Anti-CNTN4 antibodies and uses thereof

Abstract

The present invention relates to anti-CNTN4 antibodies or antigen-binding fragments thereof, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to anti-CNTN4 antibodies or antigen-binding fragments thereof, uses thereof, and pharmaceutical compositions containing them. [Background technology]

[0002] The human body has a defense system that protects it from external invaders (viruses, toxins, etc.) and internal harmful changes (cancer cell mutations). Unlike normal cells, cancer cells have specific antigens on their surface, which allows them to be destroyed by the immune system in the early stages of cancer development. When the balance of power between the endlessly proliferating cancer cells and the immune cells that attack them is disrupted, the cancer cells begin to proliferate in earnest. As cancer cells continue to proliferate, they destroy the body's immune system. At this time, some cancer cells evade the immune system by exploiting immune checkpoints in immune cells. Immune checkpoint inhibitors block these checkpoints, enhancing the power of immune cells and leading to cancer cell death.

[0003] Immune checkpoints are a group of signaling molecules possessed by immune cells, and immune checkpoint proteins are proteins involved in pathways that inhibit immune responses and proteins involved in pathways that activate immune responses.

[0004] Immune checkpoint inhibitors are drugs that activate T cells to attack cancer cells by blocking the activation of immune checkpoint proteins involved in T cell suppression, and include CTLA-4, PD-1, and PD-L1 inhibitors. Representative currently available drugs include ipilimumab (trade name: YERVOY®) as a CTLA-4 monoclonal antibody, nivolumab (trade name: OPDIVO®) and pembrolizumab (trade name: KEYTRUDA®) as PD-1 monoclonal antibodies, and atezolizumab (trade name: TECENTRIQ®) and durvalumab (trade name: IMFINZI®) as PD-L1 monoclonal antibodies.

[0005] However, there are still cancers that cannot be treated with conventional immune checkpoint inhibitors, and therefore there is a need to develop new anti-cancer therapeutic agents. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an anti-CNTN4 antibody or an antigen-binding fragment thereof that specifically binds to the CNTN4 protein. In particular, an object of the present invention is to provide an antibody or an antigen-binding fragment thereof that binds to the CNTN4 protein and neutralizes the immune evasion mechanism of CNTN4.

[0007] Another object of the present invention is to provide a use for preventing or treating a disease caused by reduced T cell activity, such as cancer, by activating T cells through blocking the immune evasion mechanism of CNTN4 using an antibody or an antigen-binding fragment thereof, or a composition for preventing or treating cancer. [Means for solving the problem]

[0008] The present invention provides anti-CNTN4 antibodies or antigen-binding fragments thereof that specifically bind to CNTN4 protein. The antibodies or antigen-binding fragments specifically bind to CNTN4 protein, e.g., human or mouse CNTN4 protein, and neutralize the immune evasion mechanism of CNTN4. Thus, the antibodies or antigen-binding fragments of the present invention can enhance the activity of T cells, e.g., CD4+ T cells or CD8+ T cells, suppressed by CNTN4.

[0009] In one embodiment, the present invention provides a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 4; a heavy chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8 to 18, and 21 to 35; a light chain CDR1 having the amino acid sequence of SEQ ID NO: 2 or 3; a light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and a light chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 19, and 20, or an antigen-binding fragment thereof.

[0010] In one embodiment, the present invention provides a heavy chain variable region having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 38 to 48, 52, and 54 to 67; and a light chain variable region having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 49 to 51, and 53, or an antigen-binding fragment thereof.

[0011] In one embodiment, the antigen-binding fragment of the present invention may be a Fab, Fab', Fab'-SH, Fv, single-chain variable fragment (scFv), F(ab')2 fragment, VL, VH, diabody, triabody, tetrabody, minibody ((scFV-CH3)2), IgGΔCH2, scFv-Fc, (scFv)2-Fc, fynomer, dual-affinity re-targeting (DART), or TRIDENT antibody comprising the CDR sequences of the present invention. The antibody of the present invention may be a chimeric, humanized, or human antibody, and a multispecific antibody.

[0012] In another embodiment, the invention provides nucleic acid molecules encoding antibodies or antigen-binding fragments thereof, and recombinant expression vectors comprising same.

[0013] The present invention also provides a composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof as an active ingredient. The pharmaceutical composition may be used in combination with an additional anti-cancer agent, such as an immune checkpoint inhibitor or a chemotherapeutic agent, or in combination with radiation therapy.

[0014] The present invention also provides a composition for analyzing or detecting CNTN4 protein, comprising an antibody or an antigen-binding fragment thereof. [Effects of the Invention]

[0015] The novel anti-CNTN4 antibodies or antigen-binding fragments thereof of the present invention bind to the CNTN4 protein with high affinity. Therefore, the anti-CNTN4 antibodies or antigen-binding fragments thereof of the present invention may be useful in preventing or treating diseases caused by reduced T cell activity, particularly cancer, by activating T cells through blocking the immune evasion mechanism of CNTN4. [Brief explanation of the drawings]

[0016] [Figure 1] The amino acid sequences of the CDRs of the scFv antibody fragment prepared according to Example 1 (Figure 1) and the amino acid sequences of its heavy chain variable region and light chain variable region (Figure 2: underlined or bolded sequences indicate the CDR sequences of each region) are shown. [Figure 2A] The amino acid sequences of the CDRs of the scFv antibody fragment prepared according to Example 1 (Figure 1) and the amino acid sequences of its heavy chain variable region and light chain variable region (Figure 2: underlined or bolded sequences indicate the CDR sequences of each region) are shown. [Figure 2B] The amino acid sequences of the CDRs of the scFv antibody fragment prepared according to Example 1 (Figure 1) and the amino acid sequences of its heavy chain variable region and light chain variable region (Figure 2: underlined or bolded sequences indicate the CDR sequences of each region) are shown. [Figure 3] The data (MFI) obtained by determining the binding degree of nine IgG antibodies (A102, A103, A104, A105, A106, A108, A109, A112, and A114) to HEK293 cells overexpressing human CNTN4 by concentration through FACS experiments are shown. [Figure 4A] The amino acid sequences of the CDRs of the scFv antibody fragment prepared according to Example 4 (Figure 4) and the amino acid sequences of its heavy chain variable region and light chain variable region (Figure 5: underlined or bolded sequences indicate the CDR sequences of each region) are shown. [Figure 4B] The amino acid sequences of the CDRs of the scFv antibody fragment prepared according to Example 4 (Figure 4) and the amino acid sequences of its heavy chain variable region and light chain variable region (Figure 5: underlined or bolded sequences indicate the CDR sequences of each region) are shown. [Figure 5A] The amino acid sequences of the CDRs of the scFv antibody fragment prepared according to Example 4 (Figure 4) and the amino acid sequences of its heavy chain variable region and light chain variable region (Figure 5: underlined or bolded sequences indicate the CDR sequences of each region) are shown. [Figure 5B] The amino acid sequences of the CDRs of the scFv antibody fragment prepared according to Example 4 (Figure 4) and the amino acid sequences of its heavy chain variable region and light chain variable region (Figure 5: underlined or bolded sequences indicate the CDR sequences of each region) are shown. [Figure 6] The data (MFI) obtained by determining the binding degree of A114-IgG and A211-IgG antibodies to HEK293 cells overexpressing human CNTN4 by concentration through FACS experiments are shown. [Figure 7A] Absolute tumor size (FIG. 7A) after administration of A114-IgG antibody and A211-IgG antibody is shown. [Figure 7B] The relative tumor size after administration of A114-IgG and A211-IgG antibodies compared to the control is shown (FIG. 7B). DETAILED DESCRIPTION OF THE INVENTION

[0017] Best form Anti-CNTN4 antibody or antigen-binding fragment thereof The present invention provides anti-CNTN4 antibodies or antigen-binding fragments thereof that specifically bind to CNTN4 protein.

[0018] In one embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the invention a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 4; a heavy chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8 to 18, and 21 to 35; a light chain CDR1 having the amino acid sequence of SEQ ID NO: 2 or 3; a light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and a light chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 19, and 20.

[0019] In yet another embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention may comprise one or more CDRs of CDR1 to CDR3 of the heavy chain or light chain described above.

[0020] In exemplary embodiments, an anti-CNTN4 antibody or antigen-binding fragment thereof of the invention may comprise one or more CDRs of the set of six CDRs of the heavy and light chains shown in Tables 1 and 4, or all six CDRs.

[0021] In one embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the invention 101) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 6, 2, 5, and 7, respectively; 102) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 8, 2, 5, and 7, respectively; 103) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 9, 2, 5, and 7, respectively; 104) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 10, 2, 5, and 7, respectively; 105) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 11, 2, 5, and 7, respectively; 106) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 12, 2, 5, and 7, respectively; 107) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 13, 2, 5, and 7, respectively; 108) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 14, 2, 5, and 7, respectively; 109) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 15, 2, 5, and 7, respectively; 110) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 16, 2, 5, and 7, respectively; 111) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 17, 2, 5, and 7, respectively; 112) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 18, 3, 5, and 7, respectively; 113) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 18, 2, 5, and 19, respectively; 114) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 18, 2, 5, and 20, respectively; 201) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 21, 3, 5, and 20, respectively; 202) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 22, 3, 5, and 20, respectively; 203) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 23, 3, 5, and 20, respectively; 204) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 24, 3, 5, and 20, respectively; 205) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 21, 2, 5, and 20, respectively; 206) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 25, 3, 5, and 20, respectively; 207) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 26, 2, 5, and 20, respectively; 208) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 27, 2, 5, and 20, respectively; 209) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 26, 3, 5, and 20, respectively; 210) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 28, 3, 5, and 20, respectively; 211) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 27, 3, 5, and 20, respectively; 212) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 9, 3, 5, and 20, respectively; 213) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 29, 2, 5, and 20, respectively; 214) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 30, 2, 5, and 20, respectively; 215) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 31, 2, 5, and 20, respectively; 216) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 32, 3, 5, and 20, respectively; 217) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 33, 2, 5, and 20, respectively; 218) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 4, 34, 3, 5, and 20, respectively; or 219) Contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 35, 2, 5, and 20, respectively.

[0022] In the present invention, each chain or variable region "comprising" a particular amino acid sequence means comprising, having, or consisting of the entire amino acid sequence.

[0023] In one example, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 6, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 2, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 7.

[0024] In another example, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 consisting essentially of the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 consisting essentially of the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR3 consisting essentially of the amino acid sequence of SEQ ID NO: 6, a light chain CDR1 consisting essentially of the amino acid sequence of SEQ ID NO: 2, a light chain CDR2 consisting essentially of the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 consisting essentially of the amino acid sequence of SEQ ID NO: 7.

[0025] In yet another example, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6, a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 7.

[0026] The terms "including," "include," and variations thereof have an open-ended meaning. In one example, an antibody or antigen-binding fragment thereof comprising an amino acid sequence listed above may include additional amino acid sequences not listed above, whether essential or not.

[0027] As used herein, the term "consisting essentially of" or variations thereof includes any of the recited elements and allows for the presence of elements that do not substantially affect the basic, novel, or functional characteristics of the embodiment. In one example, an antibody or antigen-binding fragment thereof consisting essentially of a recited amino acid sequence may contain substitutions of one or more amino acid residues that do not substantially affect the characteristics of the antibody or fragment thereof.

[0028] As used herein, the term "consisting of" or variations thereof means that each component does not exclude any element not mentioned or listed in the description of the embodiment.

[0029] Each chain or variable region of another antibody or antigen-binding fragment thereof defined herein may similarly comprise, or may consist essentially of, or may consist of, the amino acid sequences exemplified above.

[0030] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are essential for antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3. The CDRs contain most of the residues that mediate specific interactions between the antibody (or its antigen-binding fragment) and the antigen, and thus contribute to the functional activity of the antibody molecule. These are the primary determinants of antigen specificity.

[0031] In yet another embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 38 to 48, 52, and 54 to 67; and a light chain variable region having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 49 to 51, and 53.

[0032] In yet another embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention may comprise one or more variable regions of the heavy chain variable regions and light chain variable regions mentioned above.

[0033] In exemplary embodiments, the anti-CNTN4 antibodies or antigen-binding fragments thereof of the invention may comprise one or more variable regions of one heavy chain variable region and one light chain variable region pair set forth in Tables 2 and 5. In specific examples, the antibodies or antigen-binding fragments thereof of the invention may comprise: A101) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 36 and 37, respectively; A102) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 38 and 37, respectively; A103) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 39 and 37, respectively; A104) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 40 and 37, respectively; A105) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 41 and 37, respectively; A106) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 42 and 37, respectively; A107) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 43 and 37, respectively; A108) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 44 and 37, respectively; A109) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 45 and 37, respectively; A110) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 46 and 37, respectively; A111) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 47 and 37, respectively; A112) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 48 and 49, respectively; A113) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 48 and 50, respectively; A114) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 48 and 51, respectively; A201) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 52 and 53, respectively; A202) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 54 and 53, respectively; A203) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 55 and 53, respectively; A204) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 56 and 53, respectively; A205) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 52 and 51, respectively; A206) ​​Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 57 and 53, respectively; A207) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 58 and 51, respectively; A208) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 59 and 51, respectively; A209) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 58 and 53, respectively; A210) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 60 and 53, respectively; A211) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 59 and 53, respectively; A212) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 39 and 53, respectively; A213) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 61 and 51, respectively; A214) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 62 and 51, respectively; A215) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 63 and 51, respectively; A216) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 64 and 53, respectively; A217) Heavy chain and light chain variable regions having the amino acid sequences of SEQ ID NOs: 65 and 51, respectively; A218) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 66 and 53, respectively; or A219) Heavy chain variable region and light chain variable region having the amino acid sequences of SEQ ID NOs: 67 and 51, respectively may include:

[0034] In the same manner as defined above, in the present invention, a heavy or light chain variable region "comprising" a particular amino acid sequence means that it includes, has, or consists of the entire amino acid sequence.

[0035] Thus, in one example, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:36 and a light chain variable region having the amino acid sequence of SEQ ID NO:37.

[0036] In another example, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting essentially of the amino acid sequence of SEQ ID NO:36 and a light chain variable region consisting essentially of the amino acid sequence of SEQ ID NO:37.

[0037] In yet another example, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:36 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:37.

[0038] The variable region of an antibody refers to the portion of the heavy or light chain of the antibody molecule having the amino acid sequences of the CDRs and framework regions (FRs).

[0039] The term "antibody," as used herein, refers to an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, or protein, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" is used in the broadest sense and is therefore intended to broadly include not only intact polyclonal or monoclonal antibodies, but also dimers, polymers, multispecific antibodies (e.g., bispecific antibodies), antigen-binding fragments thereof, antibody fragments, fusion proteins comprising immunoglobulin molecules in any other modified configuration, including antigen recognition sites (e.g., variable regions), synthetic antibodies (e.g., "antibody mimetics"), and "FynomAb."

[0040] Antibodies are classified into five types: immunoglobulin (Ig) M, IgD, IgG, IgA, and IgE, each with a heavy chain made from the heavy chain constant region genes μ, δ, γ, α, and ε. The light and heavy chains of an antibody are divided into variable regions, whose amino acid sequences differ from antibody to antibody, and constant regions, whose amino acid sequences are the same for each antibody. The heavy chain constant region contains CH1, hinge (H), CH2, and CH3 domains. Each domain consists of two β-sheets connected by an intramolecular disulfide bond.

[0041] The antibody of the present invention may be a chimeric antibody, a humanized antibody, or a human antibody.

[0042] The term "chimeric antibody" as used herein refers to an antibody having a variable region derived from one type and a constant region derived from another type, e.g., an antibody having a variable region sequence derived from a mouse antibody and a constant region sequence derived from a human antibody. Methods for preparing chimeric antibodies are known in the art. See, for example, Morrison, Science 229:1202 (1985), the entire contents of which are incorporated herein by reference.

[0043] As used herein, the term "humanized antibody" refers to an antibody in which one or more CDR sequences derived from the germline of another mammal, such as a non-human species, e.g., a mouse or chicken, have been inserted into the framework sequences of a human immunoglobulin molecule. Additionally, the framework sequences can be remodified, for example, by mutational methods. Human Ig sequences can be found, for example, in the NCBI database (Entez Gene). By using appropriate sequences, the immunogenicity of the antibody can be reduced, or the binding, affinity, on-rate, off-rate, specificity, half-life, or any other suitable characteristic can be reduced, improved, or altered.

[0044] The term "human antibody," as used herein, refers to an antibody comprising variable regions in which both the framework and CDR regions are derived from human immunoglobulin sequences. The antibody constant regions are also derived from human immunoglobulin sequences.

[0045] The term "antigen-binding fragment" or "antibody fragment" typically includes at least a portion of the antigen-binding domain or variable region of a parent antibody (e.g., one or more CDRs), but also broadly includes any modified form containing a recognition site specific for a target antigen, for example, glycosylation variants of the antibody and amino acid sequence variants of the antibody, covalently modified antibodies, and other modified configurations of immunoglobulin molecules having a specific antigen recognition site, such as antigen-binding antibodies thereof. Specific examples of antigen-binding fragments that can be used in the present invention include Fab, Fab', Fab'-SH, Fv, single-chain antibody (scFv), F(ab')2 fragment, VL, VH, diabody, triabody, tetrabody, minibody ((scFv-CH3)2), IgGΔCH2, scFv-Fc, (scFv)2-Fc, FynomAb, FynomAb fused to antibody, dual affinity retargeting (DART), AlbudAb, bispecific T cell engager (BiTE), tandem diabody (TandAb), and bispecific antibody. Examples of antibodies include, but are not limited to, dual-variable domain Fabs (DAFs), two-in-one antibodies, small modular immunopharmaceuticals (SMIPs), anticalins, FN3 monobodies, DARPins, affibodies, affilins, affimers, affitins, alphabodies, avimers, Im7, VLRs, VNARs, trimabs, CrossMabs, TRIDENTs, nanobodies, bi-nanobodies, di-sdFvs, dual variable domain immunoglobulins (DVD-Igs), peptide-modified antibodies (CovX-bodies), duobodies, and triomAbs.

[0046] Specifically, a Fab fragment refers to a monovalent fragment consisting of the VL, VH, CL, and CH1 domains.

[0047] Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region.

[0048] Fab'-SH refers to Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group.

[0049] F(ab')2 antibody fragments are produced as pairs of Fab' fragments separated by hinge cysteines.

[0050] Fv is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment is a dimer of one heavy-chain variable region and one light-chain variable region tightly bound non-covalently. These two regions fold together to form six hypervariable loops (three loops each from the heavy and light chains), which provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable region has the ability to recognize and bind to an antigen, although with lower affinity than the entire binding site.

[0051] A single-chain antibody scFv is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected by a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains such that the scFv can form the desired structure for antigen binding. In the present invention, scFv polypeptides may also be referred to as scFv antibody fragments, antigen-binding fragments (scFv), scFv antibodies, antibody scFvs, or simply scFvs.

[0052] Diabodies refer to small antibody fragments prepared by linking scFv fragments between the VH and VL domains using a short linker (approximately 5-10 residues) such that pairing occurs within the V domains, rather than between the chains, to produce bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are heterodimers consisting of two "cross-paired" scFv fragments present on polypeptide chains each bearing different VH and VL domains of two antibodies. Similarly, triabodies and tetrabodies contain three and four polypeptide chains, either the same or different, that form three and four antigen-binding sites, respectively.

[0053] Fynomer refers to a non-immunoglobulin-derived binding polypeptide derived from the human FynSH3 domain. FynSH3-derived polypeptides are well known in the art and are described, for example, in the literature [Grabulovski et al. (2007) JBC, 282, pp. 3196-3204] and WO 2008 / 022759. Fynomers can be fused to different molecules (e.g., antibodies) in a genetically engineered manner to produce "FynomAb," a form that can be engineered to have bispecificity.

[0054] Dual affinity targeting (DART) and TRIDENT refer to antibodies designed to simultaneously bind to two or more targets. DART refers to covalently linked bispecific diabodies, such as diabodies linked via C-terminal disulfide bridges, and their specific structures and definitions are described in the literature [J.Mol.Biol.(2010)399,436-449].

[0055] Antibodies or antigen-binding fragments thereof of the present invention may be named using the symbols listed in Tables 1 to 7. For example, in the present invention, antibodies or antigen-binding fragments thereof comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 6, 2, 5, and 7, respectively, are collectively referred to as "101." In the present invention, anti-CNTN4 antibodies or antigen-binding fragments thereof comprising heavy chain variable regions and light chain variable regions having the amino acid sequences of SEQ ID NOs: 36 and 37, respectively, are collectively referred to as "A101." According to a specific embodiment, "A101" may refer to an antigen-binding fragment, particularly an scFv, and an antibody produced using this may be referred to as an A101-IgG antibody.

[0056] The anti-CNTN4 antibody or antigen-binding fragment thereof can specifically bind to a CNTN4 protein, preferably a human or mouse CNTN4 protein.

[0057] As used herein, the terms "specifically bind to" or "specific for" refer to having selectivity for the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules, and refer to a measurable and reproducible interaction, such as the binding of a target to an antibody. For example, an antibody that specifically binds to a particular target (e.g., epitope) refers to an antibody that binds to the target with greater affinity, more readily, and / or with longer duration than it binds to other targets.

[0058] The term "specifically binds to CNTN4 protein" refers to a specific binding to CNTN4 protein with a concentration of 5×10 -8 M or less, preferably 1 x 10 -8 M or less, more preferably 5 × 10 -9 Therefore, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention binds to CNTN4 protein, for example, human CNTN4 protein, with an equilibrium dissociation constant (KD) of 5×10 -8 M or less, preferably 1 x 10 -8 M or less, more preferably 5 × 10 -9 It can bind with an equilibrium dissociation constant (KD) of M or less.

[0059] The term "KD" as used herein refers to the binding equilibrium dissociation constant (KD) of a specific antibody-antigen interaction, calculated using the formula KD = Kd / Ka (where Ka is the association rate constant and Kd is the dissociation rate constant), where the constant KD has units of M. The KD value for an antibody can be measured using methods widely established in the art. A preferred method for measuring the KD value of an antibody can be surface plasmon resonance (SPR), preferably a biosensor system such as a Biacore® system, or bio-layer interferometry (BLI), for example, an Octet® system. In one specific example, the KD described in the present invention can be a value obtained through SPR.

[0060] In one embodiment, the anti-CNTN4 antibodies or antigen-binding fragments thereof of the invention are monospecific and specifically bind to a single epitope, i.e., the CNTN4 protein.

[0061] In another embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention is a multispecific, e.g., bispecific or trispecific, antibody molecule. A multispecific antibody molecule comprises multiple variable regions, each of which has binding specificity for a different epitope. In one embodiment, a first variable region of a bispecific antibody molecule has a first binding specificity for a first epitope, e.g., the CNTN4 protein, and a second variable region has a second binding specificity for a second epitope, e.g., a target protein other than the CNTN4 protein.

[0062] In one specific example, the bispecific antibody molecule may bind to an immune checkpoint protein. Immune checkpoint protein is a general term for proteins involved in pathways that inhibit immune responses and proteins involved in pathways that activate immune responses. Immune checkpoint proteins may be proteins involved in signaling pathways that inhibit the activity of regulatory T cells, or proteins involved in signaling pathways that directly stimulate effector T cells or memory T cells. Such immune checkpoint proteins include, but are not limited to, PD-L1, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, OX40, VISTA, TIGIT, 4-1BB, GITR, and A2aR.

[0063] In one particular example, the bispecific antibody molecule may specifically bind to any one of PD-L1, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, OX40, VISTA, TIGIT, 4-1BB, GITR, and A2aR, and to CNTN4.

[0064] Alternatively, the bispecific antibody molecule functions as a bispecific T cell engager capable of specifically binding to any one of CD3, 4-1BB, and CD28, as well as to CNTN4.

[0065] Alternatively, the bispecific antibody molecule may bind to a cytokine that activates the immune system and CNTN4. The cytokine that activates the immune system may be, for example, IFN-γ, TNF-α, IL-2, IL-6, or IL-12, but the present invention is not limited thereto. In one specific example, the bispecific antibody molecule may specifically bind to any one of IFN-γ, TNF-α, IL-2, IL-6, and IL-12, and CNTN4.

[0066] In a specific example, a bispecific antibody molecule may specifically bind to CNTN4 and any one of PD-L1, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, OX40, VISTA, TIGIT, 4-1BB, GITR, A2aR, CD3, 4-1BB, CD28, IFN-γ, TNF-α, IL-2, IL-6, and IL-12. In another specific example, any combination of the above-mentioned molecules may be prepared as a multispecific antibody molecule, e.g., a trispecific antibody having a first binding specificity for CNTN4 and second and third binding specificities for two or more of CTLA-4, PD-1, and PD-L1. The multispecific antibody molecules of the invention may be produced using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques).

[0067] In another embodiment, the antibody or antigen-binding fragment of the present invention may be conjugated with a drug to form an antibody-drug conjugate (ADC). As used herein, an "antibody-drug conjugate" or "ADC" may be represented by the formula M-[L-(D)m]n, where M is an antibody molecule, i.e., an anti-CNTN4 antibody or antigen-binding fragment thereof, of the present invention, L is an optional linker or linker unit, D is a suitable drug or prodrug, and each of m and n is an integer of about 1 to about 20. The drug contained in the ADC may be appropriately selected depending on the therapeutic or diagnostic use, as long as it does not interfere with the specific binding of the antibody of the present invention. In one embodiment, the drug may include an adjuvant, a cytotoxic agent (e.g., a chemotherapeutic agent), a prodrug-converting enzyme, a radioactive isotope or compound, or a toxin, but the present invention is not limited thereto. Adjuvants are substances that nonspecifically promote immune responses to antigens in the early activation process of immune cells and are referred to as agents that enhance immunity by enhancing cellular activity in the immune system. Drugs and linkers that can be used in ADCs and methods for preparing them can be according to methods known in the art. Thus, the present invention provides antibody-drug conjugates comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0068] In another embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention can bind to a CNTN4 protein (e.g., a human or mouse CNTN4 protein) with an EC50 of 100 nM or less, preferably 50 nM or less, more preferably 10 nM or less, even more preferably 1 nM or less, particularly 0.1 nM or less or 0.05 nM or less.

[0069] The term "EC50" as used herein refers to an in vitro or in vivo assay using an antibody and refers to the concentration of antibody that induces 50% of the maximal response, i.e., a response halfway between the baseline and maximal response.

[0070] Nucleic Acid Molecules and Vectors Another aspect of the invention pertains to nucleic acid molecules encoding the anti-CNTN4 antibodies or antigen-binding fragments thereof of the invention.

[0071] Nucleic acids may be present in whole cells or cell lysates, or specifically in purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" nucleic acids that have been separated and purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, such as alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods known in the art.

[0072] Nucleic acids of the invention can be, for example, DNA or RNA, and may or may not contain intron sequences. In an exemplary embodiment, the nucleic acid is a cDNA molecule.

[0073] In one embodiment, a nucleic acid molecule of the present invention encodes the heavy chain region, light chain region, or both the heavy chain region and the light chain region of an anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention, preferably the heavy chain variable region, light chain variable region, or both the heavy chain variable region and the light chain variable region. In one embodiment, a nucleic acid molecule of the present invention encodes a heavy chain variable region having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 38 to 48, 52, and 54 to 67, e.g., the heavy chain variable region of SEQ ID NO: 36, and / or a light chain variable region having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 49 to 51, and 53, e.g., the light chain variable region of SEQ ID NO: 37. Alternatively, a nucleic acid molecule of the present invention encodes the heavy chain variable regions and / or light chain variable regions of A101 to A114 and A201 to A219.

[0074] Once DNA fragments encoding the VL and / or VH regions are obtained, such DNA fragments can be further manipulated, for example, by standard recombinant DNA techniques, resulting in the conversion of the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In this engineering process, the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding a different protein, for example, an antibody constant region such as a hIgG1 Fc (hFc) region, a hIgG4 Fc region (e.g., a hIgG4 with an S228P mutation (S228P)), or a hCK region, or a flexible linker. As used herein, the term "operably linked / linked" means that the two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0075] The isolated DNA encoding the VH domain can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (CH1, H, CH2, or CH3). The heavy chain constant region can be the variable region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD. In one exemplary embodiment of the invention, the heavy chain constant region can be IgG4, specifically hIgG4(S228P).

[0076] For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0077] To produce an scFv gene, the VL- and VH-encoding DNA fragments can be operably linked to a flexible linker, for example, another fragment encoding the amino acid sequence (Gly4-Ser)3. As a result, the VL and VH sequences can be expressed as a continuous single-chain protein with the VL and VH domains connected by the flexible linker.

[0078] The nucleic acid sequences of the present invention, e.g., RNA or DNA, can be isolated from a variety of sources, genetically engineered, amplified, and / or recombinantly expressed. Any recombinant expression system can be used, including bacterial, e.g., yeast, as well as insect or mammalian systems. Nucleic acid manipulations, such as subcloning into expression vectors, probe labeling, sequencing, and hybridization, can be performed as known in the art.

[0079] Accordingly, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0080] The term "vector" as used herein refers to a DNA molecule capable of autonomous replication in prokaryotic and / or eukaryotic cells, and is used interchangeably with recombinant vector, cloning vector, and expression vector, and is generally used as an intermediate carrier for delivering genes or DNA fragments into cells. A vector usually includes an origin of replication that enables replication in prokaryotic and / or eukaryotic cells, a selectable marker gene that can confer resistance to specific conditions / substances such as antibiotic-degrading enzymes, a promoter that enables transcription of the gene in eukaryotic or prokaryotic cells, and a translatable sequence, but the present invention is not limited thereto.

[0081] One type of vector is a "plasmid," which refers to a circular double-stranded standard DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).

[0082] The present invention also provides host cells containing the vectors. Host cells include, but are not limited to, mammalian, plant, insect, fungal, or bacterial cells. Bacterial cells include Gram-positive or Gram-negative bacteria, such as some species of Escherichia, e.g., E. coli, and cells derived from the Pseudomonas genus. Among fungal cells, yeast cells are preferred. Expression in yeast can be achieved using yeast strains, such as Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha, among others. In addition, insect cells, such as Drosophila-derived and Sf9-derived cells, can be used as host cells. In addition, expression systems using mammalian cells, such as Chinese hamster ovary (CHO) cells, monkey COS cells, BHK cells, NSO cells, or Bowes melanoma cells, can be used.

[0083] Because the antibodies or antigen-binding fragments thereof of the present invention may ultimately be administered to humans, fully human expression systems are particularly preferred, in which case the host cells may be human cells, such as HeLa, 911, AT1080, A549, 293, and HEK293 cells, specifically Expi293F cells.

[0084] Preparation of antibodies or antigen-binding fragments thereof The antibodies or antigen-binding fragments thereof of the present invention can be prepared by conventional known methods.

[0085] In one embodiment, antibodies of the invention, e.g., monoclonal antibodies, can be prepared by injecting a test subject (e.g., a mouse) with CNTN4 antigen according to methods known in the art, and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics.

[0086] DNA encoding the monoclonal antibody is readily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of this DNA. Once isolated, the DNA is placed into an expression vector and then transfected into host cells, such as E. coli cells, simian COS cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to achieve monoclonal antibody synthesis in the recombinant host cells.

[0087] In another embodiment, the antibodies or antigen-binding fragments thereof of the present invention can be prepared by using antibody display technology, for example, phage library technology.

[0088] An antibody phage library is constructed, for example, by cloning human antibody heavy and light chain variable region genes fused to a phage coat protein (pll) into a phagemid vector, expressing them in E. coli, and infecting M13 helper phage with them, thereby displaying antibody fragments (scFv or Fab) having various combinations of heavy and light chain variable region sequences on the surface of the phage. Specific human monoclonal antibodies are produced by isolating antibody fragments that bind to specific antigens from this library using a panning method, characterizing the isolated antibody fragments, converting them into whole IgG forms, and overexpressing them in animal cells.

[0089] The term "phagemid vector" as used herein refers to a plasmid DNA having a phage replication origin and typically having an antibiotic resistance gene as a selection marker. The phagemid vector used for phage display contains the gill gene of M13 phage or a part thereof, and the scFv gene is ligated at the 5' end of the gill gene and expressed through transformants.

[0090] A "helper phage" is a phage that provides the genetic information necessary for phagemids to assemble into phage particles. Because phagemids contain only gIII or a portion of the phage genes, host cells transformed with the phagemid (transformants) are infected with helper phage to provide other phage genes. Helper phages include M13K07 and VCSM13, and most of them contain genes that confer resistance to antibiotics such as kanamycin, allowing transformants infected with the helper phage to be selected. In addition, due to the lack of a packaging signal, phagemid genes are preferentially assembled into phage particles over helper phage genes.

[0091] Use and Method The antibodies or antigen-binding fragments thereof of the present invention restore immune responses suppressed by the binding of the CNTN4 protein. The CNTN4 protein has been confirmed to inhibit the proliferation of T cells, particularly CD4+ T cells and CD8+ T cells. Therefore, the antibodies or antigen-binding fragments thereof of the present invention can specifically bind to the CNTN4 protein, thereby enhancing the activity of T cells, particularly CD4+ T cells or CD8+ T cells, and can therefore be used to treat diseases associated with immunosuppression.

[0092] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention enhances T cell activity. Therefore, it can be seen that the antibody or antigen-binding fragment thereof activates T cells, particularly CD4+ T cells or CD8+ T cells. In one specific example, the antibody or antigen-binding fragment thereof can enhance the proliferation of T cells suppressed by CNTN4. The antibody or antigen-binding fragment thereof of the present invention exhibits excellent CNTN4 neutralization ability.

[0093] Thus, the present invention relates to inducing T cell activation using an anti-CNTN4 antibody or an antigen-binding fragment thereof. In one embodiment, the present invention provides a method for inducing or enhancing T cell activation, comprising administering an effective amount of an anti-CNTN4 antibody or an antigen-binding fragment thereof to a subject. In yet another embodiment, the present invention provides use of an anti-CNTN4 antibody or an antigen-binding fragment thereof for inducing or enhancing T cell activation. In yet another embodiment, the present invention provides a pharmaceutical composition for inducing or enhancing T cell activation, comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0094] Thus, the present invention relates to the prevention, amelioration, or treatment of immunosuppression-related diseases using anti-CNTN4 antibodies or antigen-binding fragments thereof.

[0095] As used herein, the term "prevention" refers to any action that inhibits or slows the progression of an immunosuppression-related disease, and as used herein, "treatment" refers to any action that alleviates or beneficially alters the symptoms of an immunosuppression-related disease.

[0096] In one embodiment, the present invention provides a method for preventing, ameliorating, or treating an immunosuppression-related disease, comprising administering to a subject an effective amount of an anti-CNTN4 antibody or antigen-binding fragment thereof.

[0097] In another embodiment, the invention provides a method for preventing or treating cancer in a subject, comprising administering an anti-CNTN4 antibody, or antigen-binding fragment thereof, to the subject.

[0098] In another embodiment, the present invention provides a use of an anti-CNTN4 antibody or antigen-binding fragment thereof for preventing, ameliorating, or treating an immunosuppression-related disease.

[0099] In another embodiment, the present invention provides use of an anti-CNTN4 antibody or antigen-binding fragment thereof for preparing a medicament for preventing or treating cancer.

[0100] In another embodiment, the present invention provides a pharmaceutical composition for preventing, ameliorating, or treating an immunosuppression-related disease, comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0101] As used herein, the term "subject" refers to both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, preferably mammals, e.g., non-human primates, sheep, dogs, cats, cows, and horses. A preferred subject is a human in need of an activated or enhanced immune response.

[0102] Preferably, the anti-CNTN4 antibodies or antigen-binding fragments thereof of the present invention can activate T cells and / or enhance immune responses against cancer cells in cancer patients by blocking the binding of CNTN4 protein, thereby inhibiting the growth of cancer cells in vivo, and therefore may be useful for the prevention, amelioration, or treatment of cancer.

[0103] Preferred cancers whose growth can be inhibited using the antibodies of the present invention typically include cancers that typically respond to immunotherapy. For example, cancers of the present invention include, but are not limited to, melanoma (e.g., metastatic melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate cancer), breast cancer, colorectal cancer, rectal cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). In addition, targets treated in the present invention include refractory or recurrent malignant tumors whose growth can be inhibited using the antibodies of the present invention.

[0104] Other examples of cancers that may be treated using the methods of the present invention include gallbladder cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma and intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, sarcomas such as osteosarcoma or soft tissue sarcomas (e.g., Kaposi's sarcoma), urethral cancer, penile cancer, chronic and acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, ureter cancer, renal pelvis cancer, and central nervous system cancer. These include neoplasms of the central nervous system (CNS), primary CNS lymphomas, tumor angiogenesis, spinal cord tumors, brain stem gliomas, pituitary adenomas, basal cell carcinoma, squamous cell carcinoma, T-cell lymphomas, and environmentally induced cancers such as asbestos-related cancers, as well as combinations thereof.

[0105] In another embodiment, the cancer can be a CNTN4-expressing cancer.

[0106] In another embodiment, the cancer may be refractory or resistant to an existing immune checkpoint inhibitor (e.g., resistant to a PD-1 pathway inhibitor, a PD-L1 pathway inhibitor, or a CTLA-4 pathway inhibitor).

[0107] The antibodies or antigen-binding fragments of the invention can be used alone or in combination with another anti-cancer therapy, which can be, for example, a standard cancer therapy (e.g., chemotherapy, radiation therapy, or surgery), or another anti-cancer agent, such as a cytotoxic agent, a cell growth inhibitor, an anti-angiogenic or antimetabolite agent, a tumor-targeting agent, an immunostimulatory or immunomodulatory agent, or an antibody conjugated to a cytotoxic agent, a cell growth inhibitor, or another toxic substance, or an immune checkpoint inhibitor.

[0108] Preferably, the antibody or antigen-binding fragment of the present invention can be used in combination with another anti-cancer agent, such as an immune checkpoint inhibitor, a chemotherapeutic agent, or radiation therapy. The immune checkpoint inhibitor can be, for example, an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), or an anti-PD-L1 antibody (e.g., atezolizumab, avelumab, or durvalumab). Chemotherapeutic agents may include, but are not limited to, alkylating agents, antimetabolites, kinase inhibitors, spindle-poisoning plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens, and selective estrogen receptor modulators (SERMs), antiprogesterones, estrogen receptor downregulators (ERDs), estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists, antiandrogens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, and antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Specific examples of chemotherapeutic agents of the present invention include gemcitabine, vinorelbine, etoposide (VP-16), platinum analogs such as cisplatin or carboplatin, and taxoids such as paclitaxel, albumin-bound paclitaxel, or docetaxel.

[0109] When co-administered with another anticancer drug, the antibody or antigen-binding fragment thereof of the present invention can be administered separately or in the form of a combined product in which multiple active ingredients are present in one pharmaceutical preparation. When administered separately, the two preparations can be administered sequentially or simultaneously. When administered simultaneously, the two preparations are administered to a patient together. When administered sequentially, the two preparations can be administered to a patient with a short time difference, for example, within 12 hours or less, or within 6 hours or less.

[0110] In one embodiment, the present invention provides a method for preventing, ameliorating, or treating an immunosuppression-associated disease, e.g., cancer, comprising administering to a subject an effective amount of an anti-CNTN4 antibody or antigen-binding fragment thereof in combination with an additional anti-cancer agent. The method includes simultaneous or sequential administration of compositions comprising the antibody and the anti-cancer agent separately, and simultaneous administration of the anti-CNTN4 antibody or antigen-binding fragment thereof in combination with the additional anti-cancer agent in a single composition to a patient in need thereof.

[0111] In another embodiment, the invention provides the use of an anti-CNTN4 antibody or antigen-binding fragment thereof in combination with an additional anti-cancer agent to prevent, ameliorate, or treat an immunosuppression-related disease, e.g., cancer.

[0112] In another embodiment, the present invention provides a pharmaceutical composition or complex for preventing, ameliorating, or treating an immunosuppression-associated disease, such as cancer, comprising an anti-CNTN4 antibody or antigen-binding fragment thereof and an additional anti-cancer drug. The present invention encompasses pharmaceutical compositions or complexes comprising an anti-CNTN4 antibody or antigen-binding fragment thereof and an additional anti-cancer drug, not only when both components are physically present in a single preparation, but also when both components are administered simultaneously or sequentially in separate preparations. Here, the two drugs may be provided individually or in a single kit. Accordingly, the present invention provides a kit for preventing, ameliorating, or treating an immunosuppression-associated disease, such as cancer, comprising an anti-CNTN4 antibody or antigen-binding fragment thereof and an additional anti-cancer drug.

[0113] The additional anti-cancer agent is preferably an immune checkpoint inhibitor, more preferably an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), or an anti-PD-L1 antibody (e.g., atezolizumab, avelumab, or durvalumab).

[0114] Another preferred additional anti-cancer agent may be a chemotherapeutic agent, for example, a platinum analogue such as gemcitabine, vinorelbine, etoposide (VP-16), cisplatin or carboplatin, or a taxoid such as paclitaxel, albumin-bound paclitaxel, or docetaxel.

[0115] Another preferred additional anti-cancer agent used in combination with the antibody or antigen-binding fragment thereof of the present invention may be radiation therapy.

[0116] The present invention also provides a method for detecting the presence or amount of CNTN4 protein in a sample using an anti-CNTN4 antibody or its antigen-binding fragment as an active ingredient. The method includes contacting the antibody or its antigen-binding fragment with a sample and a control sample under conditions that allow the antibody or its antigen-binding fragment to bind to the CNTN4 protein and form a complex. Whether a complex is formed is then detected, where a difference in the degree of complex formation between the sample and the control sample is evidence of the presence of a human blood antigen in the sample (e.g., blood).

[0117] Thus, the present invention provides a composition for diagnosing cancer, comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0118] Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof. The compositions may contain inactive ingredients, i.e., pharmaceutically acceptable excipients (see, for example, Handbook of Pharmaceutical Excipients). Therapeutic and diagnostic compositions may be prepared by mixing physiologically acceptable carriers, excipients, or stabilizers in the form of lyophilized powder, slurry, aqueous solution, or suspension.

[0119] Suitable routes of administration include parenteral administration, e.g., intramuscular, intravenous, or subcutaneous administration. Administration of antibodies used in the pharmaceutical compositions or used to practice the methods of the invention can be by a variety of conventional methods, including topical application, or transdermal, subcutaneous, intraperitoneal, parenteral, intraarterial, or intravenous injection. In one embodiment, the antibodies of the invention are administered intravenously or subcutaneously.

[0120] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that the following examples are provided only to more specifically explain the present invention, and the scope of the present invention is not limited to the examples according to the gist of the present invention.

[0121] Mode of Invention Example 1: Preparation of CNTN4-specific binding scFv and confirmation of its binding affinity (1) 1.1 Construction of scFv library (1) A total of 14 scFv fragments were obtained by electroporating the purified PCR products for the CDRs in Table 1 into E. coli BL21, and the amino acid sequences of the heavy and light chain variable regions of the scFvs are shown in Figure 2 and Table 2 (the underlined and bolded sequences in Figure 2 indicate the CDR sequences in each region).

[0122] [Table 1]

[0123] [Table 2-1]

[0124] [Table 2-2]

[0125] 1.2 Confirmation of CNTN4 binding affinity of scFv (1) Confirmation of binding affinity of scFv to antigen protein through FACS FACS analysis was performed on the scFv prepared in Example 1.1.

[0126] Periplasm containing various concentrations of scFv was incubated with mouse antigen-overexpressing 293F cells at 4°C for 1 hour. After washing, PE-labeled anti-His tag antibody was added to the wells and incubated in the dark at 4°C for 0.5 hours. Blank 293F cells were used as a negative control (NC). The mean fluorescence intensity (MFI) of the cells was measured by flow cytometry.

[0127] The results are shown in Figure 3. It was confirmed that all of the scFvs in Example 1.1 exhibited excellent cell-binding affinity for CNTN4 protein at the nM level.

[0128] [Table 3]

[0129] Example 2: Preparation of anti-CNTN4 IgG antibody (1) Of the scFvs prepared in Example 1.1, nine scFvs (A102, A103, A104, A105, A106, A108, A109, A112, and A114) were selected to prepare antibodies.

[0130] Specifically, the VH genes of the nine selected scFvs were fused to the CH1, hinge, CH2, and CH3 segments of human IgG4 (S228P) for the heavy chain, and the VL gene was fused to the human Ig domain for the light chain. Plasmids carrying the heavy and light chains were co-transfected into ExpiCHO-S cells. The cells were cultured for 8 days, and the supernatant was collected. Proteins were purified by applying the cell culture supernatant to a column through the sample inlet. Specifically, a Protein A column equilibrated with wash buffer (50 mM Tris, 150 mM NaCl, pH 7.4) was used. After washing the column with 5 CV of wash buffer, gradient elution was performed using buffer B (0.1 M glycine, pH 3.4). The eluted protein was then applied to a cation exchange resin, and the final protein was purified and eluted using a NaCl gradient in sodium acetate buffer (pH 5.0). The final eluted protein concentration was determined by A280 / extinction coefficient using Nanodrop 2000. The purified protein was analyzed by SDS-PAGE and HPLC-SEC to confirm that anti-CNTN4 antibody was prepared (Table 4).

[0131] The prepared antibodies were designated scFv-IgG antibodies (for example, an antibody prepared using A102 scFv was designated A102-IgG antibody).

[0132] [Table 4]

[0133] Example 3: Confirmation of CNTN4 binding affinity of anti-CNTN4 IgG antibodies (1) 3.1 ELISA test ELISA tests were performed on the nine IgG antibodies (A102, A103, A104, A105, A106, A108, A109, A112, and A114) prepared in Example 2. In addition, a human IgG4 antibody was used as a negative control (NC). A 96-well plate was coated with anti-His antibody in coating buffer overnight at 4°C. The next day, the plate was blocked with 5% skim milk in PBS at 25°C for 1 hour. Human CNTN4 (His tag) was added to the plate and incubated at 25°C for 1 hour. After washing, the antibody was added to the wells and incubated at 25°C for 2 hours. The plate was then incubated with anti-human IgG (HRP) conjugate at 25°C for 1 hour. HRP activity was detected with tetramethylbenzidine (TMB) substrate, and the reaction was quenched with 2M HCl. The plate was read at 450 nm.

[0134] As shown in Table 5, the results indicated that all nine antibodies exhibited very high binding affinities, with EC50 values ​​at the pM level.

[0135] [Table 5]

[0136] 3.2 FACS analysis FACS analysis was performed on the nine IgG antibodies (A102, A103, A104, A105, A106, A108, A109, A112, and A114) prepared in Example 2. In addition, a human IgG4 antibody was used as a negative control (NC).

[0137] A HEK293 cell line overexpressing human CNTN4 on the cell surface was constructed. Overexpressing HEK293 cells were seeded at 1 x 10 cells / well in each well of a 96-well plate. Nine IgG antibodies, pre-diluted at different concentrations, were then added to each well containing the cells. The highest antibody concentration was set at 100 nM, and serially diluted 3-fold to 10 points. After antibody addition, the cells were incubated at 4°C for 1 hour. After washing the reagent rod twice with FACS buffer, an APC-conjugated anti-human IgG antibody was added to the cells, and the resulting cells were further incubated at 4°C for 30 minutes. The cells were washed twice, and 7-AAD was added to each sample at a concentration of 0.5 mg / mL. Each sample was transferred to a FACS tube, and the binding of the nine IgG antibodies was analyzed using a FACS Canto II flow cytometer.

[0138] The results are shown in Figure 3 and Table 6. The mean fluorescence intensity (MFI) of the nine IgG antibodies was observed to increase in a concentration-dependent manner (Figure 3), confirming that the nine IgG antibodies exhibited high binding affinity to CNTN4 expressed on the cell surface, with EC50 values ​​at the pM level (Table 6).

[0139] [Table 6]

[0140] Example 4: Preparation of CNTN4-specific binding scFv and confirmation of its binding affinity (2) 4.1 Construction of scFv library (2) The CDR sequences of the scFv selected in Example 1 were further mutated. To remove scFv post-translational modification sites, fixed mutations were introduced to generate a set of mutated CDRs, as shown in Figure 4 and Table 7.

[0141] [Table 7-1]

[0142] [Table 7-2]

[0143] 200 ng of each library DNA for the CDRs in Table 7 was electroporated into E. coli BL21 and expressed as scFv fragments. The amino acid sequences of the heavy and light chain variable regions of each of the 19 scFvs are shown in Figure 5 and Table 8 (the underlined and bolded sequences in Figure 5 indicate the CDR sequences in each region).

[0144] [Table 8-1]

[0145] [Table 8-2]

[0146] [Table 8-3]

[0147] 4.2 Confirmation of CNTN4 binding affinity of scFv (2) Confirmation of binding affinity of scFv to antigen protein through FACS FACS tests were performed in the same manner as in Example 1.2, except that the scFv prepared in Example 4.1 was used and human CNTN4-overexpressing 293F cells were used.

[0148] As shown in Table 9, the results indicated that all of the scFvs prepared in Example 4.1 exhibited high binding affinity to CNTN4 protein at the nM level.

[0149] [Table 9]

[0150] Example 5: Preparation of anti-CNTN4 IgG antibody (2) Six of the scFvs prepared in Example 4.1 (A202, A204, A206, A209, A211, and A217) were selected to prepare antibodies.

[0151] Specifically, the VH genes of the six selected scFvs were fused to the CH1, hinge, CH2, and CH3 segments of heavy chain human IgG4 (S228P), and the VL gene was fused to the light chain human Ig domain. The heavy and light chain plasmids were co-transfected into Expi293F cells. The cells were cultured for 6 hours, and the supernatant was collected. The cell culture supernatant was applied to a column through the sample inlet to purify the protein. Specifically, a Protein A column equilibrated with wash buffer (100 mM Tris, pH 7.0) was used. After washing the column with 15 CV of wash buffer, gradient elution was performed using buffer B (0.1 M glycine, pH 3.5). The protein concentration of the eluate was determined by A280 / extinction coefficient using a Nanodrop 2000. The purified protein was analyzed by SDS-PAGE and HPLC-SEC to confirm the preparation of anti-CNTN4 antibodies (Table 10).

[0152] The prepared antibodies were designated scFv-IgG antibodies (for example, an antibody prepared using A202 scFv was designated A202-IgG antibody).

[0153] [Table 10]

[0154] Example 6: Confirmation of CNTN4 binding affinity of anti-CNTN4 IgG antibodies (2) 6.1 ELISA Test ELISA tests were performed on the six IgG antibodies (A202, A204, A206, A209, A211, and A217) prepared in Example 5. A 96-well plate was coated with anti-His antibody in coating buffer overnight at 4°C. The next day, the plate was blocked with 3% BSA in PBS at 25°C for 1 hour. Human CNTN4 (His tag) was added to the plate and incubated at 25°C for 1 hour. After washing, the antibody was added to the wells and incubated at 25°C for 2 hours. The plate was then incubated with anti-human IgG (HRP) conjugate at 25°C for 1 hour. HRP activity was detected with tetramethylbenzidine (TMB) substrate, and the reaction was quenched with 2M HCl. The plate was read at 450 nm.

[0155] As shown in Table 11, the results indicated that all six antibodies exhibited very high binding affinities, with EC50 values ​​at the pM level.

[0156] [Table 11]

[0157] 6.2 Surface Plasmon Resonance (SPR) Test SPR tests were performed on the six IgG antibodies prepared in Example 5 (A202, A204, A206, A209, A211, and A217).

[0158] Specifically, an activator was prepared by mixing 400 mM EDC and 100 mM NHS (GE) immediately before injection into the chip. A CM5 sensor chip was activated with the mixture at a flow rate of 10 μL / min for 420 seconds. 30 μg / mL of Protein G in 10 mM NaAc (pH 4.0) was injected into the chip at a flow rate of 10 μL / min for 420 seconds. The chip was then deactivated with 1 M ethanolamine-HCl (GE) at a flow rate of 10 μL / min for 420 seconds.

[0159] IgG antibodies were captured on the chip using THE™ His-tag antibody in running buffer 1x HBS-EP+ at a flow rate of 10 μL / min. For the binding and dissociation steps, a series of concentrations of analyte (human CNTN4 His-tag) and running buffer were injected sequentially over the chip at a flow rate of 30 μL / min.

[0160] The chip was regenerated with 10 mM glycine at pH 1.5.

[0161] The surface channel Fc1, which did not contain any captured ligand, was used as a reference surface for reference subtraction. The final data for each interaction was subtracted from the data for the reference Fc1 and buffer channels. The experimental data were fitted by 1:1 binding in the 8K (ver. 2.0.15.12933) evaluation software.

[0162] The measured equilibrium dissociation constant (K d ) are shown in Table 12. The results confirmed that the antibodies of the present invention bind to human CNTN4 protein with high affinity.

[0163] [Table 12]

[0164] 6.3.FACS analysis FACS analysis was performed on the IgG antibody A114 prepared in Example 2, as well as the IgG antibody A211 prepared in Example 5. In addition, a human IgG4 antibody was used as a negative control (NC).

[0165] We constructed a HEK293 cell line overexpressing human CNTN4 on the cell surface. 1 × 10 overexpressing HEK293 cells were plated in each well of a 96-well plate. 5After seeding cells per well, pre-diluted A114 and A211 antibodies were prepared at different concentrations and added to each well containing cells. The highest antibody concentration was set at 100 mg / mL, and serially diluted 3-fold to 14 points. After antibody addition, the cells were incubated at 4°C for 1 hour. After washing the reagent rod twice with FACS buffer, APC-conjugated anti-human IgG antibody was added to the cells, and the resulting cells were further incubated at 4°C for 30 minutes. The cells were washed twice, and 7-AAD was added to each sample at a concentration of 0.5 mg / mL. Each sample was transferred to a FACS tube, and the binding of the A114 and A211 antibodies was analyzed using a FACS Canto II flow cytometer.

[0166] The results are shown in Figure 6 and Table 13. It was observed that the mean fluorescence intensity (MFI) of both A114 and A211 increased in a concentration-dependent manner (Figure 6), and their EC50 values ​​were observed to be 0.539 nM and 0.316 nM, respectively (Table 13).

[0167] [Table 13]

[0168] The above results confirmed that both antibodies A114 and A211 have high binding affinity to CNTN4 expressed on the cell surface.

[0169] Example 7. Confirmation of the efficacy of antibodies in inhibiting tumor growth This example was intended to confirm the tumor growth inhibitory efficacy of the antibodies of the present invention in vivo.

[0170] This experiment was performed with the IgG antibodies (A114 and A211) prepared in Examples 2 and 5, and used the CT26 syngenic mouse tumor model. The CT26 tumor model was developed using 1×10 6 CT26 cancer cells were established by subcutaneous inoculation into 6-week-old female BALB / c mice.

[0171] Tumor volume 75-150mm3 When the mice reached 100 mg / kg, they were randomly selected and divided into four groups of 10 mice each for the experiments. Each group was intraperitoneally administered 1) control (hIgG4, 3 mg / kg), 2) A114 IgG antibody (3 mg / kg), 3) A211 IgG antibody (1 mg / kg), or 4) A211 IgG antibody (3 mg / kg) every three days (q3d) for four doses over two weeks (D0, D3, D6, and D9). Growing tumor size was measured three times a week, and mouse body weights were measured twice a week. The tumor-inhibiting efficacy of the antibodies was evaluated relative to the control (human IgG4) by setting the tumor growth rate at the final drug administration (D9) as 100% (relative tumor growth, 100%).

[0172] The results are shown in FIG.

[0173] [Table 14]

[0174] In the case of A114 and A211 administration, tumor growth was statistically significantly inhibited compared to the control (hIgG4), and A211 administration exhibited dose-dependent efficacy (Figure 7A). When the control tumor growth rate at the final administration (D9) was set to 100%, the A114 (3 mg / kg) group exhibited a tumor growth rate of approximately 64.4%, the A211 (1 mg / kg) group exhibited a tumor growth rate of approximately 51.9%, and the A211 (3 mg / kg) group exhibited a tumor growth rate of approximately 42% (Figure 7B). That is, compared to the control group, the A114 (3 mg / kg) group exhibited a tumor growth inhibition rate of approximately 35.6%, and the A211 (1 mg / kg) and A211 (3 mg / kg) groups exhibited tumor growth inhibition rates of approximately 48.1% and 58.0%, respectively (Table 14). No adverse reactions, including weight changes, were observed.

[0175] The above results confirmed that the antibodies of the present invention exhibited tumor-inhibiting efficacy in vivo without adverse reactions.

Claims

1. An anti-CNTN4 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:4; a heavy chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8-18, and 21-35; a light chain CDR1 having the amino acid sequence of SEQ ID NO: 2 or 3; a light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and a light chain CDR3 having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 19, and 20.

2. 101) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 6, 2, 5, and 7, respectively; 102) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 8, 2, 5, and 7, respectively; 103) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 9, 2, 5, and 7, respectively; 104) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 10, 2, 5, and 7, respectively; 105) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 11, 2, 5, and 7, respectively; 106) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 12, 2, 5, and 7, respectively; 107) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 13, 2, 5, and 7, respectively; 108) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 14, 2, 5, and 7, respectively; 109) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 15, 2, 5, and 7, respectively; 110) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 16, 2, 5, and 7, respectively; 111) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 17, 2, 5, and 7, respectively; 112) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 18, 3, 5, and 7, respectively; 113) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 18, 2, 5, and 19, respectively; 114) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 18, 2, 5, and 20, respectively; 201) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 21, 3, 5, and 20, respectively; 202) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 22, 3, 5, and 20, respectively; 203) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 23, 3, 5, and 20, respectively; 204) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 24, 3, 5, and 20, respectively; 205) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 21, 2, 5, and 20, respectively; 206) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 25, 3, 5, and 20, respectively; 207) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 26, 2, 5, and 20, respectively; 208) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 27, 2, 5, and 20, respectively; 209) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 26, 3, 5, and 20, respectively; 210) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 28, 3, 5, and 20, respectively; 211) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 27, 3, 5, and 20, respectively; 212) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 9, 3, 5, and 20, respectively; 213) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 29, 2, 5, and 20, respectively; 214) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 30, 2, 5, and 20, respectively; 215) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 31, 2, 5, and 20, respectively; 216) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 32, 3, 5, and 20, respectively; 217) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 33, 2, 5, and 20, respectively; 218) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 34, 3, 5, and 20, respectively; or 219) The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3 having the amino acid sequences of SEQ ID NOs: 1, 4, 35, 2, 5, and 20, respectively.

3. a heavy chain variable region having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 38-48, 52, and 54-67; and a light chain variable region having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 49-51, and 53.

4. A101) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 36 and 37, respectively; A102) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 38 and 37, respectively; A103) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 39 and 37, respectively; A104) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 40 and 37, respectively; A105) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 41 and 37, respectively; A106) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 42 and 37, respectively; A107) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 43 and 37, respectively; A108) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 44 and 37, respectively; A109) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 45 and 37, respectively; A110) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 46 and 37, respectively; A111) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 47 and 37, respectively; A112) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 48 and 49, respectively; A113) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 48 and 50, respectively; A114) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 48 and 51, respectively; A201) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 52 and 53, respectively; A202) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 54 and 53, respectively; A203) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 55 and 53, respectively; A204) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 56 and 53, respectively; A205) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 52 and 51, respectively; A206) ​​A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 57 and 53, respectively; A207) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 58 and 51, respectively; A208) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 59 and 51, respectively; A209) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 58 and 53, respectively; A210) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 60 and 53, respectively; A211) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 59 and 53, respectively; A212) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 39 and 53, respectively; A213) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 61 and 51, respectively; A214) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 62 and 51, respectively; A215) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 63 and 51, respectively; A216) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 64 and 53, respectively; A217) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 65 and 51, respectively; A218) A heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 66 and 53, respectively; or A219) The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region and a light chain variable region having the amino acid sequences of SEQ ID NOs: 67 and 51, respectively.

5. The anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which specifically binds to CNTN4 protein.

6. The anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which binds to the CNTN4 protein with an equilibrium dissociation constant (KD) of 1 x 10-8 M or less.

7. The anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which binds to the CNTN4 protein with an EC50 of 100 nM or less.

8. The anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antigen-binding fragment is a Fab, Fab', Fab'-SH, Fv, single-chain variable fragment (scFv), F(ab')2 fragment, VL, VH, diabody, triabody, tetrabody, minibody ((scFv-CH3)2), IgGΔCH2, scFv-Fc, (scFv)2-Fc, Fynomer, dual affinity retargeting (DART), or TRIDENT.

9. The anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

10. An antibody-drug conjugate comprising the anti-CNTN4 antibody or antigen-binding fragment thereof of any one of claims 1 to 4.

11. A nucleic acid molecule encoding the anti-CNTN4 antibody or antigen-binding fragment thereof of any one of claims 1 to 4.

12. A recombinant expression vector comprising the nucleic acid molecule of claim 11.

13. A pharmaceutical composition for preventing or treating cancer, comprising the anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

14. The pharmaceutical composition of claim 13, wherein the cancer is a CNTN4-expressing cancer.

15. A pharmaceutical composition for preventing or treating cancer, comprising the anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and an additional anticancer agent.

16. 16. The pharmaceutical composition of claim 15, wherein the additional anti-cancer agent is an immune checkpoint inhibitor or a chemotherapeutic agent.

17. The pharmaceutical composition according to claim 16, wherein the immune checkpoint inhibitor is one or more selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody.

18. 16. The pharmaceutical composition of claim 15, wherein the anti-CNTN4 antibody or antigen-binding fragment thereof and the additional anti-cancer agent are co-administered in one preparation or administered simultaneously or sequentially in separate preparations.

19. A pharmaceutical composition for preventing or treating cancer, comprising the anti-CNTN4 antibody or antigen-binding fragment thereof of any one of claims 1 to 4, used in combination with an additional anti-cancer therapy.

20. 20. The pharmaceutical composition of claim 19, wherein the additional anticancer therapy is one or more selected from the group consisting of an immune checkpoint inhibitor, a chemotherapeutic agent, and radiation therapy.

21. A method for preventing or treating cancer in a subject, comprising administering to the subject the anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

22. Use of the anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 for preparing a medicament for preventing or treating cancer.

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