Anti-CNTN4 antibodies and uses thereof

Abstract

The present invention relates to anti-CNTN4 antibodies or antigen-binding fragments thereof, pharmaceutical compositions containing same, and their uses for activating T cells that upregulate cellular immune responses, e.g., for treating cancer.

Description

[Technical field]

[0001] The present invention relates to anti-CNTN4 antibodies or antigen-binding fragments thereof, pharmaceutical compositions containing same, and their uses for activating T cells, e.g., for treating cancer. [Background technology]

[0002] The human body has a defense mechanism to protect itself from external invaders (viruses, toxins, etc.) and internal harmful changes (cancer cell mutations). Unlike normal cells, cancer cells have specific antigens on their surface and are destroyed by the immune system at the early stage of cancer development. Then, when the balance of power between cancer cells that want to grow infinitely and immune cells that want to attack cancer cells is disturbed, the cancer cells begin to grow substantially. As the cancer cells grow further, they interfere with the immune system in the body, and some cancer cells evade the immune system by using the immune checkpoints of immune cells, and when immune checkpoint inhibitors suppress the immune checkpoints, the power of the immune cells increases, thereby killing the cancer cells.

[0003] Immune checkpoint inhibitors are drugs that attack cancer cells by activating T cells by blocking the activation of immune checkpoint proteins involved in T cell inhibition, and include CTLA-4, PD-1, and PD-L1 inhibitors. Representative drugs currently on the market include ipilimumab (product name: YERVOY®) as a CTLA-4 monoclonal antibody, nivolumab (product name: OPDIVO®) and pembrolizumab (product name: KEYTRUDA®) as PD-1 monoclonal antibodies, and atezolizumab (product name: TECENTRIQ®) and durvalumab (product name: IMFINZI®) as PD-L1 monoclonal antibodies.

[0004] However, there are still cancers that cannot be treated with existing immune checkpoint inhibitors, and therefore the development of new anti-cancer therapies is necessary. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide an anti-CNTN4 antibody or an antigen-binding fragment thereof that specifically binds to CNTN4 protein. In particular, the present invention is intended to provide an antibody or an antigen-binding fragment thereof that binds to CNTN4 protein and neutralizes the immune evasion mechanism of CNTN4.

[0006] The present invention also contemplates providing a composition for preventing or treating diseases such as cancer caused by decreased T cell activity by using an antibody or an antigen-binding fragment thereof to block the immune evasion mechanism of CNTN4 and activate T cells.

[0007] The present invention also contemplates providing compositions for analyzing or detecting CNTN4 protein using the antibodies or antigen-binding fragments thereof. [Means for solving the problem]

[0008] The present invention provides an anti-CNTN4 antibody or antigen-binding fragment thereof that specifically binds to CNTN4 protein. The antibody or antigen-binding fragment specifically binds to CNTN4 protein, e.g., human or mouse CNTN4 protein, and neutralizes the immune evasion mechanism of CNTN4. Thus, the antibody or antigen-binding fragment of the present invention can increase the activity of T cells, such as CD4+ T cells or CD8+ T cells, that are inhibited by CNTN4.

[0009] In one embodiment, the present invention provides A light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 70; A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 88; a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116; Heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:58; A heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and Heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 115 The present invention provides an anti-CNTN4 antibody or antigen-binding fragment thereof, comprising:

[0010] In one embodiment, the present invention provides a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; and A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 The present invention provides an anti-CNTN4 antibody or antigen-binding fragment thereof, comprising:

[0011] In one embodiment, the present invention provides a light chain variable region comprising the amino acid sequence of SEQ ID NO: 156; and A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 159 The present invention provides an anti-CNTN4 antibody or antigen-binding fragment thereof, comprising:

[0012] In one embodiment, the antigen-binding fragment of the invention may be an antibody fragment selected from the group consisting of a Fab, a Fab', a Fab'-SH, an Fv, a single chain antibody scFv, a (Fab')2 fragment, a single domain antibody, a bispecific antibody (dAb), or a linear antibody, and the antibody may be a chimeric antibody or a humanized antibody.

[0013] In another embodiment, the invention provides nucleic acid molecules encoding the antibody or antigen-binding fragment thereof, and recombinant expression vectors comprising the nucleic acid molecules.

[0014] In another embodiment, the present invention also provides a composition for preventing or treating cancer, comprising an antibody or an 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.

[0015] In another embodiment, the present invention also provides a composition for analyzing or detecting CNTN4 protein, the composition comprising an antibody or an antigen-binding fragment thereof.

[0016] In another embodiment, the present invention provides the use of an anti-CNTN4 antibody, or an antigen-binding fragment thereof, for preventing or treating cancer.

[0017] In another embodiment, the invention provides use of an anti-CNTN4 antibody, or an antigen-binding fragment thereof, for the preparation of a medicament for preventing or treating cancer.

[0018] In another embodiment, the invention provides a method for preventing or treating cancer, comprising administering to a subject an effective amount of an anti-CNTN4 antibody, or antigen-binding fragment thereof.

[0019] In another embodiment, the present invention provides a method for preventing or treating cancer, comprising administering to a subject an effective amount of an anti-CNTN4 antibody or antigen-binding fragment thereof, and an effective amount of an additional anti-cancer agent, which may be, for example, an immune checkpoint inhibitor or a chemotherapeutic agent. Effect of the Invention

[0020] The novel anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention activates T cells by blocking the immune escape mechanism of CNTN4, and can therefore be effectively used to prevent or treat diseases caused by decreased T cell activity, particularly cancer. Therefore, the use of the anti-CNTN4 antibody of the present invention may exert an excellent anticancer effect on cancers for which existing immunotherapies do not provide a therapeutic effect. [Brief description of the drawings]

[0021] [Figure 1a] 1 shows the amino acid sequences of the light and heavy chain variable regions of scFv antibody fragments selected from a phage display library, with the sequences indicated by solid and dashed lines representing the CDR sequences of each region. [Figure 1b] 1 shows the amino acid sequences of the light and heavy chain variable regions of scFv antibody fragments selected from a phage display library, with the sequences indicated by solid and dashed lines representing the CDR sequences of each region. [Figure 1c] 1 shows the amino acid sequences of the light and heavy chain variable regions of scFv antibody fragments selected from a phage display library, with the sequences indicated by solid and dashed lines representing the CDR sequences of each region. [Figure 1d] 1 shows the amino acid sequences of the light and heavy chain variable regions of scFv antibody fragments selected from a phage display library, with the sequences indicated by solid and dashed lines representing the CDR sequences of each region. [Figure 2a] 1 shows the amino acid sequences of the light and heavy chain variable regions of scFv antibody fragments selected from a phage display library, with the sequences indicated by solid and dashed lines representing the CDR sequences of each region. [Figure 2b] 1 shows the amino acid sequences of the light and heavy chain variable regions of scFv antibody fragments selected from a phage display library, with the sequences indicated by solid and dashed lines representing the CDR sequences of each region. [Figure 2c]1 shows the amino acid sequences of the light and heavy chain variable regions of scFv antibody fragments selected from a phage display library, with the sequences indicated by solid and dashed lines representing the CDR sequences of each region. [Figure 2d] 1 shows the amino acid sequences of the light and heavy chain variable regions of scFv antibody fragments selected from a phage display library, with the sequences indicated by solid and dashed lines representing the CDR sequences of each region. [Diagram 3] FIG. 1 shows the amino acid sequences of the variable regions used to prepare anti-CNTN4 humanized antibodies. [Figure 4] FIG. 1 shows that the antibodies A-01 and L1H2 of the present invention effectively neutralize CNTN4 protein and induce T cell proliferation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] 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.

[0023] In one embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the invention comprises: A light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 70; A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 88; a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116; Heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:58; A heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and Heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 115 Includes.

[0024] Table 1 shows the CDR sequences of each of the light and heavy chain variable regions for the anti-CNTN4 antibodies or antigen-binding fragments thereof of the present invention.

[0025] [Table 1] TIFF0007642096000002.tif207149 TIFF0007642096000003.tif210149 TIFF0007642096000004.tif210149 TIFF0007642096000005.tif205149 TIFF0007642096000006.tif122149

[0026] In another specific embodiment, an anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention may comprise each pair of light chain variable regions and heavy chain variable regions shown in Tables 4 and 5. For example, an antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (S1). Similarly, an antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (S2). Another antibody or antigen-binding fragment thereof may similarly comprise two pairs of light and heavy chain variable regions.

[0027] In a preferred embodiment, an anti-CNTN4 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:18, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0028] In another specific embodiment, an anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention may comprise each pair of light chain variable regions and / or heavy chain variable regions set forth in Table 12. For example, an antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 159 (LAH2). Similarly, an antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 156, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 159 (L1H2). Alternatively, an antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 158, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 159 (L2H2).

[0029] In a preferred embodiment, an anti-CNTN4 antibody, or antigen-binding fragment thereof, of the invention comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:156, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:159.

[0030] As used herein, a light or heavy chain variable region comprises a particular amino acid sequence means that the light or heavy chain variable region includes, has, or consists of the entire amino acid sequence.

[0031] In one example, the antibody or antigen-binding fragment thereof comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:70, a light chain CDR2 having the amino acid sequence of SEQ ID NO:88, a light chain CDR3 having the amino acid sequence of SEQ ID NO:116, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:58, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:89, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:115.

[0032] In another example, the antibody or antigen-binding fragment thereof comprises a light chain CDR1 consisting essentially of the amino acid sequence of SEQ ID NO:70, a light chain CDR2 consisting essentially of the amino acid sequence of SEQ ID NO:88, a light chain CDR3 consisting essentially of the amino acid sequence of SEQ ID NO:116, a heavy chain CDR1 consisting essentially of the amino acid sequence of SEQ ID NO:58, a heavy chain CDR2 consisting essentially of the amino acid sequence of SEQ ID NO:89, and a heavy chain CDR3 consisting essentially of the amino acid sequence of SEQ ID NO:115.

[0033] In another example, the antibody or antigen-binding fragment thereof comprises a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 70, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 88, a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 116, a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 58, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 89, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 115.

[0034] In another example, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 18 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2, or comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 156 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 159.

[0035] In another example, the antibody or antigen-binding fragment thereof comprises a light chain variable region consisting essentially of the amino acid sequence of SEQ ID NO: 18 and a heavy chain variable region consisting essentially of the amino acid sequence of SEQ ID NO: 2, or comprises a light chain variable region consisting essentially of the amino acid sequence of SEQ ID NO: 156 and a heavy chain variable region consisting essentially of the amino acid sequence of SEQ ID NO: 159.

[0036] In another example, the antibody or antigen-binding fragment thereof comprises a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2, or comprises a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 156 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 159.

[0037] As used herein, the terms "comprising," "comprise," or variations thereof, refer to the open meaning. As an example, an antibody or antigen-binding fragment thereof that comprises a recited amino acid sequence may contain additional amino acid sequences that are not necessarily or necessarily recited.

[0038] As used herein, the term "consisting essentially of" or variations thereof includes any of the recited elements and acknowledges the presence of elements that do not substantially affect the basic, novel, or functional characteristics of the embodiment. As an 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.

[0039] As used herein, the term "consisting of" or variations thereof refers to each component described herein without allowing for any element not described or listed in that description of the embodiment.

[0040] As used herein, the term "complementarity determining region" (CDR; CDR1, CDR2, and CDR3) refers to the amino acid residues in an antibody variable region that are necessary for antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3.

[0041] In another preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention may comprise a fully or partially humanized sequence in the framework region of the scFv. The partially humanized sequence may be at least about 60%, at least about 70%, or at least about 80% humanized compared to the fully humanized sequence, and some of the fully humanized sequences (e.g., 1, 2, 3, 4, or 5, etc.) may be back-mutated. The antibody or antigen-binding fragment thereof comprising a humanized sequence may have the same binding ability to CNTN-4, or may not have a substantially large difference in binding ability to CNTN-4, even when compared to a non-humanized antibody or antigen-binding fragment thereof.

[0042] 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, protein, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. The term "antibody," as used herein, encompasses not only intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof, and fusion proteins comprising antibody fragments, and any other modified form of an immunoglobulin molecule that contains an antigen recognition site.

[0043] Antibodies include five classes of immunoglobulins (Ig), M, IgD, IgG, IgA, and IgE, each of which contains 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 a variable region having a different amino acid sequence for each antibody, and a constant region having the same amino acid sequence, and the heavy chain constant region contains CH1, H (hinge), CH2, and CH3 domains. Each domain consists of two β-sheets connected by an intramolecular disulfide bond.

[0044] An antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 is referred to herein as "A-01."

[0045] Furthermore, an antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 156 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 159 is referred to herein as "L1H2."

[0046] The antibody of the present invention may be a chimeric antibody, a humanized antibody, or a human antibody.

[0047] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., the variable region sequences are derived from a mouse or chicken antibody and the constant region sequences are derived from a human antibody.

[0048] As used herein, the term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as mouse or chicken, have been grafted onto human framework sequences. The framework sequences may be further modified, for example by back mutation.

[0049] As used herein, the term "human antibody" refers to an antibody having 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.

[0050] As used herein, the term "antigen-binding fragment" or "antibody fragment" refers to antigen-binding fragments and analogs of antibodies that typically contain at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody. Antibody fragments maintain at least a portion of the binding specificity of the parent antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, Fv, F(ab')2 fragments, single chain antibodies scFv, single domain antibodies, bispecific antibodies (dAbs), or linear antibodies.

[0051] In particular, a Fab fragment refers to a monovalent fragment consisting of the VL, VH, CL, and CH1 domains.

[0052] Fab' fragments differ from Fab fragments by the addition of several residues from the antibody hinge region to the carboxyl terminus of the CH1 domain including at least one cysteine.

[0053] Fab'-SH refers to Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group.

[0054] F(ab')2 antibody fragments are produced as pairs of Fab' fragments separated by hinge cysteines.

[0055] Fv is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy chain variable region and one light chain variable region in tight non-covalent association. The folding of these two regions results in six hypervariable loops (three loops each from the heavy and light chain) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, a single variable region still has the ability to recognize and bind to an antigen, although with a lower affinity than the entire binding site.

[0056] A single chain antibody scFv is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. The scFv polypeptide herein is also referred to as scFv antibody fragment, antigen-binding fragment scFv, scFv antibody, antibody scFv, or simply scFv.

[0057] Bispecific antibodies refer to small antibody fragments prepared by constructing scFv fragments using a short linker (about 5-10 residues) between the VH and VL domains such that inter-chain, but not intra-chain, pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers composed of two "crossover" scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.

[0058] The anti-CNTN4 antibodies, or antigen-binding fragments thereof, of the present invention can specifically bind to CNTN4 protein, preferably human or mouse CNTN4 protein.

[0059] As used herein, the terms "specifically bind to" or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of a target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds to a particular target (e.g., epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets.

[0060] As used herein, the term "specifically binds to human CNTN4 protein" means a dissociation constant (Kd) of 5×10 -7 M or less, or preferably 1×10 -7 Therefore, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention can refer to an antibody that binds to human CNTN4 protein with a dissociation constant (Kd) of 5×10 -7 M or less, preferably 1×10 -7 It can bind to human CNTN4 protein with M or less.

[0061] As used herein, the term "Kd" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, the constant having units M. The Kd value of an antibody can be determined using methods well established in the art. A preferred method for determining the Kd value of an antibody is by using surface plasmon resonance (SRP), preferably using a biosensor system such as the Biacore® system.

[0062] 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.

[0063] In another embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention is a multispecific antibody molecule, such as a bispecific or trispecific antibody molecule. A multispecific antibody molecule comprises multiple variable regions, each variable region having a 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., a 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 (e.g., including but not limited to, CTLA-4, PD-1, or PD-L1). In a particular embodiment, the bispecific antibody molecule specifically binds to CNTN4 and any one of CTLA-4, PD-1, or PD-L1. In another embodiment, any combination of the above molecules may be prepared by a multispecific antibody molecule, e.g., a trispecific antibody comprising a first binding specificity for CNTN4, and a second and third binding specificity for at least two of CTLA-4, PD-1, or PD-L1. The multispecific antibody molecules of the present invention may be prepared using standard molecular biology techniques known to those skilled in the art (eg recombinant DNA and protein expression techniques).

[0064] In another embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention may form an antibody-drug conjugate (ADC). As used herein, the term "antibody-drug conjugate" or "ADC" refers to a compound of the formula M-[LD] n where M represents an antibody molecule, i.e., an anti-CNTN4 antibody or an antigen-binding fragment thereof of the present invention, L is any linker or linker unit, D is a suitable drug or prodrug, and n is an integer of about 1 to about 20. The drug contained in the ADC may be appropriately selected according to the therapeutic or diagnostic application, so long as the drug does not interfere with the specific binding of the antibody of the present invention. In one embodiment, the drug includes, but is not limited to, a cytotoxic drug (e.g., a chemotherapeutic agent), a prodrug-converting enzyme, a radioisotope or compound, or a toxin. The drug and linker that may be contained in the ADC and the method for preparing the ADC may follow methods known in the art.

[0065] In another embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention binds to a CNTN4 protein (e.g., a human or mouse CNTN4 protein) with an EC50 of 5 nM or less, preferably 3 nM or less, more preferably 2 nM or less, and even more preferably 1 nM or less, as determined by an ELISA assay.

[0066] As used herein, the term "EC50" refers to an in vitro or in vivo assay using an antibody and refers to the concentration of an antibody that induces 50% of the maximum response, i.e., a response halfway between the maximum response and the baseline.

[0067] 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.

[0068] Nucleic acids may be present in whole cells, in a cell lysate, or in a specifically purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art.

[0069] A nucleic acid of the invention may be, for example, DNA or RNA and may or may not contain intron sequences, hi a preferred embodiment, the nucleic acid is a cDNA molecule.

[0070] In one embodiment, the nucleic acid molecule of the invention encodes the light chain region, the heavy chain region, or both the light and heavy chain regions, preferably the light chain variable region, the heavy chain variable region, or both the light and heavy chain variable regions, of an anti-CNTN4 antibody or antigen-binding fragment thereof of the invention. In one embodiment, the nucleic acid molecule of the invention encodes a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 156, or encodes a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 159. Alternatively, the nucleic acid molecule of the invention encodes antibody A-01 or antibody L1H2.

[0071] Once a DNA fragment encoding the VL and / or VH region is obtained, such DNA fragment may be further manipulated by standard recombinant DNA techniques, e.g., to convert a variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, e.g., an antibody constant region such as hIgG1 Fc region (hFc) or hCκ region, or a flexible linker. As used herein, the term "operably linked" means that two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0072] The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (CH1, CH2 and CH3). The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region.

[0073] 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.

[0074] To produce an scFv gene, a DNA fragment encoding the VL and VH is operably linked to another fragment encoding a flexible linker, e.g., the amino acid sequence (Gly4-Ser)3, such that the VL and VH sequences may be expressed as a contiguous single-chain protein with the VL and VH regions linked by the flexible linker.

[0075] The nucleic acid sequences of the present invention, such as RNA or DNA, may be isolated from various sources, genetically modified, amplified, and / or recombinantly expressed. In addition to bacterial machinery, any recombinant expression machinery may be used, including, for example, yeast, insect, or mammalian machinery. Nucleic acid manipulations, such as, for example, subcloning into expression vectors, labeling probes, sequencing, and hybridization, may be performed as known in the art.

[0076] Accordingly, the invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0077] As used herein, the term "vector" refers to a DNA molecule capable of autonomous replication in prokaryotic and / or eukaryotic cells, and is used interchangeably with recombinant vector, cloning vector, or expression vector, which are commonly used as carriers for delivering genes or DNA fragments to cells, etc. A vector generally includes, but is not limited to, an origin of replication capable of replicating in prokaryotic and / or eukaryotic cells, a selectable marker gene capable of conferring resistance to a particular condition / substance, such as an antimicrobial degrading enzyme, a promoter capable of transcription of a gene in a eukaryotic or prokaryotic cell, and a translatable sequence.

[0078] One type of vector is a "plasmid", which refers to a circular double stranded 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).

[0079] The present invention also provides a host cell comprising the vector. The host cell includes, but is not limited to, cells of mammalian, plant, insect, fungal or bacterial origin. Bacterial cells include cells from Gram-positive or Gram-negative bacteria, such as Escherichia, such as E. coli, and some species of Pseudomonas. In the group of fungal cells, yeast cells are preferably used. Expression in yeast can be achieved by using yeast strains such as Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha, among others. Furthermore, insect cells, such as cells from Drosophila and Sf9, may be used as host cells.

[0080] Additionally, expression systems using mammalian cells, such as Chinese Hamster Ovary (CHO) cells, monkey COS cells, BHK cells, NSO cells or Bowes melanoma cells, may be used. Since the antibodies or antigen-binding fragments thereof of the invention are ultimately administered to humans, fully human expression systems may be particularly preferred. In this case, the host cells may be human cells, such as HeLa, 911, AT1080, A549, 293 and HEK293 cells.

[0081] Preparation of antibodies or antigen-binding fragments thereof The antibodies or antigen-binding fragments thereof of the present invention may be prepared according to conventional methods known in the art.

[0082] In one embodiment, an antibody of the invention, e.g., a monoclonal antibody, can be prepared by injecting a test subject (e.g., a mouse) with a CNTN4 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional characteristics, according to methods known in the art.

[0083] DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, and the vector is then transfected into host cells, such as E. coli cells, monkey COS cells, CHO cells, or myeloma cells, that do not otherwise produce immunoglobulin proteins, resulting in the synthesis of the monoclonal antibody in the recombinant host cells.

[0084] In another embodiment, the antibodies or antigen-binding fragments thereof of the present invention can be prepared using antibody display techniques, such as phage library techniques.

[0085] An antibody phage library is prepared by cloning human antibody heavy and light chain variable region genes fused to a phage surface protein (pIII) of a phagemid vector expressed in E. coli, then infecting M13 helper phage to prepare an antibody library in which antibody fragments (scFv or Fab) having various combinations of sequences of heavy and light chain variable regions are displayed on the surface of the phage. From this library, antibody fragments that bind to specific antigens are isolated using a panning method, the isolated antibody fragments are characterized, and then converted into a whole IgG form and expressed in large quantities in animal cells, thereby producing specific human monoclonal antibodies.

[0086] As used herein, the term "phagemid vector" refers to a plasmid DNA having a phage replication origin and usually has an antibiotic resistance gene as a selection marker. The phagemid vector used for phage display contains the gIII gene of M13 phage or a part thereof, and the ScFv gene is ligated to the 5' end of the gIII gene and expressed by the transformant.

[0087] A "helper phage" is a phage that supplies the necessary genetic information so that a phagemid can be packaged into a phage particle. Because the phagemid contains the gIII gene or a portion thereof of the phage, a host cell transformed with the phagemid (the transformant) is infected with a helper phage to supply the remaining phage genes. Helper phages include M13K07 or VCSM13, and most contain an antibiotic resistance gene, such as a kanamycin resistance gene, so that transformants infected with the helper phage can be selected. In addition, the packaging signal is incomplete, so that the phagemid genes are preferentially packaged into phage particles rather than the helper phage genes.

[0088] Use and Method The antibody or antigen-binding fragment thereof of the present invention restores immune responses inhibited by the binding of CNTN4 protein. It has been found that CNTN4 protein inhibits the proliferation of T cells, particularly CD4+ T cells and CD8+ T cells. The antibody or antigen-binding fragment thereof of the present invention may be used to treat diseases associated with immunosuppression by specifically binding to CNTN4 protein, thereby increasing T cell activity, particularly the activity of CD4+ T cells or CD8+ T cells.

[0089] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention increases T cell activity. Thus, the antibody or antigen-binding fragment thereof of the present invention may activate T cells, particularly CD4+ T cells or CD8+ T cells. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention may increase the proliferation of T cells inhibited by CNTN4. The antibody or antigen-binding fragment thereof of the present invention has excellent ability to neutralize CNTN4.

[0090] Thus, the present invention relates to inducing T cell activation by 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, the method comprising administering to a subject an effective amount of an anti-CNTN4 antibody or an antigen-binding fragment thereof. In another embodiment, the present invention provides a use of an anti-CNTN4 antibody or an antigen-binding fragment thereof for inducing or enhancing T cell activation. In another embodiment, the present invention provides a pharmaceutical composition for inducing or enhancing T cell activation, the composition comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0091] Thus, the present invention relates to the prevention, amelioration, or treatment of immunosuppression-related diseases by using anti-CNTN4 antibodies or antigen-binding fragments thereof.

[0092] In one embodiment, the invention provides a method for preventing, ameliorating, or treating an immunosuppression-related disease, the method comprising administering to a subject an effective amount of an anti-CNTN4 antibody, or antigen-binding fragment thereof.

[0093] In another embodiment, the present invention provides the use of an anti-CNTN4 antibody, or an antigen-binding fragment thereof, to prevent, ameliorate, or treat an immunosuppression-related disease.

[0094] In another embodiment, the present invention provides a pharmaceutical composition for preventing, ameliorating, or treating an immunosuppression-related disease, the composition comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof. As used herein, the term "subject" is intended to include both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, with non-human primates, sheep, dogs, cats, cows, and horses being preferred. A preferred subject is a human in need of immune response activation or enhancement.

[0095] Preferably, the anti-CNTN4 antibodies or antigen-binding fragments thereof of the present invention can block the binding of CNTN4 protein, thereby activating T cells and / or enhancing the immune response against cancer cells in cancer patients, and thus inhibiting the growth of cancer cells in vivo, and therefore can be effectively used to prevent, ameliorate, or treat cancer.

[0096] In another embodiment, the present invention provides the use of an anti-CNTN4 antibody, or an antigen-binding fragment thereof, for preventing or treating cancer.

[0097] In another embodiment, the invention provides use of an anti-CNTN4 antibody, or an antigen-binding fragment thereof, for the preparation of a medicament for preventing or treating cancer.

[0098] In another embodiment, the invention provides a method for preventing or treating cancer, comprising administering to a subject an effective amount of an anti-CNTN4 antibody, or antigen-binding fragment thereof.

[0099] In another embodiment, the invention provides a method for preventing or treating cancer, comprising administering to a subject an effective amount of an anti-CNTN4 antibody or antigen-binding fragment thereof, and an effective amount of an additional anti-cancer agent.

[0100] The additional anti-cancer agent may be an immune checkpoint inhibitor or a chemotherapeutic agent, and the immune checkpoint inhibitor may be 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.

[0101] An effective amount of an anti-CNTN4 antibody or antigen-binding fragment thereof and an effective amount of an additional anti-cancer agent may be administered simultaneously to a subject in a single formulation or may be administered simultaneously or sequentially to a subject in separate formulations.

[0102] Preferred cancers whose growth can be inhibited using the antibody of the present invention include cancers that are usually responsive to immunotherapy. For example, cancers in the present invention include, but are not limited to, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, colon cancer, rectal cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). In addition, the subject to be treated in the present invention includes refractory or recurrent malignant tumors whose growth can be inhibited using the antibody of the present invention.

[0103] Examples of other cancers that may be treated using the methods of the present invention include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, cancer of the fallopian tubes, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, acute bone marrow tumors, and / or bronchitis. chronic or acute leukemia including myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; solid tumors of childhood; lymphocytic lymphoma; bladder cancer; kidney cancer; ureteral cancer; renal pelvis cancer; central nervous system (CNS) neoplasms; primary CNS lymphoma; tumor angiogenesis; spinal axis tumors; brain stem glioma; pituitary adenoma; Kaposi's sarcoma; epidermoid carcinoma; squamous cell carcinoma; T-cell lymphoma; environmentally induced cancers including asbestos induced cancers; and combinations of these cancers.

[0104] In another embodiment, the cancer may be a CNTN4-expressing cancer.

[0105] In another embodiment, the cancer may be refractory or resistant to conventional immune checkpoint inhibitors (e.g., resistant to a PD-1 pathway inhibitor, a PD-L1 pathway inhibitor, or a CTLA-4 pathway inhibitor).

[0106] The antibodies or antigen-binding fragments of the invention may be used alone or in combination with other anti-cancer therapies, which may include, for example, standard cancer therapies (e.g., chemotherapy, radiation therapy, or surgery), or other anti-cancer agents, such as cytotoxic, cytostatic, antiangiogenic, or antimetabolite agents, tumor-targeting agents, immunostimulatory or immunomodulatory agents, or antibodies conjugated to cytotoxic, cytostatic, or other toxic agents, immune checkpoint inhibitors, and the like.

[0107] Preferably, the antibody or antigen-binding fragment of the present invention may be used in combination with other anti-cancer agents, such as immune checkpoint inhibitors, chemotherapeutic agents, or radiation therapy. The immune checkpoint inhibitor may be, for example, an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab, nivolumab), or an anti-PD-L1 antibody (e.g., atezolizumab, avelumab, durvalumab). Chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens and selective estrogen receptor modulators (SERMs), antiprogesterone agents, estrogen receptor downregulators (ERDs), estrogen receptor antagonists, luteinizing hormone releasing hormone agonists, antiandrogens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, antisense oligonucleotides that inhibit the expression of genes associated with 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, taxoids such as paclitaxel, albumin-bound paclitaxel, and doxetaxel, and the like. Other cancer therapeutic agents include probiotics that exhibit anti-cancer effects, such as Lactococcus lactis GEN3013 strain (KCTC13426BP), Lactococcus lactis GEN3033 strain (KCTC13684BP), Bifidobacterium bifidum MG731 strain (KCTC13452BP), and the like.

[0108] When the antibody or antigen-binding fragment of the present invention is used in combination with other anticancer drugs, they may be administered separately, or in the form of a combination product in which multiple active ingredients are present in a single pharmaceutical preparation.When they are administered as separate preparations, the two preparations may be administered sequentially or simultaneously.In simultaneous administration, they are administered to a patient together.In sequential administration, they may be administered at a time interval that is not long, for example, the preparations may be administered to a patient within a period of 12 hours or less, or 6 hours or less.

[0109] In one embodiment, the present invention provides a method for preventing, ameliorating, or treating an immunosuppression-related disease, such as 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 drug. The embodiment includes not only simultaneously administering to a patient in need thereof a single composition containing an anti-CNTN4 antibody or antigen-binding fragment thereof together with an additional anti-cancer drug, but also simultaneously or sequentially administering to a patient in need thereof compositions each containing an anti-CNTN4 antibody or antigen-binding fragment thereof and an additional anti-cancer drug separately.

[0110] In another embodiment, the present invention provides the use of an anti-CNTN4 antibody or antigen-binding fragment thereof for use in combination with an additional anti-cancer agent to prevent, ameliorate, or treat an immunosuppression-related disease, such as cancer.

[0111] In another embodiment, the present invention provides a pharmaceutical composition or combination for preventing, ameliorating, or treating an immunosuppression-related disease, such as cancer, the composition or combination comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof, and an additional anti-cancer drug. The pharmaceutical composition or combination herein comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof, and an additional anti-cancer drug includes not only the case where the two components are physically present together in the form of a single formulation, but also the case where the two components are administered simultaneously or sequentially as separate formulations, and the two drugs can be supplied separately or together as a single kit. Thus, the present invention provides a kit for preventing, ameliorating, or treating an immunosuppression-related disease, such as cancer, the kit comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof, and an additional anti-cancer drug.

[0112] The additional anti-cancer agent may preferably be an immune checkpoint inhibitor, more preferably an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab and nivolumab), or an anti-PD-L1 antibody (e.g., atezolizumab, avelumab, and durvalumab).

[0113] Other preferred additional anti-cancer agents may include chemotherapeutic agents such as gemcitabine, vinorelbine, etoposide (VP-16), platinum analogues such as cisplatin or carboplatin, taxoids such as paclitaxel, albumin-bound paclitaxel, or doxetaxel.

[0114] Another preferred additional anti-cancer therapy for use in conjunction with the antibody or antigen-binding fragment thereof of the present invention may include radiation therapy.

[0115] The present invention also provides a method for detecting the presence of CNTN4 protein or measuring the amount of anti-CNTN4 antibody in a sample by using an anti-CNTN4 antibody or an antigen-binding fragment thereof as an active ingredient. The method includes contacting the antibody or an antigen-binding fragment thereof with a sample and a control sample under conditions in which the antibody or the antigen-binding fragment thereof can bind to the CNTN4 protein to form a complex. Whether or not a complex is formed is then detected, and a difference in the degree of complex formation between the sample compared to the control sample is evidence that the human circulating antigen is present in the sample (e.g., blood).

[0116] Thus, the present invention provides a composition for diagnosing cancer, the composition comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0117] Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof. The composition may contain inactive ingredients, i.e., pharma- ceutically acceptable excipients (see Handbook of Pharmaceutical Excipients, etc.). Therapeutic and diagnostic formulation compositions may be prepared, for example, in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension, by mixing the formulation with a physiologically acceptable carrier, excipient, or stabilizer.

[0118] Suitable routes of administration include parenteral administration, e.g., intramuscular, intravenous, or subcutaneous administration. Administration of antibodies used in the pharmaceutical compositions of the invention or used to practice the methods of the invention may be carried out by a variety of conventional methods, e.g., topical application, or intradermal, subcutaneous, intraperitoneal, parenteral, intraarterial, or intravenous injection. In one embodiment, the antibodies of the invention are administered intravenously or subcutaneously.

[0119] Hereinafter, the present invention will be described in more detail with reference to examples. It should be clear to those skilled in the art that these examples are only intended to describe the present invention in more detail, and the scope of the present invention is not limited by these examples according to the subject matter of the present invention. EXAMPLES

[0120] Hereinafter, the present invention will be described in more detail with reference to examples. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0121] Example 1: CNTN4 specific binding scFv antibody selection 1.1 scFv antibody library preparation and antibody selection (1) Preparation of chicken scFv antibody library Fifteen chickens were divided into three groups of five chickens each, and the first group was inoculated with mouse CNTN4, the second group with human CNTN4, and the third group with mouse CNTN4 and human CNTN4 alternately, for a total of four times to produce antibodies. The amount of antigen inoculated was 20 μg / chicken for each group in the first inoculation, 10 μg / chicken after three weeks in the second inoculation, 10 μg / chicken after two weeks in the third inoculation, and 10 μg / chicken after two weeks in the fourth inoculation, and each group was immunized with those amounts. Before antigen inoculation at each time point, blood was collected with a syringe and whether or not antibodies were produced was confirmed by ELISA, which binds serum to the antigen. After the third inoculation, a total of four chickens that showed reactivity to both human CNTN4 and mouse CNTN4 in ELISA were selected, specifically, the third chicken in the first group (hereinafter referred to as 1-3), 1-4, 3-2, and 3-5 were selected. After sacrifice, blood, spleen, bone marrow, and bursa of Fabricius were extracted, and total RNA was extracted. The major RNA, 28S rRNA, was fully identified, and cDNA was synthesized by using SuperScript™ IV First Strand Synthesis System (Invitrogen, #18091050) using the extracted RNA as a template for PCR of the immune antibody library. The scFv fragments in which the light and heavy chain variable regions of the antibody were linked by a linker were obtained by PCR, and cloned into pComb3X, a phagemid vector, by using the restriction enzyme SfiI for phage display, and electroporated into E. coli ER2738 with F' fimbriae that can be infected by M13 helper phage. The complexity of the scFv library is confirmed as shown in Table 2.

[0122] [Table 2]

[0123] (2) scFv antibody selection For each of chicken numbers 1-3, 1-4, 3-2, and 3-5 in Table 2, biopanning was performed by pooling and phage amplification. More specifically, a total of four conditions (A to D) were set and performed in the following combinations: a condition in which phage was mixed with protein A and 50 μL of immunoglobulin complex and incubated at 37° C. for 1 hour to remove non-specific binding (Fc-bound substances) (pre-removal); a condition in which PBS (pH 7.4) or 50 mM sodium acetate (pH 5.5) was used as the binding buffer and incubation was performed at 37° C. for 4 hours; and a condition in which competitive elution with antigen was performed in the elution step, washed with 0.5% Tween 20 in PBS, and then 100 μL of anti-CNTN4 chicken serum was added and washed for additional elution (Table 3). For each condition, 1 to 3 rounds or up to 5 rounds were performed while controlling the stringency of the phage binding conditions with respect to the antigen (20 μg to 1 μg of antigen).

[0124] [Table 3]

[0125] To select candidate antibodies, phage rescue was performed on the output colonies of the third or fifth round, for example, a single colony was found, placed in a medium, cultured until OD600nm was 0.7-0.8, infected with helper phage, then cultured overnight, centrifuged to obtain phage culture medium, and then phage ELISA was performed. For phage ELISA, the plate was first coated with antigen (mouse CNTN4 or human CNTN4) overnight at 4°C, and then blocked with 3% BSA in PBS for 1 hour. Then, 50 μL of phage was added to the plate coated with the antigen, followed by incubation for 2 hours. After incubating the plate with the secondary antibody anti-HA-HRP for 1 hour, ABTS was added thereto to develop the color and measure the absorbance at 405 nm. A total of 950 colonies from chicken no. 1-4 and 3-2 libraries and a total of 380 colonies from chicken no. 1-3 and 3-5 libraries were subjected to ELISA screening, 138 and 306 colonies were selected as positive clones binding to the antigen CNTN4, respectively, and 26 and 35 unique scFv sequences were selected by sequencing to identify the scFv sequences.

[0126] 1.2 Expression and purification of scFv For the functional evaluation of the scFv antibodies selected in Example 1.1, cloning was performed in the form of constant human kappa (hCk)-tagged scFv in the form of fusion protein or human IgG4 (S228P) and transient transfection was performed on Expi293F cells. The secreted scFv-hCk or IgG was bound to kappa selection in the culture medium, then purified by pH elution, exchanged into PBS buffer by dialysis, and protein was quantified to obtain scFv-hCk or IgG.

[0127] As a result, a total of 25 scFv-hCk and a total of 27 IgG4 were confirmed to be expressed, and the amino acid sequences of the light chain and heavy chain variable regions of each of the expressed scFvs are shown in Figures 1a to 1d and 2a to 2d and Tables 4 and 5 (the sequences shown in Figures 1a to 1d and 2a to 2d, indicated by solid and dashed lines, represent the CDR sequences of each region).

[0128] [Table 4] TIFF0007642096000010.tif218149 TIFF0007642096000011.tif218149 TIFF0007642096000012.tif218149 TIFF0007642096000013.tif58149

[0129] [Table 5] TIFF0007642096000015.tif218149 TIFF0007642096000016.tif218149 TIFF0007642096000017.tif218149 TIFF0007642096000018.tif130149

[0130] 1.3 Confirmation of CNTN4 binding ability of selected scFv antibodies (1) Confirmation of the binding ability of scFv to antigenic proteins by ELISA ELISA tests were performed on a total of 25 scFv-hCk and a total of 27 IgG whose expression was confirmed in Example 1.2. ELISA plates were coated with 200 ng of antigen (human CNTN4-rabbit Fc or mouse CNTN4-rabbit Fc). 200 ng of negative control for tag protein (human antigen-rabbit Fc) was used. For antibody binding, anti-CNTN4 scFv-hCk was serially diluted 1 / 10x from 1 μM to 0.001 nM, and anti-CNTN4 IgG was serially diluted 1 / 10x from 10 nM to 0.00001 nM, thereby binding to the antigen. Anti-human kappa-HRP or anti-human IgG Fc-HRP was used as the secondary antibody for antibody detection. Color development was performed using ABTS, a color reagent, and the absorbance at 405 nm was measured.

[0131] As shown in Tables 6 and 7, the results showed that the reactivity against most antigens was good, and there were many clones that showed similar reactivity against both human and mouse antigens.

[0132] [Table 6]

[0133] [Table 7]

[0134] (3) Confirmation of the binding ability of scFv to antigens expressed on the cell surface by FACS In 293FT cells, human CNTN4 antigen was expressed on the cell membrane by transient transfection, and anti-CNTN4 scFv-hCk or anti-CNTN4 IgG was bound thereto at a concentration of 40 μg / mL. A dot shift was confirmed by detecting the antigen-bound anti-CNTN4 with anti-hCk-PE or anti-hFc-PE by using FACS.

[0135] As shown in Tables 8 and 9, the results show that hCk-type scFv clones 2, 4, 99, 129, 130, 131, 132, 134, 135, 137, and 138, which exhibit binding ability to human CNTN4, belong to the top group (over 15.0%) with large dot shifts, and all of the IgG-type anti-CNTN4 clones exhibit excellent binding ability of 75% or more.

[0136] [Table 8]

[0137] [Table 9]

[0138] Example 2: Preparation of anti-CNTN4 chicken-human chimeric IgG4 antibody and confirmation of its binding ability 2.1 Preparation of anti-CNTN4 chicken-human chimeric IgG4-S228P, A-01 For the generation of antibodies in the form of human chimeric IgG, S129, whose binding ability was confirmed in Example 1, was selected, and the light and heavy chain regions of S129 were cloned into a bicistronic expression vector. The DNA was transiently transfected into Expi293F cells, which were cultured to a viability of 50% to allow IgG expression, and the culture medium was bound to a kappa selection resin (kappa region capture) and eluted, and the IgG was purified for the first time. Then, anti-CNTN4 129 chimeric IgG4-S228P (hereinafter referred to as "A-01") with a purity of 72.4% by SEC-HPLC was prepared by binding to Mabselect (Fc region capture) to remove light chain impurities, eluting, and purifying the IgG for the second time.

[0139] 2.2 Confirmation of the binding ability of A-01 to antigenic proteins by ELISA ELISA test was performed on A-01 prepared in Example 2.1. ELISA plate was coated with 200ng of antigen (human CNTN4-10xHis or mouse CNTN4-10xHis). For antibody binding, A-01 was serially diluted from 1μM up to 0.0001nM, thereby binding to the antigen. Anti-human Fc-HRP was used as the secondary antibody for antibody detection. Color development was performed using ABTS, a color reagent, and the absorbance at 405nm was measured. The resulting binding capacity is shown in Table 10.

[0140] [Table 10]

[0141] 2.3 Confirmation of the binding ability of A-01 to antigenic proteins by surface plasmon resonance (SPR) An SPR test was carried out on A-01 prepared in Example 2.1.

[0142] Specifically, in order to bind anti-human IgG Fc (capture antibody) using amine coupling, a CM5 chip was placed, a running buffer was poured, the chip was activated, and then the capture antibody was bound to the chip. Dextrin to which the capture antibody did not bind was inactivated using ethanolamine, and then regeneration was performed using 3M magnesium chloride to remove analytes other than the capture antibody. A-01 prepared at a concentration of 0.05 μg / mL was poured at a speed of 10 μL / min for 30 seconds for binding. Then, human CNTN4 (400 nM to 0.78 nM) prepared by serial dilution was poured as an antigen at a speed of 30 μL / min for 3 minutes for binding, and dissociation was performed for 5 minutes. Next, in order to remove all analytes bound to the capture antibody, a regeneration solution (3M magnesium chloride) was poured at a speed of 20 μL / min for 30 seconds to perform regeneration. For each concentration of antigen, binding of A-01, binding of antigen, dissociation, and regeneration were repeated in sequence. The results are shown in Table 11.

[0143] [Table 11]

[0144] This confirmed that the antibody of the present invention binds to human CNTN4 protein with high affinity.

[0145] Example 3: Preparation of anti-CNTN4 humanized antibody and confirmation of binding ability 3.1 Preparation of anti-CNTN4 humanized antibody A humanized antibody was prepared using A-01.

[0146] Specifically, sequences (VL1 and VH1) in which the framework regions of the original sequences (VLA and VHA) of the variable regions of A-01 were completely changed to human framework regions were combined with sequences (VL2 and VH2) in which a portion of the original sequence of A-01 was left in VL1 and VH1 (Table 12 and Figure 3). When A-01 was compared with a fully humanized antibody (VL1 and VH1), A-01 showed about 78% humanization. When the fully humanized antibody (VL1 and VH1) was compared with an antibody having a combination of VL2 and VH2 with some back-mutation sequences, at least 96% of the sequences were identical. To prepare a total of eight humanized antibodies (LAH1, LAH2, L1HA, L1H1, L1H2, L2HA, L2H1, and L2H2) in the form of IgG4, excluding A-01 (LAHA), DNA was transiently transfected into Expi293F cells and cultured, but antibodies having VH1 in which the heavy chain variable region was completely human (LAH1, L1H1, and L2H1) were not expressed. Then, antibodies having VH2, LAH2, L1H2, and L2H2, were purified to obtain 4 mg, 11 mg, and 12 mg, respectively.

[0147] [Table 12]

[0148] 3.2 Confirmation of the binding ability of humanized antibodies to antigenic proteins by ELISA The LAH2, L1H2, and L2H2 antibodies prepared in Example 3.1 were subjected to ELISA testing with A-01. The ELISA plate was coated with 200 ng of antigen (human CNTN4-10xHis or mouse CNTN4-10xHis). For antibody binding, the antibody was serially diluted from a maximum of 1 μM to 0.0001 nM, thereby binding to the antigen. Anti-human Fc-HRP was used as the secondary antibody for antibody detection. Color development was performed using ABTS, a color reagent, and the absorbance at 405 nm was measured.

[0149] The resulting binding capacities are shown in Table 13. The most humanized L1H2 was identified to have a binding capacity with CNTN4 protein within 3-fold difference compared to the A-01 origin.

[0150] [Table 13]

[0151] 3.3 Confirmation of the binding ability of humanized antibodies to antigenic proteins by surface plasmon resonance (SPR) Among the antibodies prepared in Example 3.1, an SPR test was performed on L1H2, which is the most humanized at about 98%. More specifically, in order to bind anti-human IgG Fc (capture antibody) using amine coupling, a CM5 chip was placed on the chip, a running buffer was poured, the chip was activated, and then the capture antibody was bound to the chip. Dextrin to which the capture antibody did not bind was inactivated using ethanolamine, and then regeneration was performed using 3M magnesium chloride to remove analytes other than the capture antibody. L1H2 prepared at a concentration of 0.05 μg / mL was poured at a speed of 10 μL / min for 30 seconds for binding. Then, human CNTN4 (400 nM to 0.78 nM) prepared by serial dilution was poured as an antigen at a speed of 30 μL / min for 3 minutes to bind, and dissociation was performed for 5 minutes. Next, in order to remove all analytes bound to the capture antibody, a regeneration solution (3M magnesium chloride) was poured at a speed of 20 μL / min for 30 seconds to perform regeneration. For each concentration of antigen, L1H2 binding, antigen binding, dissociation, and regeneration were repeated in sequence.

[0152] The results are shown in Table 14. [Table 14]

[0153] This may indicate that the A-01 antibody has sufficiently high binding ability to the CNTN4 protein even though the A-01 antibody is 98% humanized.

[0154] Example 4: Testing the CNTN4 neutralizing ability of A-01 and L1H2 antibodies using human T cells A final 50 mL of a-CD3 antibody at a concentration of 4 mg / mL and CNTN4 recombinant protein at a concentration of 150 nM were prepared, placed in a 96-well plate, and incubated at 37° C. for approximately 16 hours.

[0155] 50 mL of blood (contained in blood collection tubes treated with sodium heparin) and PBS were mixed in a 1:1 ratio per donor, 15 mL of Ficoll was pre-contained in a 50 mL tube, and 30 mL of diluted blood was carefully placed and stacked on top of the Ficoll so that the Ficoll layer and blood layer were separated. Centrifugation (acceleration and deceleration speeds set to 0) was performed at 1,800 rpm for 25 minutes at 20°C, and the supernatant was removed so that the white buffy layer did not rise. The buffy layer was transferred to a new 50 mL tube, the tube was filled to 50 mL with PBS, and centrifuged at 1,800 rpm for 5 minutes at 4°C. 10 mL of 1X RBC lysis buffer was added, thoroughly dissolved, and left at room temperature for 5 minutes. After filling up to 50 mL with PBS, centrifugation was performed at 1,800 rpm for 5 minutes at 4°C, the supernatant was removed, the pellet was dissolved in PBS, and the cells (PBMC) were then observed under a microscope and the cell number was calculated.

[0156] 2 x 10 PBMC cells obtained from the above experiment 81,000 μL of MACS buffer was added to each cell, and then the cells were resuspended. The solution was divided into two equal parts, one in a CD4 tube and the other in a CD8 tube. 1 × 10 PBMCs from each group were 8 The antibody cocktail was resuspended in 50 μL per cell and stored at room temperature for 10 minutes. 8 100 μL of anti-biotin microbeads were resuspended per cell and stored at room temperature for 10 minutes. Meanwhile, the MACS magnetic field was disinfected and placed in a clean bench, and two LS columns (for CD4 and CD8) were installed. 3 mL of MACS buffer was poured into each column, and the buffer that had passed through each column was discarded. After the reaction was completed, the cells of each group were loaded onto two LS columns placed on the magnetic field. When the cell solution stopped falling, 3 mL of MACS buffer was poured into each column. After that, the cells in the solution that had fallen from each column were observed under a microscope, and the cell number was calculated.

[0157] 18 μL of DMSO was added to 50 μg of CFSE and pipetted to make a concentration of 5 mM for use in the cells. 1 mL of MTM medium was added to the CD4 and CD8 cells obtained in the above experiment, CFSE was added to reach a final concentration of 5 μM, suspended, and then stored in a 37°C water bath for 10 minutes. The solution was centrifuged at 1,800 rpm and 4°C for 5 minutes, and the supernatant was removed. 1 mL of MTM medium was then added thereto, resuspended, and then centrifuged again at 1,800 rpm and 4°C for 5 minutes, and the supernatant was removed. Finally, the cells were resuspended in MTM medium and plated at 2 × 10 per well. 5 The cells / 200 μL was reached and used for the experiments.

[0158] The incubated plates were then washed twice with 200 μL of PBS, and 50 mL of final volumes of 1.5 mM and 3 mM A-01 and L1H2, respectively, were added to the CNTN4-treated wells and placed in the 96-well plate. Incubation was carried out at 37° C. for approximately 4 hours.

[0159] After that, the incubated plate was washed twice with 200 mL of PBS, and 200 mL of T cells were added per well, and then stored in a 5% CO2 incubator at 37°C for 3 days. After 3 days, the T cells were obtained and transferred into FACS tubes, and the degree of differentiated cells stained with CFSE was observed using a FACS machine.

[0160] The results are shown in FIG.

[0161] As shown in FIG. 4, the CNTN4 recombinant protein inhibited the expression of CD4 + T cells and CD8 + It was confirmed that A-01 and L1H2 inhibited the proliferation of T cells. It was observed that the proliferation of T cells inhibited by CNTN4 increased with the concentration of A-01 and L1H2 administered. This indicates that A-01 and L1H2 of the present invention exhibit a remarkably excellent ability to neutralize CNTN4.

[0162] From this, it was confirmed that the CNTN4 antibody of the present invention effectively neutralizes CNTN4 and inhibits the T cell inhibitory function of CNTN4.

Claims

1. A light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 70; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 88; a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 116; A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:58; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:89; and Heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 115 An anti-CNTN4 antibody or an antigen-binding fragment thereof comprising:

2. A light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; and A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 An anti-CNTN4 antibody or an antigen-binding fragment thereof comprising:

3. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 156; and A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 159 An anti-CNTN4 antibody or an antigen-binding fragment thereof comprising:

4. The anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which specifically binds to human or mouse CNTN4 protein.

5. Dissociation constant (Kd) 1 x 10 －７ The anti-CNTN4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which binds to human CNTN4 protein with an M or less.

6. The anti-CNTN4 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, which increases T cell activity.

7. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 6, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

8. The anti-CNTN4 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody scFv, (Fab')2 fragment, bispecific antibody (dAb), and linear antibody.

9. The anti-CNTN4 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody is a chimeric antibody or a humanized antibody.

10. The anti-CNTN4 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody is a multispecific antibody.

11. The anti-CNTN4 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody is conjugated to a drug.

12. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

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

14. A pharmaceutical composition for preventing or treating cancer, comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3.

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

16. A pharmaceutical composition for preventing or treating cancer, comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, and a further anti-cancer agent.

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

18. The pharmaceutical composition of claim 17, 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.

19. 17. The pharmaceutical composition of claim 16, wherein the anti-CNTN4 antibody or antigen-binding fragment thereof and the additional anti-cancer agent are administered simultaneously in a single formulation or administered simultaneously or sequentially in separate formulations.

20. A pharmaceutical composition for preventing or treating cancer, comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, used in combination with a further anti-cancer therapy.

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

22. Use of an anti-CNTN4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3 for the preparation of a medicament for preventing or treating cancer.

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