g., heavy-chain antibodies or single-domain antibodies compared to typical full-length antibodies), resulting in higher potency and improved immunotherapy.

[0056] All scientific and technical terms used herein have the same meanings as those generally understood by those skilled in the art, unless otherwise defined. In case of conflict, the definitions herein shall apply.

[0057] The terms used herein are for the purpose of describing specific embodiments and are not intended to limit the invention. While only a few exemplary materials and methods are described herein, many similar or equivalent methods and materials can be used in the practice of this disclosure.

[0058] Unless otherwise explicitly stated, the singular forms “one,” “one,” and “the foregoing” as used herein are also intended to include the plural forms. Furthermore, while the open expressions “includes” and “inclusive” may include structural components or process steps not mentioned, it should be noted that these open expressions also include cases where the components and process steps described herein consist only of those described herein (i.e., cases where the closed expression “consists of” is also included).

[0059] Generally, the term "approximately" is used herein to refer to a 5% change above and below the stated value.

[0060] As used throughout this specification, "range" is used as an abbreviation to describe each value and all values ​​within that range. Any number within a range, such as an integer, a tenth increment (if the value at the end of the range has one decimal place), or a hundredth increment (if the value at the end of the range has two decimal places), can be selected as the endpoint of the range. For example, the range 1–10 is intended to describe all numbers within this range: 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, 10 (tenth increment), 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.

[0061] As used herein, the term “antibody” may include the entire antibody and any antigen-binding fragment thereof (i.e., “antigen-binding portion”) or a single chain. In one embodiment, “antibody” refers to a glycoprotein or its antigen-binding fragment comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V as used herein). HIt includes a light chain variable region (abbreviated as V in this specification) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region includes three domains: CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain has a light chain variable region (V in this specification). L It includes the (abbreviated as) and the light chain constant region. The light chain constant region contains one domain of CL. V H and V L The domain can be further subdivided into relatively conservative domains called framework domains (FRs) and highly variable domains called complementarity-determining domains (CDRs), which are arranged alternately. H and V L The antibody contains three CDRs and four framework regions (FRs), which are arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Unless otherwise specified, immunoglobulins may be from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. In certain species, IgG isotypes are classified into subclasses: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, such as human IgG1, exist in several allotypes that differ from each other by up to a few amino acids.

[0062] Unless otherwise specified, "antibodies" include, for example, complete antibodies, single-chain antibodies (scFv), heavy-chain antibodies, single-domain antibodies, Fab fragments, Fab' fragments, F(ab')2, Fv, Fd fragments, bispecific antibodies (BsAb), monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, and totally synthetic antibodies.

[0063] As used herein, an antibody “antigen-binding fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen to which the complete antibody binds. An “antigen-binding fragment” is any proteinaceous structure capable of exhibiting binding affinity to a particular antigen. Antigen-binding fragments include those provided by known techniques such as enzymatic cleavage, peptide synthesis, and recombination techniques. Some antigen-binding fragments consist of an antigen-binding specificity moiety that holds the parent antibody molecule of the complete antibody. For example, an antigen-binding fragment may include 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 particular antigen. Examples of suitable antigen-binding fragments include, but are not limited to, bispecific antibodies 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 WO 2007059782, bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region, Fd fragments consisting basically of VH and CH1 domains, Fv fragments consisting basically of the VL and VH domains of a single arm of the antibody, dAb fragments consisting basically of the VH domain, also called domain antibodies, alpaca or nanoantibodies, isolated complementarity-determining regions (CDRs), etc. All antibody isotypes can be used to generate antigen-binding fragments. The antigen-binding fragments may also include non-antibody protein frameworks that successfully incorporate polypeptide segments in an orientation that confers affinity to a given antigen of interest (e.g., a protein scaffold). The antigen-binding fragments may be generated by recombination or by enzymatic or chemical cleavage against a complete antibody.

[0064] As used herein, the terms “heavy chain antibody” or “heavy chain-only antibody” refer to a single-chain antibody containing only the heavy chain. A “heavy chain antibody” lacks not only the light chain but also the CH1 region, which is a constant region in a typical antibody heavy chain. A “single-domain antibody,” as used herein, lacks the Fc region compared to a heavy chain antibody.

[0065] As used herein, the term sequence “identity” refers to the relationship between two or more polynucleotide sequences or two or more polypeptide sequences. Sequences are said to be “same” at a position if a position in one sequence is occupied by the same nucleic acid base or amino acid residue at the corresponding position in the comparison sequence. Percent sequence “identity” is calculated by determining the number of positions in which the same nucleic acid base or amino acid residue appears in the two sequences, resulting in a number of “same” positions. The number of “same” positions is then divided by the total number of positions in the comparison window and multiplied by 100 to produce the percentage of sequence “identity.” The percentage of “identity” is determined by comparing two sequences that are optimally aligned within the comparison window. To optimally align and compare sequences, some of the polynucleotide or polypeptide sequences within the comparison window may contain additions or deletions called gaps, while the reference sequence remains constant. Optimal alignment is the alignment that, even with gaps, produces the maximum possible number of “same” positions between the reference and comparison sequences. For example, the percentage "identity" between two sequences can be determined using the "BLAST 2 Sequences" program, which includes the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison) obtained from the National Center for Biotechnology Information (NCBI). These programs are based on the Karlin and Altscul algorithm (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993).

[0066] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR structural domain of a non-human antibody, such as a mouse antibody, are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized antibody, some, most, or all of the amino acids outside the CDR structural domain are replaced with amino acids derived from human immunoglobulins, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not eliminate the antibody's ability to bind to a particular antigen. Humanized antibodies retain antigen specificity similar to that of the original antibody.

[0067] A "chimeric antibody" refers to an antibody in which the variable region originates from one species and the constant region from another species, for example, an antibody in which the variable region originates from a mouse antibody and the constant region from a human antibody. A "hybrid" antibody refers to an antibody that has different types of heavy and light chains, for example, a mouse (parental) heavy chain and a humanized light chain, or vice versa.

[0068] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits single-binding specificity and affinity to a specific epitope, or an antibody composition in which all antibodies exhibit single-binding specificity and affinity to a specific epitope.

[0069] As used herein, “isotype” refers to the antibody type encoded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0070] As used herein, the terms “antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated process in which nonspecific cytotoxic cells expressing FcγR (e.g., natural killer (NK) cells and monocytes such as macrophages) recognize a binding antibody (or other protein capable of binding to FcγR) on target cells, and subsequently cause degradation of those target cells. In principle, any effector cell with FcγR activation can be induced to mediate ADCC. The primary cells mediating ADCC are NK cells expressing only FcγRIII, although monocytes can express FcγRI, FcγRII, and FcγRIII depending on their activation, localization, or differentiation state.

[0071] In one embodiment, the present disclosure provides an antibody that specifically binds to CTLA4, wherein the antibody includes a heavy chain variable region, and the heavy chain variable region comprises CDR1, CDR2, and CDR3, each containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with the following sequences. (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.

[0072] In some embodiments, the heavy chain variable region includes CDR1 containing or consisting of the sequence shown in SEQ ID NO:1, CDR2 containing or consisting of the sequence shown in SEQ ID NO:2, and CDR3 containing or consisting of the sequence shown in SEQ ID NO:3.

[0073] In some embodiments, the heavy chain variable region includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:31.

[0074] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:4.

[0075] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:5.

[0076] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:7.

[0077] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:8.

[0078] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:10.

[0079] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:11.

[0080] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:31.

[0081] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:32.

[0082] In some embodiments, the heavy chain variable region includes CDR1 containing or consisting of the sequence shown in SEQ ID NO:13, CDR2 containing or consisting of the sequence shown in SEQ ID NO:14, and CDR3 containing or consisting of the sequence shown in SEQ ID NO:15.

[0083] In some embodiments, the heavy chain variable region includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:16 or SEQ ID NO:19.

[0084] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:16.

[0085] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:17.

[0086] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:19.

[0087] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:20.

[0088] In some embodiments, the heavy chain variable region includes CDR1 containing or consisting of the sequence shown in SEQ ID NO:22, CDR2 containing or consisting of the sequence shown in SEQ ID NO:23, and CDR3 containing or consisting of the sequence shown in SEQ ID NO:24.

[0089] In some embodiments, the heavy chain variable region includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:25 or SEQ ID NO:28.

[0090] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:25.

[0091] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:26.

[0092] In some embodiments, the heavy chain variable region includes or consists of SEQ ID NO:28.

[0093] In some embodiments, the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:29.

[0094] In some embodiments, the antibody or its antigen-binding fragment is selected from the group consisting of complete antibodies, single-chain antibodies (scFv), heavy-chain antibodies, single-domain antibodies, Fab fragments, Fab' fragments, F(ab')2, Fv, Fd fragments, and bispecific antibodies (BsAb).

[0095] In some embodiments, the antibody of this disclosure consists of a heavy chain only. In some embodiments, the antibody of this disclosure contains two heavy chains or consists of two heavy chains. In some embodiments, the antibody of this disclosure is a single-domain antibody.

[0096] In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0097] In some embodiments, the antibody is a monoclonal antibody.

[0098] In some embodiments, the antibody is isolated.

[0099] In some embodiments, the antibody is an isolated monoclonal antibody.

[0100] Provided herein are “conserved sequence modifications” to antibody sequences, i.e., nucleotide and amino acid sequence modifications that do not remove the binding of the antibody to the antigen, either encoded by a nucleotide sequence or containing an amino acid sequence. For example, modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis or PCR-induced mutagenesis. Conserved sequence modifications include conserved amino acid 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 are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., asparagine, glutamic acid), polar side chains with no charge (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).

[0101] In some embodiments, the antibody exhibits strong antibody-dependent cell-mediated cytotoxicity (ADCC).

[0102] In one embodiment, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody and a pharmaceutically acceptable carrier, which may be an inactive or physiologically active carrier. As used herein, the term “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial agent, and antifungal agent. Examples of suitable carriers include water, saline, phosphate-buffered saline, dextrose, glycerin, ethanol, and any combination thereof.

[0103] In one embodiment, the present disclosure provides an isolated nucleic acid molecule encoding the antibody.

[0104] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with 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.

[0105] In some embodiments, the nucleic acid molecule includes a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:6. In some embodiments, the nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:6. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:6. In some embodiments, the nucleic acid molecule includes a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:9. In some embodiments, the nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:9. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:9. In some embodiments, the nucleic acid molecule includes a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:12. In some embodiments, the nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:12. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:12. In some embodiments, the nucleic acid molecule includes a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:18. In some embodiments, the nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:18. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:18. In some embodiments, the nucleic acid molecule contains a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:21. In some embodiments, the nucleic acid molecule contains the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:21.In some embodiments, the nucleic acid molecule includes a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:27. In some embodiments, the nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:27. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:27. In some embodiments, the nucleic acid molecule includes a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:30. In some embodiments, the nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:30. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:30. In some embodiments, the nucleic acid molecule contains a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:33. In some embodiments, the nucleic acid molecule contains the nucleotide sequence of SEQ ID NO:33. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:33.

[0106] In one embodiment, the present disclosure provides an expression vector comprising the nucleic acid molecule.

[0107] In one embodiment, the present disclosure provides a host cell that expresses the antibody and / or contains the nucleic acid molecule or the expression vector.

[0108] In one embodiment, the present disclosure provides an antibody or pharmaceutical composition for treating a disease or condition which is cancer or an autoimmune disease.

[0109] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, squamous cell carcinoma, melanoma, myeloma, stomach 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.

[0110] In some embodiments, the antibody or pharmaceutical composition is used in combination with one or more other chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0111] In one embodiment, the present disclosure provides the use of the antibody or pharmaceutical composition in the manufacture of a pharmaceutical product for treating a disease or condition which is cancer or an autoimmune disease.

[0112] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, squamous cell carcinoma, melanoma, myeloma, stomach 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.

[0113] In some embodiments, the antibody or pharmaceutical composition is used in combination with one or more other chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0114] In one embodiment, the present disclosure provides a method for treating a disease or condition which is cancer or an autoimmune disease in a subject of interest, comprising administering the antibody or pharmaceutical composition in a therapeutically effective amount.

[0115] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, squamous cell carcinoma, melanoma, myeloma, stomach 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.

[0116] In some embodiments, the antibody or pharmaceutical composition is administered in combination with one or more other chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0117] "Administration" means introducing a composition containing a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. The routes of administration of the pharmaceutical compositions of this disclosure include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes by injection or infusion. As used herein, the term "parenteral administration" means a conventional form of administration by injection other than intestinal and topical administration, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intra-shearing, intralymphatic, intra-injury, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intravertebral, epidural, and intrasternal injections and infusions, as well as internal electroperforation. In some embodiments, the composition is administered by routes other than parenteral, and in some embodiments, it is administered orally. Other routes other than parenteral include topical, epidermal, or mucosal administration routes, such as nasal, vaginal, rectal, sublingual, or topical. The administration may be, for example, a single dose, multiple doses, and / or one or more extended periods.

[0118] As used herein, the term “effective dose” refers to the amount of activator required to provide a subject with therapeutic and / or prophylactic benefits.

[0119] The aforementioned "required subjects" means, but is not limited to, any mammal such as humans, horses, cattle, cats, mice, rabbits, rats, and goats. Preferably, the mammal is a human.

[0120] In one aspect, this disclosure is, a) The antibody or the pharmaceutical composition and b) Usage specifications and It provides a medicine container that includes [the necessary components].

[0121] [Table 1A] TIFF2026512775000003.tif245168TIFF2026512775000004.tif245168TIFF2026512775000005.tif242168TIFF2026512775 000006.tif242168TIFF2026512775000007.tif245168TIFF2026512775000008.tif245168TIFF2026512775000009.tif94168

[0122] [Table 1B] TIFF2026512775000011.tif190168

[0123] The sequences of the exemplary antibodies in this disclosure are shown in Table 2 below.

[0124] [Table 2]

[0125] The present invention will be further described with reference to the following non-limiting embodiments. [Examples]

[0126] The development, characterization, and in vivo antitumor effects of the eight antibodies mentioned above—namely, GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS019-4, GBD008-S004, GBD008-hS004-5, and GBD008-hS005-3-2—are described below with illustrative examples.

[0127] Example 1: Preparation of anti-CTLA 4 antibody Camelids, such as camels and alpacas, are ideal animal models for producing antibodies containing a single heavy chain variable region (VHH) and two normal CH2 and CH3 regions. VHH antibodies naturally lack a light chain but maintain structural stability and effective antigen-binding properties. Methods for obtaining antigen or epitope-specific VHH domains were previously described, for example, in WO 2006 / 040153 and WO 2006 / 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, Deffer et al., African Journal of Biotechnology Vol 8 (12), pp. 2645-2652, 17 Jun. 2009 and WO 94 / 04678. By immunizing alpacas with human CTLA4 / CD153 protein, an anti-CTLA4 VHH antibody sequence (Acro Biosystem, Catalog#CT4-H5229) was obtained. The VHH sequence obtained from alpacas was sequence-optimized by replacing the amino acids in the original VHH sequence with common amino acids found in the human antibody VH domain.

[0128] Example 2: FACS analysis of the binding specificity of anti-CTLA4 antibodies to human CTLA4 protein on the cell surface. CHOK 1-CTLA4 cells (1 x 10 5 The cells were washed twice with FACS buffer (PBS + 2% BSA), 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 with FACS buffer, and the bound antibody was detected by incubation with APC anti-human IgG Fc (Biolegend, Catalog #410712) at 4°C for 1 hour. After washing the cells twice with FACS buffer, they were collected and analyzed using BD LRFortesa (BD Biosciences).

[0129] As shown in Figures 1A and B, the results revealed that the antibodies GBD008-S004, GBD008-hS005, GBD008-S019, the humanized GBD008-hS005-3, GBD008-hS005-5, GBD008-hS019-4, GBD008-hS004-5, and GBD008-hS005-3-2 all specifically bound to the CTLA4 protein. In particular, the mean fluorescence intensity (MFI) trends of GBD008-S019 and GBD008-hS019-4 were close to those of ipilimumab, indicating that their binding specificity was comparable to that of ipilimumab. The mean fluorescence intensity (MFI) trends for GBD008-S005, GBD008-hS005-3, GBD008-hS005-3-2, and GBD008-hS005-5 are as follows, while the mean fluorescence intensity (MFI) trends for GBD008-S004, GBD008-hS004-5, and CTLA4 are weaker.

[0130] Example 3: FACS analysis showing that anti-CTLA4 antibody blocks the binding of CD80 and CD86 to CTLA 4. CHOK 1-CTLA4 cells (1 x 10 5 Cells were washed twice with FACS buffer (PBS + 2% BSA), resuspended in 100 μl of FACS buffer containing serially diluted (1:5) anti-CTLA4 mAb and either a CD80 biotinylation tag (SinoBiological, Catlog#10698-H49H-B) or a CD86 biotinylation tag (SinoBiological, Catlog#10699-H08H-B), and incubated at 4°C for 0.5 hours. Subsequently, cells were washed twice with FACS buffer, and conjugated antibodies were detected by incubation with Streptavidi-Alexa Flour 488 (Invitrogen, Catlog#S32354) at 4°C for 0.5 hours. After washing cells twice with FACS buffer, they were collected and analyzed using BD LRFortesa (BD Biosciences).

[0131] As shown in FIGS. 2A-F, as a result, it was revealed that all of the anti-CTLA4 antibodies GBD008-S004, GBD008-hS005, GBD008-S019, humanized GBD008-hS005-3, GBD008-hS005-5, GBD008-hS005-3-2, GBD008-hS019-4 and GBD008-hS004-5 partially inhibited the binding of CD80 or CD86 to CTLA4. In particular, the average fluorescence intensity (MFI) trends of GBD008-S019 and GBD008-hS019-4 were almost equivalent to those of ipilimumab, indicating that their blocking ability was comparable to that of ipilimumab. The average fluorescence intensity (MFI) trends of GBD008-S005, GBD008-hS005-3, GBD008-hS005-3-2 and GBD008-hS005-5 were next, indicating that their blocking ability was next, and the average fluorescence intensity (MFI) trends of GBD008-S004, GBD008-hS004-5 and CTLA4 were weaker, indicating that their blocking ability was weaker.

[0132] Example 4 Affinity of CTLA-4 Antibody Fc Fusion Protein for CTLA-4 (Octet Method) The binding kinetics of CTLA-4 antibody Fc fusion protein and recombinant human CTLA-4 were qualitatively and quantitatively analyzed for protein interaction by the Octet method (Octect RH-16, Sartorius). The association rate (k on ) and the dissociation rate (k off ) were calculated using a simple 1:1 binding model (Octet evaluation software version 12.2). The equilibrium dissociation constant (k D ) was calculated as the ratio koff / kon. The results are shown in Table 3. The results in Table 3 revealed that all CTLA4 antibody Fc fusion proteins bound to the CTLA4 antigen with strong affinity.

[0133]

Table 3

[0134] Example 5 Measurement of SEB by Analyzing IL2 Production with CTLA4 Antibodies Effector human peripheral blood monocytes obtained from individual donors (Milestone® Biotechnologies) and cultured in RPMI 1640 medium (Gibco, Catlog#A10491-01) containing 10% FBS (Gibco, Catlog#10099-141) were supplemented with serially diluted antibodies and Staphylococcus enterotoxin B (SEB) (Sigma-Aldrich, Catlog#S4881). After culturing the cells at 37°C for 4 days, the sample supernatant was collected and mixed with pre-mixed IL2-HTRF antibody (PerkinElmer, Catlog#62HIL02PET). The sample plates were sealed and incubated at room temperature for 3 hours. The plate seal was then removed and read using an EnVision 2105 multimode reader (PerkinElmer).

[0135] As shown in Figures 3A and 3B, the results showed that GBD008-S004, GBD008-hS005, GBD008-S019, humanized GBD008-hS005-3, GBD008-hS005-3-2, GBD008-hS005-5, GBD008-hS019-4, and GBD008-hS004-5 antibodies all significantly promoted IL2 production in effector cells in a concentration-dependent manner, and among these, the IL2 production by GBD008-S019 and GBD008-hS019-4 was comparable to that of ipilimumab. IL-2 production by GBD008-S004, GBD008-hS005, and the humanized GBD008-hS005-3, GBD008-hS005-3, and GBD008-hS005-5 is as follows, while IL-2 production by GBD008-hS004-5 and CTLA4 is lower. These results indicate that the antibodies of this disclosure have the potential to treat cancer or autoimmune diseases.

[0136] Example 6: Binding ability of anti-CTLA4 antibody to cynomolgus monkey and mouse CTLA4 (by ELISA) 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) antibody. The plates were then blocked at room temperature using blocking buffer. Subsequently, serial dilutions of anti-CTLA4 antibody were added, and the mixture was reacted at room temperature for 1 hour. Goat anti-human IgG Fc(HRP) (Abcam, Catlog#ab97225) was added, and the mixture was reacted at room temperature for 1 hour. After adding a chromogenic solution, the reaction was terminated, and absorbance was read at 450 nm wavelength using an EnVision 2105 multimode reader (PerkinElmer). The results are shown in Figure 4 AC.

[0137] The absorbance data in Figures 4A-F clearly shows that the GBD008-S004, GBD008-hS005, GBD008-S019, humanized GBD008-hS005-3, GBD008-hS005-3-2, GBD008-hS005-5, GBD008-hS019-4, and GBD008-hS004-5 antibodies all have binding ability to human and cynomolgus monkey CTLA4, but do not bind to mouse CTLA4.

[0138] Example 7: ADCC killing test of anti-CTLA 4 antibody Cytotoxic activity was evaluated using FACS analysis. Effector cells, human peripheral blood monocytes, were obtained from individual human donors (Milestone® Biotechnologies) and cultured overnight with 10 ng / ml hIL-2 (PeproTech, 200-02). Target Raji cells expressing full-length human CTLA4 were labeled with 5 μl of DELFIA BATDA reagent in 3 ml of detection buffer in the EuTDA kit (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 an effector-to-target cell ratio of 20:1. Serially diluted anti-CTLA4 mAbs were added to the designated rows of the detection plates. After incubation at 37°C for 2 hours, the supernatant was taken from 20 μl after centrifugation and co-incubated with 200 μl of europium solution at room temperature for 10 minutes, and read using an EnVision 2105 multimode reader (PerkinElmer). The results are shown in Figure 5.

[0139] As a result, it was revealed that the anti-CTLA4 antibody of this disclosure effectively induces degradation and death of target cells.

[0140] Example 8: In vivo antitumor effect of anti-CTLA4 antibody in MC38 colorectal cancer model Colon cancer cells were subcutaneously inoculated into MC38 (NTCC-MC 38) C57 BL / 6 human CTLA4 knock-in mice (n=6 in each group, female, 7-8 weeks old). Tumor formation (approximately 80 mm) 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 administered twice a week for three weeks. Tumor growth was monitored every three days and reported as the mean tumor volume. The results are shown in Figure 6.

[0141] Figure 6 shows that the mean tumor volume of mice treated with the anti-CTLA4 antibody of this disclosure was significantly smaller than that of the IgG1 control group. Furthermore, at the end of the experiment, the mean tumor volume of mice decreased by 62% at a dose of 0.15 mpk and by 99% at a dose of 1.5 mpk.

[0142] Example 9: Toxicity (arthritis) of anti-CTLA4 antibody in the hCTLA4 / hPD1 Balb / C mouse model. Human CTLA4 / hPD1 knock-in Balb / C mice (n=8 in each group, female, 4 weeks old) were administered IgG1 (30 mg / kg), ipilimumab and nivolumab in combination (15 mg / kg + 15 mg / kg), or GBD008-hS005-3-2 and nivolumab in combination (7.5 mg / kg + 15 mg / kg) via intraperitoneal injection. Treatment was administered twice a week until mouse arthritis (paw and joint inflammation) disappeared. Mouse body weight was monitored every 3 or 4 days, and mouse arthritis was scored. The results are shown in Figures 7A-C.

[0143] Figure 7B shows that arthritis in mice treated with the anti-CTLA4 antibody (GBD008-hS005-3-2) and nivolumab was significantly lower than in the IgG1 control group and the group treated with ipilimumab and nivolumab. Furthermore, Figure 7C shows that the incidence of arthritis in mice treated with GBD008-hS005-3-2 and nivolumab was also significantly lower than in the IgG1 control group and the group treated with ipilimumab and nivolumab.

[0144] The above examples confirmed that the anti-CTLA4 antibodies of this disclosure inhibit the binding of CD80 / CD86 to CTLA4 to varying degrees while simultaneously exhibiting a strong ADCC reaction. Due to varying degrees of blocking ability against CD80 / 86 and retention of the antibody's Fc function, the anti-CTLA4 antibodies have varying degrees of IL2 reaction rates. Furthermore, the anti-CTLA4 antibodies of this disclosure showed excellent in vivo antitumor efficacy and insignificant toxicity in a mouse model.

[0145] Although various embodiments of the present invention have been described above, it should be understood that these are provided only as examples and are not limiting. Without departing from the spirit and scope of the invention, various modifications and improvements are possible, and any such modifications and improvements will fall within the scope of the invention for which protection is sought. The scope of protection sought by the invention is defined by the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment that specifically binds to CTLA 4, wherein the antibody includes a heavy chain variable region, and the heavy chain variable region comprises CDR1, CDR2, and CDR3, each containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with the following sequences. (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 according to claim 1, wherein the heavy chain variable region comprises CDR1 comprising or consisting of the sequence shown in SEQ ID NO: 1, CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 2, and CDR3 comprising or consisting of the sequence shown in SEQ ID NO:

3.

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

31.

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

4.

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

5.

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

7.

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

8.

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

10.

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

11.

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

31.

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

32.

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

15.

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

19.

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

16.

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

17.

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

19.

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

20.

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

24.

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

28.

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

25.

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

26.

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

28.

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

29.

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

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

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

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

28. The antibody or its antigen-binding fragment 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 or antigen-binding fragment thereof exhibits significant antibody-dependent cell-mediated cytotoxicity (ADCC) according to any one of claims 1 to 28.

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

31. An isolated nucleic acid molecule encoding an antibody or its antigen-binding fragment according to any one of claims 1 to 29.

32. The nucleic acid molecule according to claim 31, wherein the nucleic acid molecule comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with 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 described in claim 31 or 32.

34. A host cell that expresses an antibody or antigen-binding fragment according to any one of claims 1 to 29, and / or a nucleic acid molecule according to claim 31 or 32, or an expression vector according to claim 33.

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

36. The method according to claim 35, wherein the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, squamous cell carcinoma, melanoma, myeloma, stomach 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 according to claim 35 or 36, wherein the antibody or its antigen-binding fragment or pharmaceutical composition is administered in combination with one or more other chemotherapeutic agents, radiotherapeutic agents, cytokines or other antibodies.

38. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 29 or a pharmaceutical composition according to claim 30 in the manufacture of a pharmaceutical product for treating a disease or condition that is cancer or an autoimmune disease.

39. The use according to claim 38, wherein the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, squamous cell carcinoma, melanoma, myeloma, stomach 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 according to claim 38 or 39, wherein the antibody or its antigen-binding fragment or pharmaceutical composition is used to be administered in combination with one or more other chemotherapeutic agents, radiotherapeutic agents, cytokines or other antibodies.

41. a) The antibody or antigen-binding fragment according to any one of claims 1 to 29 or the pharmaceutical composition according to claim 30 b) Usage specifications and A medicine box, including a pillbox.

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