Humanized anti-MYCT1 antibody and its uses

Abstract

This invention relates to a humanized anti-MYCT1 antibody and its applications.

Description

Technical Field

[0001] The present invention relates to a humanized anti-MYCT1 antibody and its uses.

[0002] The present invention claims priority based on Patent Application No. 2023-0082857 filed in Korea on June 27, 2023, and Patent Application No. 2023-0094413 filed in Korea on July 20, 2023, the contents of which are incorporated herein by reference.

Background Art

[0003] Angiogenesis is a physiological process in which new blood vessels develop from existing blood vessels. Angiogenesis is known to play its role in both normal and pathological processes. The regulation of angiogenesis is highly regulated by angiogenic factors and anti-angiogenic factors, but this process is disrupted and dysregulated in cancer. Hypoxia caused by tumors increases the expression of angiogenic factors that induce the formation of new blood vessels essential for tumor survival and growth. The VEGF family, composed of six growth factors (VEGFA-F), binds to the receptors VEGFR1-3 and neuropilin and plays the most important role in angiogenesis. Also, angiogenesis can be mediated by the angiopoietin (Ang1-2) / Tie-2 pathway independently of the VEGF pathway. As a result, in the past decade, as a strategy to deprive tumors of nutrients and suppress tumor growth, drug development has focused on anti-angiogenesis. However, despite the weak activity of these agents as single agents or in combination with chemotherapy, tumors still have the problem of overcoming their effects and developing resistance.

[0004] Cancer immunotherapy has emerged as an effective treatment for various cancers following the discovery of immune checkpoints. Immune checkpoint inhibitors (ICIs) have been proven to have long-lasting clinical activity against malignant tumors. ICIs block additional mechanisms hijacked by "immune exhaustion" of tumors and activate immune effector cells against cancer. Primary resistance to ICIs appears in tumors lacking tumor infiltrating lymphocytes. Also, tumors that initially respond to ICIs may develop secondary resistance due to defects in antigen presentation mechanisms and overexpression of co-inhibitory molecules.

[0005] Therefore, it is necessary to overcome the problems of VEGF inhibitors, normalize tumor blood vessels, and reprogram the tumor microenvironment (TME) so that immune cells can move well into the TME.

[0006] For this purpose, the development of drugs targeting MYCT1 (MYC Target 1) is necessary. To date, no anti-MYCT1 antibody has been approved as a therapeutic agent for human diseases. Therefore, there is a need to develop anti-MYCT1 antibodies with strong binding affinity to MYCT1, excellent specificity, strong ADCC and CDC activities, and excellent anti-tumor and anti-angiogenic activities through TME reprogramming. [Non-Patent Document] J.Cell.Mol.Med., 2016 Vol20, No3, pp.471-481 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] An object of the present invention is to provide a humanized antibody or antigen-binding fragment that binds to MYCT1.

[0008] Another object of the present invention is to provide nucleic acid molecules encoding the antibody or antigen-binding fragment, an expression vector containing the same, and host cells transduced by the vector.

[0009] A further object of the present invention is to provide an immune complex comprising the antibody or antigen-binding fragment and a cytotoxic agent.

[0010] A further object of the present invention is to provide the antibody or antigen-binding fragment for use in the diagnosis or treatment of MYCT1-related diseases.

[0011] Another object of the present invention is to provide the antibody or antigen-binding fragment for use in diagnosing susceptibility to immunosuppressant drugs.

[0012] A further object of the present invention is to provide a chimeric antigen receptor (CAR) containing the antigen-binding fragment and immune effector cells expressing it.

[0013] However, the technical problems of the present invention are not limited to those mentioned above, and other technical problems not mentioned can be clearly understood by an ordinary person skilled in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0014] To solve the above problems, one aspect of the present invention is: It includes at least one variable heavy chain region (VH) selected from the group consisting of SEQ ID NOs. 41, 43-60; and at least one variable light chain region (VL) selected from the group consisting of SEQ ID NOs. 42, 61-66; or The present invention provides an isolated anti-MYCT1 humanized antibody or its antigen-binding fragment, comprising at least one variable heavy chain region (VH) selected from the group consisting of SEQ ID NOs. 92 and 94-101, and at least one variable light chain region (VL) selected from the group consisting of SEQ ID NOs. 93 and 102-115.

[0015] In one specific example, VH may include CDR1, CDR2, and CDR3 having the amino acid sequences represented by SEQ ID NOs: 3, 4, and 5, respectively, and VL may also include CDR1, CDR2, and CDR3 having the amino acid sequences represented by SEQ ID NOs: 6, 7, and 8, respectively.

[0016] In other specific examples, VH may include CDR1, CDR2, and CDR3 having amino acid sequences represented by SEQ ID NOs. 9, 10, and 11, respectively, and VL may include CDR1, CDR2, and CDR3 having amino acid sequences represented by SEQ ID NOs. 12, 13, and 14, respectively.

[0017] In other specific examples, the antibody or its antigen-binding fragment is Does it contain the VH sequence of sequence number 43 and the VL sequence of sequence number 42? Does it contain the VH sequence of sequence number 44 and the VL sequence of sequence number 42? Does it contain the VH sequence of sequence number 45 and the VL sequence of sequence number 42? Does it contain the VH sequence of sequence number 41 and the VL sequence of sequence number 61? Does it contain the VH sequence of sequence number 46 and the VL sequence of sequence number 62? Does it contain the VH sequence of sequence number 41 and the VL sequence of sequence number 63? Does it contain the VH sequence of sequence number 44 and the VL sequence of sequence number 63? Does it contain the VH sequence of sequence number 47 and the VL sequence of sequence number 64? Does it contain the VH sequence of sequence number 48 and the VL sequence of sequence number 64? Does it contain the VH sequence of sequence number 48 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 49 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 50 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 51 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 52 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 53 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 54 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 55 and the VL sequence of sequence number 64? Does it contain the VH sequence of sequence number 55 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 56 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 57 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 58 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 41 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 58 and the VL sequence of sequence number 42? It contains the VH sequence of sequence number 59 and the VL sequence of sequence number 66; or It may also include the VH sequence of sequence number 60 and the VL sequence of sequence number 66.

[0018] In other specific examples, the antibody or its antigen-binding fragment is Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 102? Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 103? Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 104? Does it contain the VH sequence of sequence number 94 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 95 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 96 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 97 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 98 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 106? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 107? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 108? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 109? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 110? Does it contain the VH sequence of sequence number 95 and the VL sequence of sequence number 110? Does it contain the VH sequence of sequence number 100 and the VL sequence of sequence number 110? Does it contain the VH sequence of sequence number 100 and the VL sequence of sequence number 111? Does it contain the VH sequence of sequence number 101 and the VL sequence of sequence number 111? Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 111? Does it contain the VH sequence of sequence number 101 and the VL sequence of sequence number 112? Does it contain the VH sequence of sequence number 95 and the VL sequence of sequence number 113? It includes the VH sequence of sequence number 100 and the VL sequence of sequence number 114; or It may also include the VH sequence of sequence number 95 and the VL sequence of sequence number 115.

[0019] The antigen-binding fragment may include Fd, Fab, Fab', F(ab')2, dsFv, scFv, and single-domain antibodies (sdAb), but is not limited to these; any fragment possessing at least antigen-binding properties can be used without restriction.

[0020] In another embodiment, the present invention provides a nucleic acid molecule encoding the antibody or its antigen-binding fragment.

[0021] In yet another embodiment, the present invention provides a recombinant expression vector comprising the nucleic acid molecule; and a host cell transduced by the expression vector.

[0022] In yet another embodiment, the present invention provides an immune complex comprising the antibody or antigen-binding fragment and a cytotoxic agent.

[0023] In yet another embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of MYCT1-related diseases, comprising the antibody or antigen-binding fragment as an active ingredient.

[0024] In one specific example, the MYCT1-related disease may be angiogenesis-related disease or cancer.

[0025] In other specific examples, the pharmaceutical composition may be administered in combination with at least one further therapeutic agent or therapeutic procedure.

[0026] The aforementioned at least one further therapeutic agent or therapeutic procedure may be selected from one or more of the following: chemotherapy, targeted anticancer therapy, oncolytic agents, cytotoxic agents, immunotherapy, cytokines, surgical procedures, radiation procedures, vaccines, or cell therapies.

[0027] In yet another embodiment, the present invention provides a diagnostic composition for MYCT1-related diseases comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0028] In yet another embodiment, the present invention provides a pharmaceutical composition for enhancing sensitivity to immunosuppressant agents in cancerous tumors, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0029] In yet another embodiment, the present invention is: The present invention provides a chimeric antigen receptor (CAR) containing an antigen-binding domain that specifically binds to MYCT1. Specifically, the antigen-binding domain is: The antigen-binding fragment may include at least one variable heavy chain (VH) region selected from the group consisting of SEQ ID NOs: 41, 43-60, 92 and 94-101; and at least one variable light chain (VL) region selected from the group consisting of SEQ ID NOs: 42, 61-66, 93 and 102-115.

[0030] In yet another aspect, the present invention provides immune effector cells comprising the chimeric antigen receptor.

[0031] In yet another embodiment, the present invention provides a method for treating MYCT1-related disease, comprising the step of administering a composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment to a subject.

[0032] In yet another embodiment, the present invention provides a method for enhancing the sensitivity of an individual to an immunosuppressant, comprising the step of administering a composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment to a subject resistant to the immunosuppressant.

[0033] However, the technical problems of the present invention are not limited to those mentioned above, and other technical problems not mentioned can be clearly understood by an ordinary person skilled in the art to which the present invention pertains from the following description. [Effects of the Invention]

[0034] The antibody or antigen-binding fragment provided in the present invention specifically binds to MYCT1, exhibiting excellent affinity and binding affinity to MYCT1. Therefore, antibody-drug conjugates, bispecific antibodies, or chimeric antigen receptors (CARs) containing the antibody or antigen-binding fragment can be developed. Furthermore, the antibody or antigen-binding fragment according to the present invention can be usefully applied to the treatment or diagnosis of MYCT1-related diseases, such as cancer and angiogenesis-related diseases. Moreover, the antibody or antigen-binding fragment according to the present invention can be used in combination with immunosuppressant agents to enhance sensitivity to immunosuppressant agents.

[0035] The effects of a composition according to one embodiment of the present invention are not limited to those mentioned above, and other effects not mentioned can be clearly understood by a person of the ordinary skill from the following description. [Brief explanation of the drawing]

[0036] [Figure 1] This is a result of confirming the presence or absence of expression of parental antibody (2B4) and humanized antibody in the supernatant during small-scale production using SDS-PAGE gel. [Figure 2] This is a result of confirming the presence or absence of expression of parental antibody (11B6) and humanized antibody in the supernatant during small-scale production using SDS-PAGE gel. [Figure 3a] This is an SDS-PAGE gel of the parent antibody (2B4) purified under non-reducing and reducing conditions during small-scale production (R: reducing). [Figure 3b] This is an SDS-PAGE gel of the parent antibody (2B4) purified under non-reducing and reducing conditions during small-scale production (R: reducing). [Figure 3c] This is an SDS-PAGE gel of the parent antibody (2B4) purified under non-reducing and reducing conditions during small-scale production (R: reducing). [Figure 4a] This is an SDS-PAGE gel of the parent antibody (11B6) purified under non-reducing and reducing conditions during small-scale production (R: reducing). [Figure 4b] This is an SDS-PAGE gel of the parent antibody (11B6) purified under non-reducing and reducing conditions during small-scale production (R: reducing). [Figure 5a] These are SEC-HPLC chromatograms of purified parental antibody (2B4), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 5b] These are SEC-HPLC chromatograms of purified parental antibody (2B4), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 5c] These are SEC-HPLC chromatograms of purified parental antibody (2B4), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 5d] These are SEC-HPLC chromatograms of purified parental antibody (2B4), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 5e] These are SEC-HPLC chromatograms of purified parental antibody (2B4), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 5f]These are SEC-HPLC chromatograms of purified parental antibody (2B4), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 6a] These are SEC-HPLC chromatograms of purified parental antibody (11B6), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 6b] These are SEC-HPLC chromatograms of purified parental antibody (11B6), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 6c] These are SEC-HPLC chromatograms of purified parental antibody (11B6), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 6d] These are SEC-HPLC chromatograms of purified parental antibody (11B6), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 6e] These are SEC-HPLC chromatograms of purified parental antibody (11B6), humanized antibody, and PTM hazardous removal mutations during small-scale production. [Figure 7] This is an SDS-PAGE gel of parent antibody (2B4) and lead antibodies (z2-p7-uIgG1K and z8-p7-uIgG1K) purified under non-reducing and reducing conditions during small-scale production (R: reducing). [Figure 8] These are SEC-HPLC chromatograms of purified parent antibody (2B4) and lead antibodies (z2-p7-uIgG1K and z8-p7-uIgG1K) during small-scale production. [Figure 9] This is an SDS-PAGE gel of parent antibody (11B6) and lead antibodies (z10-p3, z12-p3, and z16-p3) purified under non-reducing and reducing conditions during small-scale production (R: reducing). [Figure 10] These are SEC-HPLC chromatograms of purified parent antibody (11B6) and lead antibodies (z10-p3, z12-p3, and z16-p3) during small-scale production. [Figure 11]This is an SDS-PAGE gel of the parent antibody (11B6) and lead antibodies (z10-p3, z12-p3, and z16-p3) purified under non-reducing and reducing conditions during scale-up production (R: reducing). [Figure 12] These are SEC-HPLC chromatograms of purified parent antibody (11B6) and lead antibodies (z10-p3, z12-p3, and z16-p3) during scale-up production. [Figure 13a] This is an SPR sensorgram of the overall curve for the parental antibody (2B4) and PTM risk removal mutations against human MYCT1 (hMYCT1). [Figure 13b] This is an SPR sensorgram of the overall curve for the parental antibody (2B4) and PTM risk removal mutations against human MYCT1 (hMYCT1). [Figure 13c] This is an SPR sensorgram of the overall curve for the parental antibody (2B4) and PTM risk removal mutations against human MYCT1 (hMYCT1). [Figure 14a] This is an SPR sensorgram of the overall curve for the parental antibody (2B4) against hMYCT1 and for PTM risk removal mutations. [Figure 14b] This is an SPR sensorgram of the overall curve for the parental antibody (2B4) against hMYCT1 and for PTM risk removal mutations. [Figure 14c] This is an SPR sensorgram of the overall curve for the parental antibody (2B4) against hMYCT1 and for PTM risk removal mutations. [Figure 14d] This is an SPR sensorgram of the overall curve for the parental antibody (2B4) against hMYCT1 and for PTM risk removal mutations. [Figure 14e] This is an SPR sensorgram of the overall curve for the parental antibody (2B4) against hMYCT1 and for PTM risk removal mutations. [Figure 14f]This is an SPR sensorgram of the overall curve for the parental antibody (2B4) against hMYCT1 and for PTM risk removal mutations. [Figure 15] This is an SPR sensorgram of the overall curve for the parent antibody (2B4) and lead antibodies (z2-p7 and z8-p7) with humanized and PTM risk elimination combinations against hMYCT1. [Figure 16] This is an SPR sensorgram of the overall curve for the parental antibody (11B6) against hMYCT1 and for PTM risk removal mutations. [Figure 17] This is an SPR sensorgram of the overall curve for the parent antibody (11B6) and lead antibodies (z10-p3, z12-p3, and z16-p3) against hMYCT1. [Figure 18] These are the DSF profiles of the parent antibody (2B4) and the lead antibody. [Figure 19a] These are the DSF profiles of the parent antibody (11B6) and the lead antibody. [Figure 19b] These are the DSF profiles of the parent antibody (11B6) and the lead antibody. [Figure 20] This is the result of evaluating antibody toxicity using one embodiment of the present invention. [Figure 21a] This is the result of an analysis of antibody tube formation using one embodiment of the present invention. [Figure 21b] This is the result of an analysis of antibody tube formation using one embodiment of the present invention. [Figure 22] This is a schematic diagram illustrating the antitumor activity experiment of an antibody in a colorectal cancer cell line model according to one embodiment of the present invention. [Figure 23a] This result demonstrates the cancer growth inhibitory effect of an antibody according to one embodiment of the present invention in a colorectal cancer cell line model (Chimeric ATN001 is an 11B6 antibody, and hzATN001 is an 11B6-z16-p3 antibody). [Figure 23b]This result demonstrates the cancer growth inhibitory effect of an antibody according to one embodiment of the present invention in a colorectal cancer cell line model (Chimeric ATN001 is an 11B6 antibody, and hzATN001 is an 11B6-z16-p3 antibody). [Modes for carrying out the invention]

[0037] The antibody or antigen-binding fragment according to the present invention specifically binds to MYCT1 and can overcome the limitations of existing immunosuppressant drugs by ultimately reprogramming the tumor microenvironment (TME) through i) suppression of angiogenesis, ii) normalization of blood vessels, iii) increased activity of immune cells in the tumor microenvironment (TME), and iv) increased infiltration of immune cells in the TME.

[0038] The aforementioned MYCT1 (hMYCT1) sequence is as follows (sequence number 1). JPEG2026521923000002.jpg34155

[0039] Sequence ID 1 is an example of a MYCT1 sequence, and any known MYCT1 sequence may be included in the present invention. Specifically, the human antibody provided in the present invention that specifically binds to MYCT1 can bind to amino acid sequences 49 to 67 of the aforementioned sequence (MANNTTSLGSPWPENFWED, Sequence ID 2).

[0040] Antibody or antigen-binding fragment In this specification, the term "antibody" means a protein molecule that acts as a ligand specifically recognizing an antigen, including immunoglobulin molecules that are immunologically reactive with a particular antigen, and includes polyclonal antibodies, monoclonal antibodies, full-length (whole) antibodies, and antibody fragments. In this invention, the antibody also includes chimeric antibodies, bivalent antibodies, bispecific molecules, minibodies, domain antibodies, bispecific or multispecific antibodies, immune cell engage bispecific or multispecific antibodies, antibody mimes, unibodies, diabodies, triabodies, or tetrabodies. The term further includes single-chain antibodies, scabs, derivatives of antibody-invariant regions, and artificial antibodies based on protein scaffolds that possess the ability to bind to FcRn (neonatal Fc receptor). The total antibody has a structure comprising two full-length light chains (LCs) and two full-length heavy chains (HCs), with each light chain linked to a heavy chain by a disulfide bond. The total antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG includes subtypes such as IgG1, IgG2, IgG3, and IgG4. In one embodiment, the antibody provided in the present invention may be an IgG antibody.

[0041] In the present invention, "humanized antibody" means an antibody that exhibits reduced immunogenicity in humans or is non-immunogeneic. The humanized antibody may be produced, for example, by conjugating complementarity determining regions (CDRs) derived from a non-human individual (non-human species) to framework regions (FRs) among the invariant regions and variable regions derived from a human antibody. The humanized antibody may be produced by transplanting CDRs of a non-human antibody between the FR sequences of a human antibody through a CDR-grafting method.

[0042] In this invention, the term "monoclonal antibody" refers to an antibody molecule with a single molecular composition obtained from substantially the same antibody population, and such monoclonal antibodies exhibit single-binding specificity and affinity to a specific antigen-determining site (epitope). Typically, immunoglobulins have a heavy chain and a light chain, each of which contains an invariant region and a variable region (these regions are also known as domains). The variable regions of the light and heavy chains include three multivariable regions and four structural regions, denoted as complementarity-determining regions (CDRs).

[0043] In this specification, the terms “complementarity determining region,” “CDR region,” or “CDR” refer to a highly variable region within the sequence of an antibody variable region, which either forms / forms a structurally defined loop ("hypervariable loop") or contains an antigen contact residue ("antigen contact site"). CDRs are primarily responsible for binding to antigen epitopes. The complementarity determining regions are located between relatively conserved regions called invariant regions (FRs). Each heavy chain variable region (VH) and light chain variable region (VL) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The heavy chain variable regions (CDRs) may be designated as HCDR1, HCDR2, and HCDR3; the light chain variable regions (CDRs) may be designated as LCDR1, LCDR2, and LCDR3; the heavy chain variable regions (FRs) may be designated as HFR1, HFR2, HFR3, and HFR4; and the light chain variable regions (FRs) may be designated as LFR1, LFR2, LFR3, and LFR4. In this case, the numbers are assigned sequentially from the N-terminus.The precise amino acid sequence boundaries of each CDR in the given light chain and heavy chain variable regions are determined by, for example, Chothia (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)) based on the three-dimensional structure of the antibody and the arrangement of the CDR ring, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USD Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (imgt.cines.fr / on the World Wide Web), and North CDR based on affinity propagation clustering using numerous determination structures. This can be determined using one or a combination of known antibody CDR assignment systems, including definitions.

[0044] The antibody of the present invention may contain an invariant region, in which case the invariant region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof, but is not limited to these. Here, the term "combination" means that when forming a dimer or polymer, a polypeptide encoding a single-chain immunoglobulin invariant region of the same origin forms a bond with a single-chain polypeptide of a different origin. For example, a dimer or polymer can be formed from two or more invariant regions selected from the group consisting of the invariant regions of IgG, IgA, IgD, IgE, and IgM. Furthermore, the term "hybrid" refers to the presence of two or more sequences corresponding to immunoglobulin heavy chain invariant regions of different origins within a single-chain immunoglobulin heavy chain invariant region. For example, a hybrid domain consisting of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG, IgA, IgD, IgE, and IgM is possible.

[0045] In this specification, the term "full-length antibody" refers to a structure having two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond, and including IgA, IgD, IgE, IgM, and IgG. The IgG includes IgG1, IgG2, IgG3, and IgG4 as its subtypes.

[0046] In this specification, the terms “fragment,” “polypeptide-bound fragment,” and “antibody fragment” refer to any fragment of the antibody of the present invention that possesses the antigen-binding activity of the antibody, and are used interchangeably. Exemplary antibody fragments include, but are not limited to, Fd, Fab, Fab', F(ab')2, dsFv, scFv, and / or single-domain antibodies (sdAb). Fd refers to the heavy chain portion contained in the Fab fragment. In this invention, the term “Fab” usually refers to a fragment containing a heavy chain variable domain and a light chain variable domain, further comprising a light chain invariant domain and a heavy chain first invariant domain (CH1). The term “Fab” usually refers to a fragment distinct from Fab by adding a few residues (including one or more cysteines from the antibody hinge region) to the carboxyl terminus of the heavy chain CH1 domain. The term “F(ab')2” usually refers to a dimeric antibody fragment of Fab' containing two Fab fragments linked via a disulfide crosslink at the hinge region. The term "Fv" typically refers to the smallest antibody fragment that contains complete antigen recognition and binding sites. In certain cases, the fragment may consist of a dimer in which the heavy chain variable region and the light chain variable region are closely non-covalently bonded. The term "dsFv" typically refers to an Fv fragment in which the disulfide bond is stable, and the bond between its single light chain variable region and single heavy chain variable region is a disulfide bond. The term "dAb fragment" typically refers to an antibody fragment composed of VH domains. In this invention, the term "scFv" typically refers to a monovalent molecule formed by the covalent linkage of the heavy chain variable domain and the light chain variable domain of an antibody via a flexible peptide linker. Such scFv molecules may have the general structure of NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. The term "single-domain antibody" is also known as a nanobody (e.g., sdAb, sdFv, nanobody) and refers to an antibody fragment composed of a single monomeric variable antibody domain.

[0047] As used herein, the terms “humanized anti-MYCT1 antibody,” “anti-MYCT1 antibody,” “anti-MYCT1,” “MYCT1 antibody,” or “antibody that binds to MYCT1” refer to an antibody that can bind to MYCT1 with sufficient affinity so that it can be used as a therapeutic agent targeting MYCT1. In one embodiment of the present invention, the MYCT1 antibody binds to MYCT1 with high affinity in vitro or in vivo. Herein, the binding may be determined, for example, by radioimmunoassay (RIA), biolayer interferometry (BLI), MSD analysis, surface plasmon resonance (SPR), or flow cytometry.

[0048] The humanized anti-MYCT1 antibody or antigen-binding fragment of the present invention comprises at least one variable heavy chain region (VH) selected from the group consisting of SEQ ID NOs: 41, 43-60; and at least one variable light chain region (VL) selected from the group consisting of SEQ ID NOs: 42, 61-66; or

[0049] It comprises at least one variable heavy chain region (VH) selected from the group consisting of SEQ ID NOs. 92 and 94-101, and at least one variable light chain region (VL) selected from the group consisting of SEQ ID NOs. 93 and 102-115.

[0050] In one specific example, VH may include CDR1, CDR2, and CDR3 having the amino acid sequences represented by SEQ ID NOs: 3, 4, and 5, respectively, and VL may also include CDR1, CDR2, and CDR3 having the amino acid sequences represented by SEQ ID NOs: 6, 7, and 8, respectively.

[0051] In other specific examples, VH may include CDR1, CDR2, and CDR3 having amino acid sequences represented by SEQ ID NOs. 9, 10, and 11, respectively, and VL may include CDR1, CDR2, and CDR3 having amino acid sequences represented by SEQ ID NOs. 12, 13, and 14, respectively.

[0052] In other specific examples, the antibody or its antigen-binding fragment is Does it contain the VH sequence of sequence number 43 and the VL sequence of sequence number 42? Does it contain the VH sequence of sequence number 44 and the VL sequence of sequence number 42? Does it contain the VH sequence of sequence number 45 and the VL sequence of sequence number 42? Does it contain the VH sequence of sequence number 41 and the VL sequence of sequence number 61? Does it contain the VH sequence of sequence number 46 and the VL sequence of sequence number 62? Does it contain the VH sequence of sequence number 41 and the VL sequence of sequence number 63? Does it contain the VH sequence of sequence number 44 and the VL sequence of sequence number 63? Does it contain the VH sequence of sequence number 47 and the VL sequence of sequence number 64? Does it contain the VH sequence of sequence number 48 and the VL sequence of sequence number 64? Does it contain the VH sequence of sequence number 48 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 49 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 50 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 51 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 52 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 53 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 54 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 55 and the VL sequence of sequence number 64? Does it contain the VH sequence of sequence number 55 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 56 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 57 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 58 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 41 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 58 and the VL sequence of sequence number 42? It contains the VH sequence of sequence number 59 and the VL sequence of sequence number 66; or It may also include the VH sequence of sequence number 60 and the VL sequence of sequence number 66.

[0053] In other specific examples, the antibody or its antigen-binding fragment is Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 102? Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 103? Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 104? Does it contain the VH sequence of sequence number 94 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 95 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 96 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 97 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 98 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 106? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 107? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 108? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 109? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 110? Does it contain the VH sequence of sequence number 95 and the VL sequence of sequence number 110? Does it contain the VH sequence of sequence number 100 and the VL sequence of sequence number 110? Does it contain the VH sequence of sequence number 100 and the VL sequence of sequence number 111? Does it contain the VH sequence of sequence number 101 and the VL sequence of sequence number 111? Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 111? Does it contain the VH sequence of sequence number 101 and the VL sequence of sequence number 112? Does it contain the VH sequence of sequence number 95 and the VL sequence of sequence number 113? It includes the VH sequence of sequence number 100 and the VL sequence of sequence number 114; or It may also include the VH sequence of sequence number 95 and the VL sequence of sequence number 115.

[0054] In one specific example, the antibody provided in the present invention may be a monoclonal antibody.

[0055] In one specific example, the antigen-binding fragment provided in the present invention may be an scFv.

[0056] The amino acid sequences described above may include variants having different sequences due to deletions, insertions, substitutions, or combinations thereof of amino acid residues, as long as they do not affect the structure, function, or activity of the polypeptide containing them. Furthermore, the amino acid sequences may include amino acids that have undergone common modifications known in the art, such as phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation.

[0057] The antibodies or antigen-binding fragments of the present invention include not only those containing the amino acid sequence described above, but also those having substantially the same amino acid sequence or variants thereof. The meaning of having substantially the same amino acid sequence is not limited to those containing an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homology with the amino acid sequence described above.

[0058] The antibody or antigen-binding fragment of the present invention may further include a linker between the heavy chain variable region and the light chain variable region. Here, the linker may be a peptide linker and may have a length of about 10 to 25 aa. For example, hydrophilic amino acids such as glycine and / or serine may be included, but are not limited to these.

[0059] Specifically, in the present invention, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (where n and m are each 1 to 10), and preferably includes GGGGS.

[0060] The antibody or antigen-binding fragment of the present invention may include not only the sequences of the anti-MYCT1 antibody or antigen-binding fragment of the present invention described herein, but also its biological equivalents, within the range that it can specifically recognize MYCT1. For example, further modifications can be made to the amino acid sequence of the antibody to further improve the antibody's binding affinity and / or other biological properties. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues in the antibody or antigen-binding fragment. Such amino acid mutations are made based on the relative similarity of amino acid side-chain substitutions, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, morphology, and type of amino acid side-chain substitutions reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar morphologies. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0061] Considering the aforementioned variants with equivalent biological activity, the antibodies or antigen-binding fragments of the present invention and the nucleic acid molecules encoding them are interpreted to include sequences that exhibit substantial identity with the sequences described in the sequence numbers. Substantial identity means sequences that, when aligned to the maximum extent possible with any other sequence and analyzed using algorithms commonly used in the art, exhibit a minimum of 90% homology, most preferably a minimum of 95% homology, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology. Alignment methods for sequence comparison are publicly known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI and other sources and can be used online in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx. BLAST is accessible at www.ncbi.nlm.nih.gov / BLAST / . The method for comparing sequence homology using this program is described at www.ncbi.nlm.nih.gov / BLAST / blast_ You can check this in help.html.

[0062] Based on this, the antibody or antigen-binding fragments of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher homology with the explicitly described sequence or whole as described in the specification. Such homology may be determined by sequence comparison and / or alignment by methods known in the art. For example, the percentage sequence homology of the nucleic acid or protein of the present invention can be determined using sequence comparison algorithms (i.e., BLAST or BLAST2.0), manual alignment, or visual inspection.

[0063] The binding affinity of the anti-MYCT1 antibody or antigen-binding fragment provided in the present invention to MYCT1 is 10 -5 M~10 -12It is within the range of M. For example, the binding affinity of the anti-MYCT1 antibody or antigen-binding fragment to MYCT1 is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M may be, but is not limited to these.

[0064] Another embodiment of the present invention relates to a nucleic acid molecule encoding an antibody or antigen-binding fragment provided in the present invention.

[0065] In this specification, the term "nucleic acid" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the nucleotides, which are the basic building blocks of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base sites. The sequences of nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include the addition, deletion, or non-conservative or conserved substitution of nucleotides.

[0066] In the present invention, the DNA encoding the antibody or antigen-binding fragment can be readily separated or synthesized using conventional molecular biological techniques (for example, by using oligonucleotide probes that can specifically bind to the DNA encoding the heavy and light chains of the antibody), the nucleic acid is separated, inserted into a replicable vector, and then cloned (DNA amplification) or expressed.

[0067] The present invention, according to further embodiments, relates to a recombinant expression vector comprising a nucleic acid molecule provided in the present invention.

[0068] In the present invention, the term "vector" refers to a recombinant vector that can be transduced into a suitable host cell and express a target protein, and is a gene construct containing essential regulatory elements that are operably linked to express the gene insert. Here, "operably linked" means that the nucleic acid expression regulatory sequence and the nucleic acid sequence encoding the target protein are functionally linked to perform a general function. The operable linkage with the recombinant vector can be manufactured using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linking can be easily performed using enzymes and the like that are generally known in the art.

[0069] In the present invention, various forms of vectors can be used as recombinant expression vectors for inserting the foreign gene, such as nanoparticles, plasmids, viruses, and cosmids. The type of recombinant vector is not particularly limited as long as it functions to express the desired gene and produce the desired protein in various host cells of prokaryotic and eukaryotic cells. Specifically, a vector can be used that has a potent promoter and strong expression capacity, while also being able to produce large quantities of foreign proteins in a form similar to that in nature.

[0070] Various gene transfer vehicles are publicly known in the art and include both viral and non-viral (e.g., naked DNA, plasmid) vectors. Viral vectors suitable for gene transfer are generally known to the art. Non-limiting examples of the aforementioned viral vectors may include retroviral vectors (derived from Moloney's mouse leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (derived from, for example, HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors including replicable, non-replicating, and apathetic forms thereof, adeno-associated virus (AAV) vectors, Simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, varicella virus vectors, harby mouse sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, maravivirus vectors, and group B adenovirus / enadenotucirev vectors.

[0071] Non-viral vectors for gene transfer include naked DNA, plasmids, transposons, and mRNA. Unrestrictive examples include pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmid (Invitrogen, San Diego, Calif.), and pMAL plasmid (New England Biolabs, Beverly, Mass.).

[0072] In the present invention, the vector can be introduced into many suitable host cells by methods disclosed or cited herein, or otherwise known to the ordinary art in the relevant field.

[0073] A suitable expression vector of the present invention may include, in addition to expression regulatory elements such as promoters, start codons, stop codons, polyadenylation signals, or enhancers, a nucleotide sequence encoding a signal peptide for membrane targeting or secretion. The start and stop codons are generally considered to be part of a nucleotide sequence encoding an immunogenic target protein, and must exert an effect in the organism when the gene construct is administered, and are required to be in frame with the coding sequence.

[0074] In this specification, the term “promoter” refers to any sequence that regulates the expression of a coding sequence, such as a gene, as used herein. A promoter may be, for example, constitutive, inductive, repressive, or tissue-specific. A promoter is a regulatory sequence, which is a region of polynucleotide sequence in which the initiation and rate of transcription are controlled. In the present invention, non-limiting examples of the promoter include the Rous sarcoma virus (RSV) LTR promoter (selectively having an RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, EF1α short (EFS) promoter, human polypeptide chain elongation factor (EF1a) promoter, P5 promoter, Ubc promoter, CAG promoter, TRE promoter, UAS promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, Gal1 promoter, TEF1 promoter, GDS promoter, ADH1 promoter, CaMV35S promoter, ubiquitin (Ubi) promoter, for example, ubiquitin C (UbiC), H1 promoter, U6 promoter, α-1-antitrypsin promoter, spleen focus-forming virus (SFFV) promoter, and the like.

[0075] Furthermore, in the present invention, the promoter may be coupled to an enhancer to increase transcription efficiency. In this case, non-limiting examples of the enhancer include, but are not limited to, an RSV enhancer, a CMV enhancer, or an α-fetal protein MERII enhancer. The vector of the present invention may be fused with other sequences to facilitate the purification of antibodies expressed therefrom. Examples of sequences to be fused include, but are not limited to, glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0076] The vector of the present invention includes, as a selection marker, an antibiotic resistance gene commonly used in the art, which may, but is not limited to, resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0077] A further embodiment of the present invention relates to host cells transduced by an expression vector provided in the present invention.

[0078] In this specification, the term “host cell” includes individual cells or cell cultures that are or were recipients of a vector for incorporating polypeptide inserts. Host cells include offspring of a single host cell, which are not necessarily entirely identical (morphologically or in terms of genomic DNA complements) to the original parent cell due to spontaneous, accidental, or intentional mutations. Host cells include cells transduced in the body by the polypeptide of this application.

[0079] In the present invention, the host cell may include mammalian, plant, insect, fungal, or cell-derived cells, for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella tiphimurium; fungal cells such as yeast cells and Pichia pastris; insect cells such as Drosophila and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bose melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), or PERC.6 (human retinal cells); or plant cells, but is not limited thereto; any cell that is commonly known to the technician as a host cell is available.

[0080] In the present invention, the transduction method can include any known method that allows the injection of a target vector into a host cell, such as, but is not limited to, methods using CaCl2, electroporation, microinjection, calcium phosphate precipitation, liposome-mediated transduction, DEAE-dextran method, gene bombardment, and virus-based transduction.

[0081] The present invention relates to a method for producing an antibody or antigen-binding fragment that specifically binds to MYCT1, further comprising the steps of: culturing the host cells to produce an antibody or antigen-binding fragment; and separating and purifying the produced antibody or antigen-binding fragment.

[0082] In the present invention, the host cells can be cultured in various culture media. Any commercially available culture medium can be used without limitation. All other essential supplements known to the ordinary technician may also be included in appropriate concentrations. Culture conditions, such as temperature and pH, have already been used with the host cells selected for expression, and this will be obvious to the ordinary technician.

[0083] In the present invention, the recovery of the antibody or its antigen-binding fragment can be performed by removing impurities, for example, by centrifugation or ultrafiltration, and the resulting product can be purified using, for example, affinity chromatography. Further other purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography, can also be used.

[0084] Further embodiments of the present invention relate to a bispecific or multispecific antibody comprising an antibody or antigen-binding fragment provided in the present invention.

[0085] In this specification, the term "bispecific antibody" means an antibody that has the ability to bind to or antagonize one or more targets, and means either an antibody in which two antibodies having the ability to bind to or antagonize each other's targets are bound, or an antibody in which an antibody having the ability to bind to one target is bound to a substance having the ability to antagonize another target.

[0086] In this specification, the term "multispecific antibody" means an antibody that has binding specificity to at least three different antigens. A multispecific antibody may include antibodies that are trispecific or higher, such as trispecific antibodies, tetraspecific antibodies, or antibodies that target more than three specific targets.

[0087] In the present invention, antibodies belonging to the category of bispecific or multispecific antibodies can be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies. In the case of bispecific or multispecific antibodies, two or more signals can be suppressed or amplified simultaneously, which may be more effective than suppressing / amplifying a single signal. Compared to treating each signal with its respective signal inhibitor, lower doses can be administered, and two or more signals can be suppressed / amplified in the same time and space.

[0088] Methods for producing bispecific or multispecific antibodies in the present invention are widely known. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two or more immunoglobulin heavy / light chain pairs under conditions in which two or more heavy chains have different specificities.

[0089] In the present invention, in the case of bispecific or multispecific antibodies based on scFv, a diabody can be produced by combining the VL and VH of different scFvs with each other to create a hybrid scFv in a heterodimeric form; a tandem scFv can be produced by linking different scFvs together; a heterodimeric miniantibody can be produced by expressing the CH1 and CL of Fab at the ends of each scFv; and a minibody in the heterodimeric scFv form can be produced by substituting some amino acids in the CH3 domain, which is the homodimeric domain of Fc, to change it into a heterodimeric structure in the form of a "knob into hole," and then expressing these modified CH3 domains at different scFv ends.

[0090] In the present invention, in the case of bispecific or multispecific antibodies based on Fab, individual Fab's for specific antigens can be combined with each other using disulfide bonds or mediators to produce heterodimeric Fab forms. By expressing scFv for different antigens at the end of the heavy or light chain of a specific Fab, two antigen-binding valencies can be created, or by placing a hinge region between the Fab and scFv, a homodimeric form with four antigen-binding valencies can be produced. Furthermore, by fusing scFv for different antigens to the light and heavy chain ends of a Fab, a bitarget bibody with three antigen-binding valencies can be created. By fusing different scFv for different antigens to the light and heavy chain ends of a Fab, a triplicate bibody with three antigen-binding valencies can be created. These can also be obtained by chemically linking three different Fabs.

[0091] In the present invention, for IgG-based bispecific or multispecific antibodies, a method is known by Trion Pharma to produce hybrid hybridomas, also known as quadromas, by further crossing mouse and rat hybridomas, and then produce bispecific antibodies. Alternatively, bispecific antibodies can be produced in the so-called "Holes and Knob" form by sharing the light chain portion while modifying some amino acids of the CH3 homodimeric domain of Fc to different heavy chains, resulting in a heterodimeric form. In addition to heterodimeric bispecific antibodies, they can also be produced as homodimeric (scFv)4-IgG by fusing two different scFvs to the constant domains instead of the variable domains of the IgG light and heavy chains, respectively. Furthermore, ImClone has reported the production of a bispecific antibody based on IMC-1C11, a chimeric monoclonal antibody against human VEGFR-2, by fusing only a single variable region for mouse platelet-derived growth factor α receptor to the light chain amino terminus of this antibody. In addition, antibodies with multiple antigen-binding titers for CD20 can be produced through the so-called "dock and lock (DNL)" method using the dimerization and docking domain (DDD) of the protein kinase A (PKA) R subunit and the anchor domain of PKA.

[0092] In the present invention, a wide variety of recombinant antibody formats have been developed, such as bivalent or multivalent antibodies with two or more valents, three or more valents, or four or more valents. This includes, for example, the bivalent, trivalent or quadrivalent antibodies described in International Patent Application Publications WO2001 / 077342, WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, WO2010 / 145793, and WO2011 / 117330. Antibodies with two or more valent, three or more valent, or four or more valent valents indicate that the antibody molecule contains two or more binding domains, three or more binding domains, or four or more binding domains, respectively.

[0093] The bispecific or multispecific antibodies according to the present invention may specifically include the above-mentioned anti-MYCT1 antibody or antigen-binding fragment in the form of a complete IgG antibody or its fragment, for example, a single-stranded Fv, VH domain and / or VL domain, Fab or (Fab)2. Furthermore, the antibody may also include antibodies that bind to targets different from the antibody targeting MYCT1, such as antibodies that target one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137(4-1BB), VISTA, CD258(LIGHT), TIGIT, CD134(OX40), CD28, CD278(ICOS), CD27, CD154(CD40L), CD357(GITR), CD30, DR3, CD226(DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO. Specifically, this may include a complete IgG antibody or a fragment thereof, for example, in the form of a single-stranded Fv, VH domain and / or VL domain, Fab, or (Fab)2.

[0094] The present invention allows for the securing of further binding specificity induced or mediated by targets other than MYCT1 through the use of bispecific or multispecific antibodies.

[0095] For example, the bispecific antibodies according to the present invention include MYCT1 and PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD3, CD20, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, and Transferrin. One or more targets selected from the group consisting of receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, MARCO, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, NKG2D, CD3, CD16a, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Step1, and TenB2 can be targeted simultaneously.

[0096] For example, the multispecific antibodies according to the present invention include MYCT1 and PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD3, CD20, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, and Transferrin. Two or more targets selected from the group consisting of receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, MARCO, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, NKG2D, CD3, CD16a, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1, and TenB2 can be targeted simultaneously.

[0097] Further embodiments of the present invention relate to an immune cell engage bispecific or multispecific antibody comprising one or more antigen-binding fragments (scFv) and scFvs that bind to immune cell-activating antigens, as provided in the present invention.

[0098] In this invention, the use of the aforementioned immune cell engagement bispecific or multispecific antibody temporarily induces cytolytic synapses between cytotoxic T cells or NK cells and cancer target cells, thereby releasing toxic substances.

[0099] In the present invention, the T cell activating antigen may be, but is not limited to, CD3, TCRα, TCRβ, TCRγ, TCRξ, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226.

[0100] In the present invention, the NK cell activating antigen may be, but is not limited to, NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160.

[0101] Other immune cell engagers are specifically described in U.S. Patent Application Publication US 2017 / 0368169 and may be introduced by reference in this invention.

[0102] Specifically, the aforementioned immune cell engagement bispecific or multispecific antibody comprises a series of scFvs and can bind to the following antigens and surface antigens on cancer cells. The surface antigen on cancer cells is MYCT1, which is targeted by the antibody according to the present invention.

[0103] In the present invention, the immune cell engagement bispecificity or multispecificity antibody may include, for example, structures in the form of VL(MYCT1)-VH(MYCT1)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(MYCT1)-VL(MYCT1)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(CD3 or CD16A)-VL(CD3 or CD16A)-VH(MYCT1)-VL(MYCT1), or VH(CD3 or CD16A)-VL(CD3 or CD16A)-VL(MYCT1)-VH(MYCT1).

[0104] In the present invention, a linker may be further included between the heavy chain variable region and the light chain variable region. Here, the linker may be a peptide linker and may have a length of about 10 to 25 aa. For example, hydrophilic amino acids such as glycine and / or serine may be included, but are not limited to these.

[0105] Specifically, in the present invention, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (where n and m are each 1 to 10), preferably GGGGS, and preferably GGGGSGGGGSGGGGS, but is not limited to these.

[0106] Examples of the immune cell engagement bispecific or multispecific antibodies in the present invention include blinatumomab (Amgen) that binds to CD3 and CD19; solitomab (Amgen) that binds to CD3 and EpCAM; MEDI565 (MedImmune, Amgen) that binds to CD3 and CEA; and BAY2010112 (Bayer, Amgen) that binds to CD3 and PSMA. Exemplary DARTs include MGD006 (Macrogenics) that binds to CD3 and CD123; and MGD007 (Macrogenics) that binds to CD3 and gpA33. Exemplary TandAbs may include AFM11 (Affimed Therapeutics) that binds to CD3 and CD19; and AFM13 (Affimed Therapeutics) that binds to CD30 and CD16A.

[0107] immune complex According to yet another embodiment of the present invention, the present invention relates to an immune complex in which the antibody or antigen-binding fragment provided herein is combined with a cytotoxic agent.

[0108] In the present invention, the cytotoxic agent may be one or more selected from the group consisting of chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins or fragments thereof derived from bacteria, fungi, plants or animals), and radioisotopes.

[0109] In one embodiment, the immune complex provided in the present invention may be an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment is bound to a drug.

[0110] In this specification, the term "antibody-drug conjugate (ADC)" refers to a form in which a drug and an antibody are chemically linked without reducing the biological activity of the antibody and the drug. In the present invention, the antibody-drug conjugate refers to a form in which the drug is bound to the N-terminal amino acid residue of the heavy chain and / or light chain of the antibody, specifically, a form in which the drug is bound to the N-terminal α-amine group of the heavy chain and / or light chain of the antibody.

[0111] In the present invention, the antibody or antigen-binding fragment may be bound to a drug via a linker. The linker is a site that connects the antibody and the drug, enabling cleavage under intracellular conditions, i.e., allowing the drug to be released from the antibody in the intracellular environment, and reflecting the antibody's long half-life, the antibody remains stable in systemic circulation, and the binding of the linker to the drug should not affect the antibody's stability or pharmacokinetics.

[0112] In the present invention, the linker may include, for example, a cleavable linker or a non-cleavable linker. In the case of a cleavable linker, like a peptide linker, it may be cleaved by an intracellular peptidase or protease enzyme, such as a lysosome or endosomal protease, while in the case of a non-cleavable linker, for example, a thioether linker, the drug may be released after the antibody is non-selectively degraded by intracellular hydrolysis.

[0113] In the present invention, the cleavable linker may include a peptide linker. The peptide linker has a length of at least two amino acids. For example, it may include Val-Cit, Val-Ala, or a dipeptide of Val-Cit, or Phe-Leu or Gly-Phe-Leu-Gly. Examples of linkers are specifically described in International Patent Application Publication WO2004 / 010957, which can be introduced by reference in the present invention.

[0114] In the present invention, the antibody-drug conjugate is formed when the antibody domain of the ADC binds to the antigen of the target cancer cell, forming an ADC-antigen conjugate, which is then encapsulated into the cancer cell along the endosomal-lysosome pathway. In this case, the intracellular release of the cytotoxic drug is regulated by the internal environment of the endosome / lysosome.

[0115] In the present invention, the cleavable linker is pH-sensitive and may be sensitive to hydrolysis at a specific pH value. Generally, a pH-sensitive linker indicates that it can be hydrolyzed under acidic conditions. For example, acid-unstable linkers that can be hydrolyzed in lysosomes may include hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, and ketals.

[0116] In the present invention, the linker can also be cleaved under reducing conditions, for example, a disulfide linker. Various disulfide bonds can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene). Such disulfide linkers can be degraded by disulfide exchange with the thiol of intracellular glutathione.

[0117] In the present invention, the drug and / or drug-linker may be randomly conjugated via the lysine of the antibody or via cysteine ​​exposed when the disulfide bond chain is reduced. Optionally, the linker-drug may be conjugated via a genetically engineered tag, e.g., cysteine ​​present in a peptide or protein. The genetically engineered tag, e.g., a peptide or protein, may include, for example, an amino acid motif that can be recognized by an isoprenoid transferase. The peptide or protein may have a deletion at its carboxyl terminus or an addition to its carboxyl (C) terminus via a covalent bond of a spacer unit.

[0118] In the present invention, the peptide or protein can be immediately covalently bonded to an amino acid motif or covalently bonded to a spacer unit and linked to the amino acid motif. The amino acid spacer unit consists of 1 to 20 amino acids, among which glycine units are preferred.

[0119] In the present invention, the linker may include a β-glucuronide linker that is recognized and hydrolyzed by β-glucuronidase, which is abundant in lysosomes or overexpressed in some tumor cells. Unlike peptide linkers, it has the advantage of being highly hydrophilic, and therefore can increase the solubility of the antibody-drug conjugate when bound to drugs with high hydrophobic properties. In connection with this, a β-glucuronide linker disclosed in International Patent Application Publication WO2015 / 182984, for example, a β-glucuronide linker containing a self-immolative group, may be used, and the above document is introduced by reference.

[0120] In the present invention, the linker may be, for example, a non-cleavable linker, and the drug is released only through the intracellular antibody hydrolysis step, producing, for example, an amino acid-linker-drug complex. Such types of linkers may be a thioether group or a maleimidocaproyl group, and can maintain stability in the blood.

[0121] In the present invention, linker-drugs can be randomly bound via cysteine ​​exposed when the disulfide bond chain of the antibody or antigen-binding fragment is reduced, or by introducing an antibody-terminally bound peptide having the sequence GGGGGGGCVIM.

[0122] In the present invention, the drug is a formulation that exhibits pharmacological effects, can be bound to an antibody, and specifically may be a chemotherapeutic agent, a toxin, microRNA (miRNA), siRNA, shRNA, or a radioisotope. The chemotherapeutic agent may be, for example, a cytotoxic formulation or an immunosuppressant. Specifically, it may include a microtubulin inhibitor, a mitotic inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent that can function as a DNA intercalator. It may also include drugs for the treatment of cancer, inflammation, or immune diseases. For example, it may include immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, anthelmintic agents, or combinations thereof.

[0123] In the present invention, the anticancer agent is, for example, meitansinoid, auristatin (including MMAE and MMAF), aminopterin, actinomycin, bleomycin, thalisomycin, camptothecin, N8-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazide, esperamycin, etoposide, 6-mercaptopurine, dorastatin, trichothecene, calichemycin, taxol, taxane, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocalmycin L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustardMustard, cytoxan, etoposide, 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparagine NASE), vinorelbine, chlorambucil, melphalan, carmustine, lomustine, busulfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, crisnatol, mitomycin C C), Trimetrexate, Mycophenolic acid, Thiazofurin, Ribavirin, EICAR (5-ethynyl-1-beta-Dribofuranosylimidazole-4-carboxamide), Hydroxyurea, Deferoxamine, Floxuridine, Doxifluridine, Raltitrexed, Cytarabine (AR C)), Cytosine ArabinosideArabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrelin A, interferon-α, interferon-γ, tumor necrosis factor factor), gemcitabine, velcade, revamid, thalamid, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2 B2) may be one or more selected from the group consisting of peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, thapsigargin, nucleolytic enzymes, and toxins derived from bacteria, plants, or animals.

[0124] In the present invention, the anti-inflammatory agent may include, but is not limited to, steroid preparations that bind to glucocorticoid receptors to reduce inflammation and swelling, non-steroidal anti-inflammatory drugs (NSAIDs) that relieve pain by acting in conjunction with cyclic oxygen saccharinase (COX) that synthesizes prostaglandins that cause inflammation, or immunospecific anti-inflammatory drugs (ImSAIDs) that alter the activation and transport of inflammatory cells.

[0125] The present invention may include, but is not limited to, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, mycophenolate, azathioprine, or methotrexate as the immunotherapy agent for the immune disease.

[0126] In the present invention, the drug may, in some cases, contain one or more nucleophilic groups selected from the group consisting of amines, thiols, hydroxyls, hydrazides, oximes, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazide groups that can react to form a covalent bond with an electrophilic group on the linker and the linker reagent.

[0127] Chimeric antigen receptor (CAR) Further embodiments of the present invention relate to a chimeric antigen receptor (CAR) comprising an antigen-binding fragment provided in the present invention.

[0128] In this specification, the terms “chimeric antigen receptor” or “CAR” mean an engineered receptor comprising an extracellular antigen-binding domain and an intracellular signaling domain. The most common type of CAR includes single-stranded variable fragments (scFv) derived from monoclonal antibodies fused to the transmembrane and intracellular domains of T cell co-receptors such as CD3ζ chains, but the invention as described herein is not limited to these domains.

[0129] As used herein, “chimeric antigen receptor” or “CAR” means any receptor engineered to express any intracellular signaling molecule and an extracellular antigen-binding domain fused to or ligated thereto.

[0130] In the present invention, the binding domain may include an antigen-binding fragment provided in the present invention, preferably an scFv.

[0131] The chimeric antigen receptor of the present invention may further include at least one of a hinge region (or spacer) and a signaling domain.

[0132] In the present invention, the hinge region is a part that connects the antigen-binding domain and the membrane-passing domain, and is also called a "spacer," and has the purpose of extending the antigen-binding domain from the T cell membrane or NK cell membrane.

[0133] In the present invention, the hinge region may be obtained from any suitable sequence from any genus including human or a part thereof, or may include, but is not limited to, a hinge region of a human protein containing all or part of CD8, D28, 4-1BB, OX40, CD3 zeta (ζ) chains, T cell receptor α or β chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof.

[0134] Furthermore, in the present invention, the hinge region is not limited to immunoglobulins and may include, but is not limited to, one selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD).

[0135] In the present invention, the signaling domain means a portion of the chimeric antigen receptor that is found inside a T cell or has been manipulated to be found inside a T cell. In the present invention, the signaling domain may or may not include a membrane-passing domain that plays a role in fixing the chimeric antigen receptor to the plasma membrane of the T cell.

[0136] In the present invention, the transmembrane domain and the signal transduction domain may originate from the same protein (e.g., CD3ζ), or they may originate from different proteins (e.g., the transmembrane domain of CD28 and the intracellular signal transduction domain of the CD3ζ molecule, or vice versa).

[0137] In the present invention, the membrane-transmitting domain includes a hydrophobic polypeptide that crosses the cell membrane. In particular, the membrane-transmitting domain may cross from one side of the cell membrane (extracellular) through the other side of the cell membrane (intracellular or cytoplasmic).

[0138] In the present invention, the membrane-passing domain may be in the form of an α-helix, a β-barrel, or a combination thereof. Furthermore, in the present invention, the membrane-passing domain may comprise a multi-component protein having multiple membrane-passing fragments, each an α-helix, a β-sheet, or a combination thereof.

[0139] In the present invention, the membrane-passing domain may include, but is not limited to, T cell receptor α or β chains, all or part of CD3 zeta (ζ) chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof.

[0140] In the present invention, the membrane-passing domain, for example, an artificially designed one, may be a polypeptide mainly containing hydrophobic residues such as leucine and valine. In one embodiment of the present invention, a triplet of phenylalanine, tryptophan, and valine may be found at each terminus of the synthetic membrane mutation domain.

[0141] In the present invention, at least one co-stimulatory domain may be further included between the membrane-passing domain and the signal transduction domain. In the present invention, the co-stimulatory domain is the site on which the co-stimulatory signal is transmitted and can transmit signals to cause CAR-T cells or CAR-NK cells that recognize a specific antigen bound to the antigen-binding domain to trigger an immune response, aid in self-proliferation, and increase the time they remain in the body.

[0142] In the present invention, the co-stimulatory domain may include, but is not limited to, functional signaling domains derived from polypeptides containing 4-1BB(CD137);OX40;CD27;CD28;CD30;CD40;PD-1;CD2;CD7;CD258;Natural Killer Group 2 Member C(NKG2C);Natural Killer Group 2 Member(NKG2D);B7-H3;CD83;ICAM-1;LFA-1(CD11a / CD18) or ligands that bind to ICOS; active fragments thereof; functional derivatives thereof; or combinations thereof.

[0143] In the present invention, the signal transduction domain may include a polypeptide that provides activation of immune cells in order to stimulate or activate at least a part of the immune cell signal transduction pathway.

[0144] In the present invention, the signal transduction domain may include, but is not limited to, functional signal transduction domains derived from polypeptides containing all or part of CD3 zeta (ζ), common FcR gamma (FcER1G), Fc gamma RIIIa, FcR beta (Fc epsilon receptor), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX activating protein 10 (DAP10), DNAX activating protein 12 (DAP12), its active fragment, its functional derivative, or combinations thereof, and such signal transduction domains are known in the art.

[0145] Furthermore, the chimeric antigen receptor of the present invention may further contain a signal peptide.

[0146] In the present invention, the "signal peptide" comprises a peptide sequence, which is a peptide of any type of secreted or transmembrane-transmissible protein that can direct the transport of the chimeric antigen receptor of the present invention to the cell membrane and cell surface, and provide precise positioning of the chimeric antigen receptor of the present invention. In particular, in the present invention, the signal peptide causes the chimeric antigen receptor of the present invention to be supported on the cell membrane, in which case the extracellular portion of the chimeric antigen receptor is displayed on the cell surface, the transmembrane-transmissible portion crosses the plasma membrane, and the active domain is located in the cytoplasm or inside the cell.

[0147] In the present invention, the signal peptide can be any peptide that performs the above function without limitation. For example, it may be an N-terminal CD8α signal peptide, but is not limited to these.

[0148] Another embodiment of the present invention relates to a nucleic acid molecule encoding a chimeric antigen receptor according to the present invention.

[0149] In the present invention, the nucleic acid molecule encoding the chimeric antigen receptor can be readily produced from the amino acid sequence of the chimeric antigen receptor identified by any conventional method. The nucleotide sequence encoding the amino acid sequence can be obtained for the amino acid sequence of each domain from the aforementioned NCBI RefSeq ID or GenBenk enrollment number, and the nucleic acids disclosed herein can be produced using standard molecular biological and / or chemical procedures. For example, polynucleotides based on the nucleotide sequence can be synthesized, and the nucleic acid molecules disclosed herein can be produced by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0150] The nucleic acid molecules of the present invention may be, but are not limited to, a gene or part of an expression or cloning cassette.

[0151] According to yet another embodiment of the present invention, the present invention relates to an expression cassette containing nucleic acid molecules.

[0152] The expression cassette of the present invention may include expression regulatory sequences such as promoters, enhancers, polyadenylation signals, transcription termination elements, or internal ribosome entry sites (IRESs) that can regulate the expression of the nucleic acid molecule of the present invention.

[0153] In the present invention, the promoter may include, but is not limited to, the SFFV promoter, the elongation factor 1α (EF 1a) promoter, or the CAG (chicken β-actin promoter having a CMV enhancer) promoter.

[0154] The expression cassette of the present invention further comprises a gene sequence encoding a surrogate marker useful for monitoring its expression, such as a cell surface marker (e.g., a cleavage-type cell surface marker), which can be used to confirm the transduction or manipulation of cells expressing a receptor.

[0155] In the present invention, exemplary marker genes may include, but are not limited to, truncated epidermal growth factor receptor (EGFRt), prostate-specific membrane antigen (PSMA), CD34, nerve growth factor receptor (NGFR), or nucleotide sequences encoding modified forms thereof.

[0156] Furthermore, the marker gene in the present invention may include, but is not limited to, nucleotide sequences encoding fluorescent proteins such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as superfolder GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), as well as species variants, monomer variants, and codon-optimized and / or enhanced variants of fluorescent proteins.

[0157] Furthermore, in the present invention, the marker gene is or includes an enzyme, such as luciferase, the Escherichia coli-derived lacZ gene, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyl transferase (CAT). Exemplary luminescence reporter genes may include, but are not limited to, nucleotide sequences encoding luciferase (luc), β-galactosidase, chloramphenicol acetyl transferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0158] Furthermore, in the present invention, the marker gene is a gene that encodes a polypeptide that confers resistance to an exogenous preparation or drug, and may be, but is not limited to, a puromycin resistance gene, a hygromycin resistance gene, a blasticidine resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeosin resistance gene or a modified form thereof.

[0159] Furthermore, in the present invention, the marker gene and one end of the polynucleotide of the present invention may be linked by a polynucleotide encoding a cleavable peptide.

[0160] In the present invention, the cleavable peptide corresponds to the self-cleavage site and may be, but is not limited to, P2A, F2A, E2A, T2A peptides or combinations thereof.

[0161] According to yet another embodiment of the present invention, the present invention relates to a recombinant expression vector comprising an expression cassette provided herein.

[0162] In this invention, the description of the recombinant expression vector overlaps with that described above for the antibody or antigen-binding fragment, and therefore, that description will be omitted below.

[0163] A further embodiment of the present invention relates to immunoeffector cells transduced by a recombinant expression vector provided in the present invention.

[0164] In the present invention, the immune effector cells may be allogeneic or autoimmune cells. Furthermore, the immune effector cells in the present invention may be derived from, for example, T cells, dendritic cells, killer dendritic cells, mast cells, NK cells, NKT cells, macrophages, mononuclear cells, B cells, and stem cells, but are not limited to these.

[0165] In this specification, the term “Natural Killer Cells (NK cells)” is defined as large granular lymphocytes (LGLs) and constitutes three types of cells that differentiate from common lymphoid progenitor cells—progenitor B and T lymphocytes. NK cells are known to differentiate from bone marrow, lymph nodes, spleen, tonsils, and thymus, mature, and enter the circulatory system. In this invention, the NK cells may include, without limitation, any type of NK cell, such as cultured NK cells, such as primary NK cells, NK cells from cultured NK cell lines, or NK cells obtained from mammals. When NK cells are obtained from mammals, they may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. NK cells may be concentrated and purified. NK cells are preferably human NK cells (for example, they can be isolated from humans). NK cell lines are available from, for example, the ATCC (American Type Culture Collection) and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408), or derivatives thereof.

[0166] A further embodiment of the present invention relates to polypeptides expressed from immunoeffector cells provided in the present invention.

[0167] therapeutic use Further embodiments of the present invention relate to an antibody or antigen-binding fragment provided in the present invention; or a pharmaceutical composition for the prevention, improvement, or treatment of MYCT1-related diseases comprising the antibody or antigen-binding fragment.

[0168] The antibody or antigen-binding fragment of the present invention exhibits excellent affinity and binding affinity to MYCT1. Specifically, it specifically binds to tumor endothelial cells expressing MYCT1 and reprograms the tumor microenvironment (TME), thereby being usefully applied to the treatment of diseases associated with MYCT1 overexpression, such as cancer, particularly solid tumors.

[0169] The MYCT1-related disease may be a disease associated with the expression or overexpression of MYCT1, and may be, for example, cancer. Therefore, the MYCT1-related disease is preferably cancer or a tumor, but is not limited thereto.

[0170] In this specification, the term "cancer" refers to or indicates a physiological condition in mammals characterized by typically uncontrolled cell growth.

[0171] The cancers or carcinomas that can be treated with the compositions of the present invention are not particularly limited and include all solid cancers and hematological cancers. Examples of such cancers may be selected from, but are not limited to, the group consisting of skin cancers such as melanoma, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0172] More preferably, the cancer is characterized by the expression of the MYCT1 protein and may be, but is not limited to, breast cancer, pancreatic cancer, prostate cancer, lung cancer, thyroid cancer, stomach cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer, or bladder cancer. The cancer may be primary or metastatic.

[0173] In the present invention, the MYCT1-related disease may be an angiogenesis-related disease. The angiogenesis-related disease may be one or more selected from the group consisting of rheumatoid arthritis, osteoarthritis, septic arthritis, psoriasis, corneal ulcer, age-related macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, retinopathy of prematurity, ophthalmic inflammation, keratoconus, Sjögren's syndrome, myopic eye tumor, corneal transplant rejection, impaired wound healing, bone disease, proteinuria, abdominal aortic aneurysm, degenerative cartilage loss associated with traumatic joint injury, demyelinating diseases of the nervous system, cirrhosis, renal glomerulosis, premature rupture of membranes of the fetus, inflammatory bowel disease, periodontal ligament disease, arteriosclerosis, restenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, and skin aging.

[0174] In this specification, the term “angiogenesis” is used to encompass all aspects of the maintenance and development of blood vessels. Therefore, angiogenesis includes not only the maintenance and control of conventional vascular structures and small vessels, but also the formation of new capillaries leading to neovascularization. Angiogenesis is a complex process involving a series of sequential steps, including endothelial cell-mediated degradation of the vascular basement membrane and interstitial matrix, endothelial cell migration, endothelial cell proliferation, and the formation of capillary loops by endothelial cells. Angiogenesis includes the growth and / or development of new blood vessels (also known as neovascularization), dilation of small vessels, excessive or prolonged vascular growth, and the maintenance of conventional vascular structures. MYCT1 is known to be involved in the regulation of angiogenesis (see Non-Patent Literature).

[0175] In this specification, the term “angiogenic disorders” is used to mean specific pathological processes in humans in which angiogenesis is abnormally prolonged. This further includes neovascular diseases and illnesses such as diseases and illnesses associated with, caused by, or related to angiogenesis. Non-limiting examples of such diseases include various forms of cancer and metastasis characterized by angiogenesis / neovascularization. The antibodies and antigen-binding fragments disclosed herein can be used to treat angiogenic disorders by binding to MYCT1 and inhibiting angiogenesis.

[0176] In the present invention, the term "prevention" may be limited to all actions that can block, suppress, or delay symptoms resulting from MYCT1-related disease using the compositions of the present invention.

[0177] In the present invention, the terms “improvement” and “treatment” can be, without limitation, any action that enables or may be beneficial in improving symptoms resulting from MYCT1-related diseases using the compositions of the present invention.

[0178] The pharmaceutical compositions of the present invention, while not limited thereto, can be used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injection solutions, by conventional methods. Preferably, the pharmaceutical compositions can be formulated for intra-organ administration, inhalation administration, or injection, but are not limited thereto.

[0179] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, fragrances, etc. For injectable preparations, it may be a mixture of buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. For topical administration, it may include bases, excipients, lubricants, preservatives, etc. The dosage forms of the pharmaceutical composition of the present invention can be manufactured in various ways by mixing it with the pharmaceutically acceptable carrier described above. For example, for oral administration, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. For injectable preparations, it can be manufactured in unit dose ampoules or multi-dose forms. Other dosage forms include solutions, suspensions, tablets, capsules, and sustained-release formulations.

[0180] Examples of carriers, excipients, and diluents suitable for formulation according to the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. The invention may also further contain fillers, anti-flocculants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like.

[0181] The administration routes of the pharmaceutical composition of the present invention are not limited to those listed above, but include oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. It may be administered orally or parenterally, preferably orally, but is not limited to these.

[0182] The parenteral administration of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-sacral, intrasternal, intradural, intrafocal, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0183] The pharmaceutical composition of the present invention can vary considerably depending on various factors, including the activity of the specific compound used, age, weight, general health, sex, diet, administration time, route of administration, excretion rate, drug formulation, and the severity of the specific disease being prevented or treated. The dosage of the pharmaceutical composition can be appropriately selected by a skilled technician, depending on the patient's condition, weight, severity of the disease, drug form, route of administration, and duration, and can be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration can be once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention can be in the form of pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, or suspensions.

[0184] A further specific example of the present invention relates to a method for preventing, improving, or treating MYCT1-related diseases, comprising the step of administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment provided in the present invention; or a bispecific or multispecific antibody containing the antibody or antigen-binding fragment.

[0185] In the present invention, the subject is a subject that has developed or is suspected of developing a MYCT1-related disease (particularly a disease caused by overexpression of MYCT1), and the subject suspected of developing the disease means all animals including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs, but any subject that can be treated with the active substance provided in the present invention is included without limitation.

[0186] In this specification, the term “pharmaceutical effective dose” is an amount sufficient to discontinue or reduce the physiological effects on a subject or patient induced by MYCT1-related disorders. An appropriate effective dose may be determined by the treating physician within the correct range of medical judgment and may be administered in one dose or in several divided doses. However, for the purposes of the present invention, it is preferable that the specific therapeutic effective dose for a particular patient be applied differently depending on various factors, including the type and degree of response to be achieved, the specific composition containing the active ingredient including whether other formulations are used, the patient’s age, weight, general health status, sex and diet, administration time, route of administration, number of administrations and the secretion rate of the composition containing the active ingredient, duration of treatment, and drugs used with or concurrently with the specific composition, as well as similar factors well known in the pharmaceutical field.

[0187] The total effective dose of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol using multiple doses over a longer period. The active ingredient content of the composition of the present invention can vary depending on the severity of the disease. Specifically, a preferred total dose of the composition of the present invention may be approximately 0.0001 mg to 500 mg per kg of patient body weight per day. However, the effective dose of the composition is determined by considering various factors such as the patient's age, weight, health condition, sex, severity of the disease, diet, and excretion rate, as well as the route of administration and number of treatments of the pharmaceutical composition. Considering these points, a person with ordinary skill in the art can determine an appropriate effective dose of the composition of the present invention for a specific use. The pharmaceutical composition according to the present invention is not particularly limited in its dosage form, route of administration, and method of administration, as long as it exhibits the effects of the present invention.

[0188] Diagnostic use Further embodiments of the present invention relate to an antibody or antigen-binding fragment provided in the present invention; or a diagnostic composition for MYCT1-related diseases comprising the antibody or antigen-binding fragment.

[0189] In the present invention, by detecting MYCT1 with the antibody or antigen-binding fragment, it can be used as a diagnostic marker for evaluating the progression of angiogenesis-related diseases, such as cancer or diseases characterized by neovascularization, and for assessing the prognosis before and after treatment.

[0190] The antibody or antigen-binding fragments of the present invention can be used in any known analytical method, such as competitive binding analysis, direct and indirect sandwich analysis, and immunoprecipitation analysis.

[0191] The antibody or antigen-binding fragment of the present invention may be labeled with a label having the following functions: (i) the function of providing a detectable signal; (ii) the function of reacting with a first or second label, e.g., a second label to modify a detectable signal provided by FRET (Fluorescence Resonance Energy Transfer); (iii) the function of affecting motility by charge, hydrophobicity, morphology, or other physical parameters, e.g., electrophoretic motility; or (iv) the function of providing a capture moisture such as affinity, antibody / antigen, or ion complex. Suitable labels include fluorescent labels, luminescent labels, chromophore labels, radioactive labels, isotope labels, isodenuclear labels, enzyme labels, particle labels, especially metal particle labels, magnetic particle labels, polymer particle labels, small organic molecules such as biotin, receptor ligands, or binding molecules such as cell adhesion proteins or lectins, and labels-sequences containing nucleic acids and / or amino acid residues that are detectable by the use of a binder.

[0192] In the present invention, the specific types of the labeled material are not particularly limited, but for example, enzymes using hydrogen peroxide to oxidize dye precursors, such as HRP, lactoperoxidase or microperoxidase, biotin / avidin, spin labeling, bacteriophage labeling, radioisotopes associated with stable free radicals such as 32P, 14C, 125I, 3H and 131I, fluorophores, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, For example, this includes, but is not limited to, firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharido oxidase, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, and heterocyclic oxidases, such as uricase and xanthine oxidase.

[0193] Another embodiment of the present invention relates to a diagnostic kit for MYCT1-related diseases comprising the diagnostic composition of the present invention.

[0194] In the present invention, the "kit" refers to a tool that allows for the evaluation of the biomarker expression level by labeling a probe or antibody that specifically binds to a biomarker component with a detectable label. This includes not only direct labeling of a detectable substance related to the probe or antibody by reaction with a substrate, but also indirect labeling by binding of a chromogenic label that reacts with other directly labeled reagents. The kit may include a chromogenic substrate solution that reacts with the label, a washing solution, and other solutions, and can be prepared by including the reagent components used. In the present invention, the kit may include essential elements necessary for performing RT-PCR, and in addition to specific primer pairs for each marker gene, it may also include a test tube, reaction buffer, deoxyribonucleotides (dNTPs), Taq polymerase, reverse transcriptase, DNase, RNase inhibitor, sterile water, etc. The kit may also be a kit for detecting genes for cancer diagnosis that includes essential elements necessary for performing DNA chipping. A DNA chip kit includes a substrate to which cDNA corresponding to a gene or fragment is attached as a probe, and the substrate may include cDNA corresponding to a quantitative control group gene or fragment. The kits of the present invention are not limited to those known in the art.

[0195] In the present invention, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0196] The kit of the present invention may further include one or more other component compositions, solutions, or apparatus suitable for the analytical method. For example, the kit in the present invention may further include essential elements necessary for carrying out a reverse transcription polymerase chain reaction. The reverse transcription polymerase chain reaction kit includes a primer pair specific to the gene encoding a marker protein. The primers are nucleotides having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include primers specific to the nucleic acid sequence of a control gene. Other components of the reverse transcription polymerase chain reaction kit may include a test tube or other suitable container, reaction buffer (various pH and magnesium concentrations), deoxyribonucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC water, sterile water, etc.

[0197] Furthermore, the diagnostic kit of the present invention may include essential elements necessary for DNA chipping. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or fragment of a gene are attached, and reagents, formulations, enzymes, etc., for producing a fluorescently labeled probe. The substrate may also include cDNA or oligonucleotides corresponding to a control group gene or fragment of a control group.

[0198] Furthermore, the diagnostic kit of the present invention may include essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific to the protein. The antibody is one that has high specificity and affinity for the marker protein and little cross-reactivity to other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. The ELISA kit may also include an antibody specific to a control group protein. The ELISA kit may also include reagents capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, enzymes (e.g., conjugated with the antibody) and their substrates, or other substances capable of binding to the antibody.

[0199] In the present invention, the fixative for the antigen-antibody binding reaction can be a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, a glass slide, or the like, but is not limited to these.

[0200] In the present invention, the washing solution preferably contains a phosphate buffer solution, NaCl, and Tween 20, and more preferably a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20. After the antigen-antibody binding reaction and the reaction of the antigen-antibody complex with the secondary antibody, an appropriate amount of the washing solution is added to the fixative and washed 3 to 6 times. A sulfuric acid solution (H2SO4) can preferably be used as the reaction stop solution.

[0201] A further specific example of the present invention relates to an information-providing method for diagnosing MYCT1-related diseases, comprising the step of contacting a biological sample separated from a target organism with an antibody or antigen-binding fragment provided in the present invention; or a bispecific or multispecific antibody containing the antibody or antigen-binding fragment.

[0202] In the present invention, the subject is a subject in which a MYCT1-related disease (particularly a disease caused by overexpression of MYCT1) has occurred or is suspected to have occurred. The subject in which the onset of the disease is suspected includes all animals including monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, including humans in which the disease has occurred or may occur. However, the subjects treatable with the active substance provided in the present invention are included without limitation.

[0203] In the present invention, the biological sample may include, but is not limited to, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0204] The method for providing information of the present invention may include the step of measuring the expression level of MYCT1 protein in the biological sample using the antibody or antigen-binding fragment provided in the present invention.

[0205] In the present invention, the measurement of the expression level of the protein can be carried out by Western blotting, ELISA (enzyme linked immunosorbent assay), competitive binding assay or a method similar thereto.

[0206] The information providing method of the present invention may include a step of predicting that when the expression level of the MYCT1 protein measured in the biological sample is increased compared to the control group, an MYCT1-related disease has occurred or the possibility of onset is high. Here, the control group may be the expression level of MYCT1 in a healthy normal control group, but is not limited thereto.

[0207] Combined therapy According to still another specific example of the present invention, there is provided a combined therapy in which a pharmaceutical composition containing, as an active ingredient, an antibody or antigen-binding fragment provided in the present invention; an immune complex; a chimeric antigen receptor; or an immune effector cell expressing a chimeric antigen receptor is administered in combination with at least one additional therapeutic agent or therapeutic procedure.

[0208] The at least one additional therapeutic agent or therapeutic procedure may be selected from one or more of chemotherapy, targeted anticancer therapy, oncolytic drugs, cytotoxic agents, immune-based therapy, cytokines, surgical procedures, radiation procedures, vaccines, or cell therapy, but is not limited thereto.

[0209] The at least one therapeutic agent may be an immune anticancer agent. <​The aforementioned at least one therapeutic agent may be a PD-1 inhibitor, such as a PD-1 antibody. Specifically, the PD-1 inhibitor may be selected from, but is not limited to, PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, or AMP-224.

[0211] The aforementioned at least one therapeutic agent may be a PD-L1 inhibitor, such as a PD-L1 antibody. Specifically, it may be selected from, but is not limited to, FAZ053, atezolizumab, avelumab, durvalumab, or BMS-936559.

[0212] In this invention, the term "combined administration" should be understood to mean simultaneous, individual, or sequential administration. When the administration is sequential or individual, the interval between administrations of the secondary components must be such that the beneficial effects of the combined administration are not lost.

[0213] Uses for enhancing sensitivity of immunosuppressant cancer drugs Further embodiments of the present invention relate to the use of the antibody or antigen-binding fragment provided in the present invention to enhance the sensitivity of immunosuppressant agents in various cancers.

[0214] In this specification, the term "sensitivity" means whether or not a particular drug (e.g., an immunosuppressant) is effective against an individual patient's cancer.

[0215] In this specification, the term "resistance," as used in reference to sensitivity, means not reacting sensitively to the drug and therefore not being affected by its efficacy.

[0216] For example, the aforementioned specific drugs are mainly immunosuppressants, and these anticancer drugs may or may not be effective depending on the type of cancer. Furthermore, even for cancers for which an anticancer drug is recognized as effective, it is known that it may or may not be effective in individual patients. Whether or not an anticancer drug is effective for an individual patient's cancer is called anticancer drug sensitivity. Therefore, if the present invention allows for the prediction of patients who are expected to respond (responders) and patients who are not expected to respond (non-responders) before the start of treatment, it is possible to realize chemotherapy with high efficacy and safety.

[0217] A composition containing a therapeutically effective amount of the antibody or antigen-binding fragment of the present invention can be administered to a target organism resistant to immunosuppressants to enhance the organism's sensitivity to immunosuppressants.

[0218] The terms used in the embodiments are for illustrative purposes only and should not be construed as intended to be limiting. Singular expressions include plural expressions unless otherwise clearly stated. In this specification, terms such as “includes” or “having” are intended to specify the existence of features, figures, stages, operations, components, parts or combinations thereof as described in the specification, and should not be understood as preemptively excluding the existence or possibility of adding one or more other features, figures, stages, operations, components, parts or combinations thereof.

[0219] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the art to which the embodiments belong. Terms as defined in commonly used dictionaries should be interpreted as having the meaning consistent with their meaning in the portal of the relevant art, and not as an ideal or overly formal meaning unless expressly defined herein.

[0220] When describing the components of an embodiment, terms such as First, Second, A, B, (a), (b), etc., may be used. Such terms are merely for distinguishing a component from other components and do not limit the nature, procedure, or order of that component. When it is stated that any component is “linked,” “joined,” or “connected” to another component, it should be understood that the component may be directly linked or connected to other components, but further other components may also be “linked,” “joined,” or “connected” between each component.

[0221] The embodiments are described in detail below with reference to the attached drawings. However, various modifications can be made to the embodiments, and the scope of the patent application is not limited or restricted by such embodiments. All modifications, equivalents, and substitutions to the embodiments should be understood to be included within the scope of the patent.

[0222] Furthermore, when explaining with reference to the attached drawings, regardless of the reference numerals, identical components will be assigned the same reference numerals, and redundant explanations related thereto will be omitted. When describing embodiments, if it is determined that a specific explanation of related known technology could unnecessarily obscure the gist of the embodiment, such detailed explanation will be omitted.

[0223] The present invention can be subjected to various transformations and has a wide range of embodiments. Specific embodiments will be illustrated in the drawings and described in detail below. However, this should not be understood as limiting the present invention to specific embodiments, but rather as encompassing all transformations, equivalents, and substitutions included in the spirit and technical scope of the present invention. In describing the present invention, if a specific description of related well-known technology is deemed to obscure the gist of the invention, such detailed description will be omitted.

[0224] Example 1. Method for producing and analyzing humanized anti-MYCT1 antibody

[0225] 1-1. Production of Humanized Anti-MYCT1 Antibody The inventors attempted to produce a humanized antibody based on a mouse antibody. Specifically, an anti-MYCT1 mouse parental antibody (2B4 clone) having a heavy chain variable region (VH) containing SEQ ID NO: 41 and a light chain variable region (VL) containing SEQ ID NO: 42; and an anti-MYCT1 mouse parental antibody (11B6 clone) having a heavy chain variable region (VH) containing SEQ ID NO: 92 and a light chain variable region (VL) containing SEQ ID NO: 93 were obtained. In Table 1 below, CDRs (italicized and bold) were annotated using the Kabat CDR definition.

[0226] [Table 1]

[0227] By analyzing such mouse antibody sequences and humanizing specific sequences while maintaining specific sequences, a humanization process was performed to reduce the possibility of immunogenicity while maintaining the binding ability as much as possible. An antibody consists of a variable region and a constant region. The variable region is further composed of complementarity determining regions (CDRs, which directly bind to an antigen) and framework regions (FRs, which support the CDR loops). A humanized antibody is primarily produced by a CDR grafting method that grafts the CDR loops of a mouse antibody onto a human antibody (chimerization). However, when only CDR grafting is performed, the affinity of the humanized antibody decreases. By substituting some of the major framework region amino acid residues in the mouse antibody, which are considered to affect the structure of the CDR, a process was carried out to increase the affinity to a level similar to that of the original mouse antibody (humanization).

[0228] The specific implementation involved commissioning Wuxi Biologics to humanize the antibodies, resulting in the production of 47 different humanized antibodies. The IgG antibody plasmids, synthesized by Genewiz, included plasmids containing VH, where the VH domain is fused to the human IgG1 CH1, hinge, CH2, and CH3 segments in the heavy chain; and plasmids containing VL, where the VL domain is fused to the human Ig kappa CK domain in the light chain. Each structure contains identical invariant segments, various VH or VL domains, and various site mutations. A human IgK monopeptide was fused to the 5' end of either VH or VL for secretory expression.

[0229] The CDR sequences of the antibodies produced in this manner are shown in Table 2, and the sequences of the heavy chain variable region and light chain variable region are shown in Tables 2-6 below.

[0230] [Table 2]

[0231] [Table 3]

[0232] [Table 4]

[0233] [Table 5] JPEG2026521923000008.jpg221144JPEG2026521923000009.jpg141141

[0234] [Table 6] JPEG2026521923000011.jpg220145JPEG2026521923000012.jpg75144

[0235] 1-2. Expression of humanized anti-MYCT1 antibody Day 1: Generally, 6.0 × 10⁶ cells have a survival rate of 95% or more in 100 mL of cell culture medium. 6 ExpiCHO cells with a viability of 95% or more (at a concentration of 3 / mL or higher) were prepared for transduction. 71.2 μg of plasmid DNA and 228.5 μL of ExpiFectamine were added. TM The CHO reagents are 2.54 mL and 2.51 mL of OptiPRO, respectively. TM After diluting and mixing with SFM, the solution was added to ExpiCHO cell culture solution and then cultured using a platform shaker set to 110 rpm, 37°C, and 8% CO2.

[0236] Day 2: 18–22 hours after transduction, 428.5 μL of enhancer and 21.5 mL of feed were added to the culture medium to support high-density transient transduction and improve protein production.

[0237] After culturing for 10 days, cells were pelleted by centrifugation at 4000 rpm (3724 g, BECKMAN ALLERGA X-15 CENTRIFUGE, catalog number: B31016) at 25°C for 10 minutes. The supernatant was collected and used for purification and gel electrophoresis. SDS-PAGE samples were prepared by mixing 15 μL of supernatant with 5 μL of 4X loading buffer and boiling for 5 minutes. 15 μL of the sample was then processed using NuPAGE. TM The proteins were loaded onto 4-12% Bis-Tris Protein Gels (ThermoFisher) and run at a constant voltage of 200V for 35 minutes using MES running buffer. The proteins in the gel were then stained with Coomassie blue. (PageRuler) TM The molecular weight of the antibody was measured using an unstained protein ladder (ThermoFisher) along with the antibody sample.

[0238] 1-3. Purification of humanized anti-MYCT1 antibody The Protein A column was pre-packaged in 5 mL of MabSelectSure resin (GE Lifesciences, Cat.# 17543803). Before loading the cell culture supernatant containing expressed IgG, the column was equilibrated with 0.1 M Tris, pH 7.0. After loading, the column was washed with 10 CV of 0.1 M Tris, pH 7.0, and then eluted with 0.1 M Glycine, pH 3.5. The eluate was then neutralized with 0.1 M Tris, pH 9.0 and dialyzed against 1X PBS buffer (Sangon Biotech, B548117-0500).

[0239] Purification process (Protein A column):

[0240] A. Buffer preparation 1) Buffer A: 0.1M Tris, pH7.0 2) Buffer B: 0.1M Glycine pH3.5 3)PBS: 137mM NaCl, 2.68mM KCl, 1.76mM KH2PO4, 10mM Na2HPO4

[0241] B. Purification 1) A collection tube was prepared. 2) The column was equilibrated with washing buffer A at a rate of 1 mL / min over 10 CV. 3) The samples were loaded into the column. 4) The column was washed with 10 CV of buffer A. 5) Proteins were eluted with 10 CV of buffer B. Fractions with UV levels of 100 mAU or higher were collected. 1 mL and 2-3 mL fractions were collected from each fraction, confirmed by SDS-PAGE detection, and then combined.

[0242] C. Dialysis The protein was dialyzed against PBS buffer.

[0243] 1-4. Characterization of humanized anti-MYCT1 antibodies Antibody concentration was measured by absorbance at 280 nm using nanodrops. Antibody purity was evaluated by SDS-PAGE and SEC-HPLC. For SDS-PAGE samples, 15 μL of protein sample was mixed with 5 μL of 4X loading buffer and boiled for 5 minutes. 15 μL of the mixture was placed on a NuPAGE Bis-Tris Mini Gels 4-12% gel and advanced at 200 V for 35 minutes using MES running buffer. The protein on the gel was then stained with Coomassie blue. For SEC-HPLC, 80 μL of purified antibody was loaded onto a TSKgel G3000SWxL column linked to an HPLC system 1260 Infinity II using a running buffer of 50 mM sodium phosphate, 150 mM NaCl, pH 7.0. The running time was 20 minutes. Protein peaks were monitored with a UV detector at 280 nm. Peak maintenance time was analyzed using ChemStation software (V2.99.2.0).

[0244] 1-5. Measurement of binding affinity by SPR (Surface Plasma Resonance)

[0245] 1-5-1.k off (Dissociation rate constant) ranking The activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The CM5 sensor tip was activated with the mixture at a flow rate of 10 μL / min for 420 seconds. Next, 30 μg / mL of anti-human Fc IgG was injected into the tip at a flow rate of 10 μL / min for 420 seconds in 10 mM NaAc (pH 4.5). The tip was deactivated with 1 M ethanolamine-HCl (GE) at a flow rate of 10 μL / min for 420 seconds.

[0246] In running buffer 1X HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween® 20, pH 7.4), 13–20 μg / mL of parental antibody, humanized and PTM (Post-translational modification) removal variants were captured to Fc2 via anti-human Fc IgG. Fc IgG antigen, MYCT1 (10 nm), and running buffer were injected into channels 1–8 at a flow rate of 100 μL / min during a 120-second binding phase at a rate of 100 μL / min, followed by a 300-second dissociation phase. After each dissociation phase, 10 mM glycine pH 1.5 was injected as a regeneration buffer. The tips were regenerated with 10 mM glycine pH 1.5.

[0247] Surface channel Fc1 without captured ligands was used as a control surface for reference defects. Final data for each interaction were subtracted from reference Fc1 and buffer channel data. Experimental data were analyzed using Biacore 8K (version 3.0.12.15655) evaluation software to match a 1:1 binding model.

[0248] 1-5-2. SPR Overall Dynamics Test The activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The CM5 sensor tip was activated with the mixture at a flow rate of 10 μL / min for 420 seconds. Next, 30 μg / mL of anti-human Fc IgG was injected into the tip at a flow rate of 10 μL / min for 420 seconds in 10 mM NaAc (pH 4.5). The tip was deactivated with 1 M ethanolamine-HCl (GE) at a flow rate of 10 μL / min for 420 seconds.

[0249] 3 μg / mL parental antibody and lead molecules, diluted in 1X HBS-EP+ running buffer, were captured to Fc2 via anti-human Fc IgG at a flow rate of 10 μL / min for 150 seconds. Multiple cycle dynamics were used for analysis. Analytes and running buffer at various concentrations (0.625, 1.25, 2.5, 5, and 10 nm) were sequentially injected into Fc1-Fc2 at a flow rate of 100 μL / min during a 120-second binding phase, followed by a 300-second dissociation phase. After each dissociation phase, 10 mM glycine pH 1.5 was injected as a regeneration buffer. The tips were regenerated with 10 mM glycine pH 1.5.

[0250] Surface channel Fc1 without captured ligands was used as a control surface for reference defects. Final data for each interaction were subtracted from reference Fc1 and buffer channel data. Experimental data were analyzed using Biacore 8K (version 3.0.12.15655) evaluation software to match a 1:1 binding model.

[0251] 1-6. Thermal stability using DSF (Differential scanning fluorimetry)

[0252] QuantStudio TM The Tm of the lead antibody was investigated using a 7 Flex Real-Time PCR system (Applied Biosystems). 19 μL of antibody solution was mixed with 1 μL of 62.5X SYPRO Orange solution (Invitrogen) and transferred to a 96-well plate (Biosystems). The plate was heated from 26°C to 95°C at a rate of 0.9°C / min, and the generated fluorescence data was collected. The negative derivatives of the fluorescence changes at different temperatures were calculated, and the maximum value was defined as the melting temperature Tm. Data collection and Tm calculation were performed using operating software (QuantStudio). TM The procedure was performed automatically using Real Time PCR software v1.3.

[0253] Example 2. Analysis Results

[0254] 2-1. Expression of parental and humanized antibodies Supernatant expression was detected by SDS-PAGE (Figure 1). Twenty antibodies (11B6) were expressed using a pcDNA3.4 expression vector and ExpiCHO cells. Supernatant expression was detected by SDS-PAGE (Figure 2).

[0255] All culture supernatants, including the parent antibody, humanized antibody, and PTM hazard-free variant, were further purified using protein A. Protein concentration was detected by nanodrop at 280 nm. Protein purity was evaluated by SDS-PAGE and SEC-HPLC. After purification, antibodies were characterized by SDS-PAGE (Figures 3a, 3b, 3c, 4a, and 4b) and SEC-HPLC (Figures 5a, 5b, 5c, 5d, 5e, 5f, and 6a, 6b, 6c, 6d, and 6e). The antibodies moved to an apparent molecular weight of 150 kDa under non-reducing conditions in SDS-PAGE. The antibodies moved to apparent molecular weights of 50 and 25 kDa under reducing conditions in SDS-PAGE, corresponding to the heavy and light chains, respectively.

[0256] 2-2. SPR of parental antibody and humanized antibody that bind to MYCT1 off Ranking Analysis k for ranking humanized and PTM risk-eliminating variants off To determine the ratio, SPR k off Rank analysis was performed, and the data was processed using a 1:1 binding model.

[0257] In the 2B4 clone, PTM risk removal mutations were fused to humanized mutants z2 and z8 (2B4-z2-p7-uIgG1K and 2B4-z8-p7-uIgG1K) to generate final lead candidate molecules. For further evaluation, the parental antibody and the two leads were expressed in 15 mL ExpiCHO cell lines.

[0258] In the 11B6 clone, the PTM risk removal variant at the "NG" site exhibited superior binding affinity to MYCT1. Z12 showed the best binding affinity to MYCT1 among all humanized mutants. To generate final lead candidate molecules, humanized mutants z10, z12, and the novel mutant z16 were conjugated with the PTM risk removal mutation (p3) (11B6-z10-p3-uIgG1K, 11B6-z12-p3-uIgG1K, and 11B6-z16-p3-uIgG1K). For further evaluation, the parental antibody and the three leads were expressed in 15 mL ExpiCHO cells.

[0259] The culture supernatant was further purified using Protein A. Protein concentration was detected at 280 nm using a Nano Drop. Protein purity was evaluated by SDS-PAGE and SEC-HPLC. After purification, the antibodies were characterized by SDS-PAGE and SEC-HPLC (Figures 7-10). The antibodies moved to an apparent molecular weight of 150 kDa under non-reducing SDS-PAGE conditions. The antibodies moved to apparent molecular weights of 50 and 25 kDa under reducing SDS-PAGE conditions, corresponding to the heavy and light chains, respectively.

[0260] 2-3. Production of lead substances Based on overall SPR dynamics, DSF results, and in vitro cell analysis results, we determined the scale-up production of the parent antibody and two leads, 11B6-z12-p3-uIgG1K and 11B6-z16-p3-uIgG1K. For further evaluation, the parent antibody and the two leads were expressed in ExpiCHO cells.

[0261] The culture supernatant was further purified using Protein A. Protein concentration was detected at 280 nm using a Nano Drop. Protein purity was evaluated by SDS-PAGE (Figure 11) and SEC-HPLC (Figure 12). After purification, the antibodies were characterized by SDS-PAGE and SEC-HPLC. The antibodies were identified with a molecular weight of 150 kDa by SDS-PAGE under non-reducing conditions, and the antibodies were identified with apparent molecular weights of 50 and 25 kDa by SDS-PAGE under reducing conditions, corresponding to the heavy and light chains, respectively.

[0262] 2-4.SPR overall dynamics analysis

[0263] 2-4-1.2B4 clone To determine binding affinity for ranking humanized and PTM risk-eliminating mutants, a total SPR dynamics analysis was performed. The data were fitted using steady-state affinity models (Figures 13a, 13b, 13c, 14a, 14b, 14c, 14d, 14e, 14f, Tables 7 and 8).

[0264] [Table 7]

[0265] [Table 8]

[0266] SPR k off Rank analysis was performed. In the case of humanized mutants, the parental antibody (K D Compared to the parent antibody (=0.181 mM), z2 showed a similar binding affinity, and z8 showed the highest binding affinity to MYCT1 among all humanized variants of the 2b4 clone. To produce the final lead molecule, humanized variants z2 and z8 were bound to the PTM hazard-eliminating variant (p7:p1+p6).

[0267] Overall SPR dynamics analysis was performed to determine the lead molecules (z2-p7 and z8-p7) for the combination of parental antibody binding affinity and humanization and PTM risk elimination against hMYCT1. The data were fitted using a steady-state affinity model (Figure 15 and Table 9).

[0268] Parent antibody (K D Compared to the bond affinity of the two reads (K in the range of 0.484~0.586 mM), the K = 0.281 mM D ) showed similar binding affinity to the antigen.

[0269] [Table 9]

[0270] 2-4-2.11B6 clone SPR k off Rank analysis was performed. The data was fitted using 1:1 binding (Figure 16 and Table 10).

[0271] [Table 10]

[0272] In the case of humanized mutants, the parental antibody (k d Compared to (4.56E-03 1 / s), z10 showed similar affinity to the parent antibody in the 11B6 clone. z12 showed the highest binding affinity to MYCT1 among all humanized variants.

[0273] To produce the final lead molecule, humanized mutants z10, z12, and the novel z16 mutant were conjugated with the PTM hazard-eliminating mutant (p3).

[0274] To determine the binding affinity of the parental antibody and lead antibodies (z10-p3, z12-p3, and z16-p3) for humanized and PTM risk elimination combinations to MYCT1, overall SPR dynamics analysis was performed (Figure 17 and Table 11). Data were confirmed using a 1:1 binding model. Parental antibody (K D Compared to the three reads (K in the range of 2.35~3.34 nM) (=1.20 nM), D The binding affinity of ) showed a similar binding affinity to that of the antigen.

[0275] JPEG2026521923000017.jpg49150

[0276] 2-5. Thermal stability using DSF

[0277] 2-5-1.2B4 clone The Tm of the parent antibody and two lead molecules was investigated using the DSF test (Figure 18 and Table 12). z8-p7 showed a Tm1 similar to that of the parent antibody. z2-p7 showed a Tm1 that was 2 degrees lower compared to that of the parent antibody.

[0278] [Table 12]

[0279] 2-5-2.11B6 clone The Tm of the parent antibody and three lead molecules was investigated using the DSF test (Figures 19a, 19b, and Table 13). The lead molecules showed a higher Tm1 than the parent antibody, but the humanized antibody showed even better thermal stability.

[0280] [Table 13]

[0281] Example 3. Evaluation of antibody toxicity NCI-H460 cell (lung cancer), HppG2 (liver cancer), MC38 (colon cancer), and ACHN (renal cell) were purchased from ATCC. Each cell line was placed in a 96-well plate in a 1x10⁶ layer. 4 Cells were added at a concentration of cells / well and cultured for 12 hours. After that, they were treated with antibodies of various concentrations and cultured for 24 hours. Subsequently, the cells were treated with MTT (3-(4,5-dimethylthiaol-2-yl)-2,5-diphenyltetrazolium bromide) and cultured at 37°C for 2-4 hours. To measure cell viability, absorbance at 570 nm was measured using a microplate reader (BioTek, Winooski, VT, USA).

[0282] As a result, it was confirmed that ATN001 (11B4-z16-p3-uIgG1K) was non-toxic from the highest concentration of 1 mM down to the lowest concentration of 1 μM through serial dilution (Figure 20).

[0283] Example 4. Tube formation assay HUVEC cells were starved for 6 hours in M199 medium containing 1% FBS, then 250 μL of ice-cooled Matrigel (BD Bioscience) was added to each 24-well plate and solidified at 37°C for 30 minutes. After starvation, 1 × 10⁶ cells were collected. 5 Cell concentration was adjusted using cells / well, and 1% FBS M199 medium mixed with antibodies (anti-human VEGF Ab(Avastin), m2B4, m11B6, 11B6-z12-p3, 11B6-z16-p3) was added. After 12 hours of incubation, the degree of tube formation was observed using a light microscope (×100).

[0284] As a result, when treated with 20 μM of the antibody of the present invention, the efficacy was found to be equivalent to or greater than that of the comparison drug compared to the control group (Figures 21a and 21b).

[0285] Example 5. Antitumor effect The experiment was conducted under the conditions shown in Figure 22 and Table 14 below. Specifically, the 11B6-z16-p3(hzATN001) antibody was administered to colorectal cancer-induced mice on days 4, 7, 11, and 18, and the tumors were removed on day 19. The antitumor effect of 11B6-z16-p3(hzATN001) was confirmed, and the tumor growth inhibition rate is shown in Table 15.

[0286] [Table 14]

[0287] [Table 15]

[0288] As can be seen from Table 15 above, the anti-MYCT1 antibody of the present invention exhibits excellent antitumor effects.

[0289] Example 6. Tumor growth inhibitory effect in a syngeneic mouse model (CT26) To study the in vivo efficacy of antibodies, we attempted to confirm their antitumor effects using the colorectal cancer cell line CT26. The CT26 cell line (ATCC, CRL-2638) was cultured at 37°C and 5% CO2 using modified RPMI1640 medium containing 10% FBS (fetal bovine serum) and 1x antibiotic-antifungal agents. Cell transplantation involved 5 × 10⁶ CT26 cells. 6 Dilute in DPBS to a concentration of cells / mL, and dispense 100 μL per individual (5 × 10). 5 Each cell was subcutaneously transplanted laterally. After transplantation of the CT26 cell line, the tumor volume was calculated using the following formula. Under the conditions in Table 16, antibodies 11B6(chATN001) and 11B6-Z16-p3(hzATN001) and the anti-PD-1 antibody ((anti-Mouse CD279(PD-1)(Clone RMP1-14)-Purified in vivo PLATINUM) were used. TM The functional grade was administered to mice.

[0290] Tumor volume (mm 3 ) = [length (mm) × width (mm)] 2 ] × 0.5

[0291] Furthermore, the mice were separated into groups based on their body weight and tumor size, and the tumor growth rate was measured using the following formula, based on the tumor size at the time of group separation and the tumor size on the measurement day.

[0292] Tumor growth rate (%) = (Tumor size on measurement day - Tumor size at group separation) / Tumor size at group separation × 100

[0293] [Table 16]

[0294] The experimental results showed that when 11B6-Z16-p3 (hzATN001) was administered in combination with an anti-PD-1 antibody, tumor volume decreased by 77.1%, and tumor weight also decreased significantly (Figures 23a and 23b).

[0295] As described above, the embodiments have been explained based on limited drawings, but a person with ordinary skill in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are carried out in a different order than described, or if the components of the described systems, structures, devices, circuits, etc. are combined or assembled in a different manner than described, or replaced or substituted by other components or equivalents, suitable results can still be achieved.

[0296] Therefore, other embodiments, other examples, and those equivalent to the claims shall also be included in the claims.

[0297] This invention was completed with funding from the Korean government (Ministry of SMEs and Startups) and support from the Korea Small and Medium Enterprise Technology Information Promotion Agency and the Startup Growth Technology Development Program (TIPS) (Project Number: 00238993). [Industrial applicability]

[0298] This invention relates to an antibody or antigen-binding fragment that specifically binds to MYCT1 and exhibits excellent affinity and binding affinity to MYCT1. Therefore, the antibody or antigen-binding fragment that specifically binds to MYCT1 according to this invention can be developed as an antibody-drug conjugate, a bispecific antibody, or a chimeric antigen receptor (CAR). Furthermore, the antibody or antigen-binding fragment according to the present invention can be usefully used in the treatment or diagnosis of MYCT1-related diseases, such as cancer and angiogenesis-related diseases. In addition, the antibody or antigen-binding fragment according to the present invention can be used in combination with immunosuppressant agents and can enhance sensitivity to immunosuppressant agents.

Claims

1. It comprises at least one variable heavy chain region (VH) selected from the group consisting of SEQ ID NOs: 41, 43-60; and at least one variable light chain region (VL) selected from the group consisting of SEQ ID NOs: 42, 61-66; or An isolated anti-MYCT1 humanized antibody or its antigen-binding fragment comprising at least one variable heavy chain region (VH) selected from the group consisting of SEQ ID NOs. 92 and 94-101; and at least one variable light chain region (VL) selected from the group consisting of SEQ ID NOs. 93 and 102-115.

2. The anti-MYCT1 humanized antibody or its antigen-binding fragment according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having amino acid sequences represented by SEQ ID NOs: 3, 4, and 5, respectively, and the VL comprises CDR1, CDR2, and CDR3 having amino acid sequences represented by SEQ ID NOs: 6, 7, and 8, respectively.

3. The anti-MYCT1 humanized antibody or its antigen-binding fragment according to claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 having amino acid sequences represented by SEQ ID NOs: 9, 10, and 11, respectively, and the VL comprises CDR1, CDR2, and CDR3 having amino acid sequences represented by SEQ ID NOs: 12, 13, and 14, respectively.

4. Does it contain the VH sequence of sequence number 43 and the VL sequence of sequence number 42? Does it contain the VH sequence of sequence number 44 and the VL sequence of sequence number 42? Does it contain the VH sequence of sequence number 45 and the VL sequence of sequence number 42? Does it contain the VH sequence of sequence number 41 and the VL sequence of sequence number 61? Does it contain the VH sequence of sequence number 46 and the VL sequence of sequence number 62? Does it contain the VH sequence of sequence number 41 and the VL sequence of sequence number 63? Does it contain the VH sequence of sequence number 44 and the VL sequence of sequence number 63? Does it contain the VH sequence of sequence number 47 and the VL sequence of sequence number 64? Does it contain the VH sequence of sequence number 48 and the VL sequence of sequence number 64? Does it contain the VH sequence of sequence number 48 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 49 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 50 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 51 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 52 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 53 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 54 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 55 and the VL sequence of sequence number 64? Does it contain the VH sequence of sequence number 55 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 56 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 57 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 58 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 41 and the VL sequence of sequence number 65? Does it contain the VH sequence of sequence number 58 and the VL sequence of sequence number 42? It includes the VH sequence of sequence number 59 and the VL sequence of sequence number 66; or The anti-MYCT1 humanized antibody or its antigen-binding fragment according to claim 1, comprising the VH sequence of SEQ ID NO: 60 and the VL sequence of SEQ ID NO:

66.

5. Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 102? Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 103? Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 104? Does it contain the VH sequence of sequence number 94 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 95 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 96 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 97 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 98 and the VL sequence of sequence number 105? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 106? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 107? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 108? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 109? Does it contain the VH sequence of sequence number 99 and the VL sequence of sequence number 110? Does it contain the VH sequence of sequence number 95 and the VL sequence of sequence number 110? Does it contain the VH sequence of sequence number 100 and the VL sequence of sequence number 110? Does it include the VH sequence of sequence number 100 and the VL sequence of sequence number 111? Does it contain the VH sequence of sequence number 101 and the VL sequence of sequence number 111? Does it contain the VH sequence of sequence number 92 and the VL sequence of sequence number 111? Does it contain the VH sequence of sequence number 101 and the VL sequence of sequence number 112? Does it contain the VH sequence of sequence number 95 and the VL sequence of sequence number 113? It includes the VH sequence of sequence number 100 and the VL sequence of sequence number 114; or The anti-MYCT1 humanized antibody or its antigen-binding fragment according to claim 1, comprising the VH sequence of SEQ ID NO: 95 and the VL sequence of SEQ ID NO:

115.

6. The anti-MYCT1 humanized antibody or its antigen-binding fragment according to claim 1, wherein the antigen-binding fragment comprises Fd, Fab, Fab', F(ab')2, dsFv, scFv, and a single-domain antibody (sdAb).

7. A nucleic acid molecule encoding an anti-MYCT1 humanized antibody or its antigen-binding fragment according to any one of claims 1 to 6.

8. A recombinant expression vector comprising the nucleic acid molecule described in claim 7.

9. A host cell transduced by the expression vector described in claim 8.

10. An immune complex comprising an anti-MYCT1 humanized antibody or antigen-binding fragment and a cytotoxic agent according to any one of claims 1 to 6.

11. A pharmaceutical composition for the prevention or treatment of MYCT1-related disease, comprising an anti-MYCT1 humanized antibody or antigen-binding fragment as described in any one of claims 1 to 6, as an active ingredient.

12. The pharmaceutical composition according to claim 11, wherein the MYCT1-related disease is angiogenesis-related disease or cancer.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition is administered in combination with at least one further therapeutic agent or therapeutic procedure.

14. The pharmaceutical composition according to claim 13, wherein the at least one further therapeutic agent or therapeutic procedure is selected from one or more of chemotherapy, targeted anticancer therapy, oncolytic agents, cytotoxic agents, immunotherapy, cytokines, surgical procedures, radiotherapy, vaccines, or cell therapies.

15. A diagnostic composition for MYCT1-related disease comprising an anti-MYCT1 humanized antibody or antigen-binding fragment as described in any one of claims 1 to 6 as an active ingredient.

16. A pharmaceutical composition for enhancing sensitivity to immunosuppressant agents in cancerous tumors, comprising an anti-MYCT1 humanized antibody or antigen-binding fragment as an active ingredient according to any one of claims 1 to 6.

17. A chimeric antigen receptor (CAR) containing an antigen-binding domain that specifically binds to MYCT1, The antigen-binding domain is A chimeric antigen receptor comprising an antigen-binding fragment having at least one variable heavy chain (VH) region selected from the group consisting of SEQ ID NOs: 41, 43-60, 92 and 94-101; and at least one variable light chain (VL) region selected from the group consisting of SEQ ID NOs: 42, 61-66, 93 and 102-115.

18. An immune effector cell expressing the chimeric antigen receptor according to claim 17.

19. A method for treating MYCT1-related disease, comprising the step of administering to a subject a composition comprising a therapeutically effective amount of an anti-MYCT1 humanized antibody or antigen-binding fragment according to any one of claims 1 to 6.

20. A method for enhancing the sensitivity of an individual to an immunosuppressant, comprising the step of administering a therapeutically effective amount of a composition comprising an anti-MYCT1 humanized antibody or antigen-binding fragment according to any one of claims 1 to 6 to a target individual resistant to the immunosuppressant.

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