Antibodies specific to CNTN4 and their use

Abstract

The present invention relates to anti-CNTN4 antibodies or antigen-binding fragments thereof and their use to activate T cells that upregulate cellular immune responses, e.g., to treat cancer.

Description

Technical Field

[0001] The present invention relates to an anti-CNTN4 specific antibody or an antigen-binding fragment thereof, a therapeutic composition containing the same, and its use for activating T cells, for example, for treating cancer.

Background Art

[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 initial stage of cancer development. Then, when the balance between the infinitely proliferating cancer cells and the immune cells that want to attack the cancer cells is disrupted, the cancer cells start to substantially proliferate. As the cancer cells grow further, they interfere with the immune system in the body, and some cancer cells avoid immunity by using the immune checkpoint of immune cells. When immune checkpoint inhibitors suppress the immune checkpoint, the power of immune cells increases, thereby killing 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, including CTLA-4, PD-1, PD-L1 inhibitors, etc. Representative drugs currently on the market include ipilimumab as a CTLA-4 monoclonal antibody (product name: YERVOY (registered trademark)), nivolumab as a PD-1 monoclonal antibody (product name: OPDIVO (registered trademark)) and pembrolizumab (product name: KEYTRUDA (registered trademark)), and atezolizumab as a PD-L1 monoclonal antibody (product name: TECENTRIQ (registered trademark)) and durvalumab (product name: IMFINZI (registered trademark)).

[0004] However, there are still cancers that are not treated by existing immune checkpoint inhibitors, and thus the development of new anti-cancer treatments is necessary.

[0005] Korean Patent Application Publication No. 10-2019-0116930 discloses that CNTN4 can be used as a new target for cancer treatment using the human immune system. The above Korean patent application discloses that CNTN4 inhibits T cell activity.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] 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, the present invention intends to provide an antibody or an antigen-binding fragment thereof that binds to the CNTN4 protein to neutralize the immune evasion mechanism of CNTN4.

[0008] The present invention also intends to provide a composition for preventing or treating diseases such as cancer caused by a decrease in T cell activity by using an antibody or an antigen-binding fragment thereof, to block the immune evasion mechanism of CNTN4, and to activate T cells.

[0009] The present invention also intends to provide a composition for analyzing or detecting the CNTN4 protein by using an antibody or an antigen-binding fragment thereof.

Means for Solving the Problems

[0010] The present invention provides an anti-CNTN4 antibody or an antigen-binding fragment thereof that specifically binds to the CNTN4 protein. The antibody or antigen-binding fragment specifically binds to the CNTN4 protein, such as human or mouse CNTN4 protein, and neutralizes the immune evasion mechanism of CNTN4. Therefore, 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, inhibited by CNTN4.

[0011] In one embodiment, the present invention 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 to provide an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0012] In one embodiment, the antibody of the present invention may be a monoclonal antibody.

[0013] In another embodiment, the present invention provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof, and a recombinant expression vector containing the nucleic acid molecule.

[0014] The present invention also provides a composition for preventing or treating cancer, which contains an antibody or an antigen-binding fragment thereof as an active ingredient. The pharmaceutical composition may be used in combination with additional anti-cancer agents such as immune checkpoint inhibitors or chemotherapeutic agents, or may be used in combination with radiotherapy.

[0015] The present invention also provides a composition for analyzing or detecting the CNTN4 protein, which contains an antibody or an antigen-binding fragment thereof.

Advantages of the Invention

[0016] The inventors of the present invention confirmed that the CNTN4 protein is an immune checkpoint protein, and the degree of immune escape due to the inhibition of T cell activity is stronger than that of PD-L1, which is known as a conventional immune checkpoint protein.

[0017] Furthermore, the novel anti-CNTN4 antibody or its antigen-binding fragment of the present invention can be effectively used to activate T cells by blocking the immune escape mechanism of CNTN4, and thus to prevent or treat diseases caused by the decrease in T cell activity, particularly cancer. Therefore, when the anti-CNTN4 antibody of the present invention is used, it may exert an excellent anti-cancer effect on cancers for which the therapeutic effect cannot be achieved by existing immunotherapies.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0019] Anti-CNTN4 antibody or antigen-binding fragment thereof The present invention provides an anti-CNTN4 antibody or an antigen-binding fragment thereof that specifically binds to the CNTN4 protein.

[0020] In one embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2.

[0021] As used herein, the statement that a light chain or heavy chain variable region contains a specific amino acid sequence means that the light chain or heavy chain variable region contains, has, or consists of the entire amino acid sequence.

[0022] In one example, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2. 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: 1 and a heavy chain variable region consisting essentially of the amino acid sequence of SEQ ID NO: 2.

[0023] As 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: 1 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2.

[0024] As used herein, the term "comprising", "comprise" or a modified form thereof refers to an open meaning. As an example, an antibody or antigen-binding fragment thereof comprising the recited amino acid sequence may or may not necessarily include additional amino acid sequences not recited.

[0025] As used herein, the term "consisting essentially of" or a modified phrase 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. As an example, an antibody or antigen-binding fragment thereof consisting essentially of the recited amino acid sequence may include substitutions of one or more amino acid residues that do not substantially affect the characteristics of the antibody or its fragment.

[0026] As used herein, the term "consisting of" or a modified phrase thereof refers to the case where each component described herein does not admit any element not described or recited in its description of the embodiment.

[0027] As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, protein, etc. through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only complete polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof, and fusion proteins containing antibody fragments, and any other modified forms of immunoglobulin molecules containing antigen recognition sites.

[0028] Antibodies include five classes of immunoglobulins (Ig) M, IgD, IgG, IgA, and IgE, which contain heavy chains made from the heavy chain constant region genes μ, δ, γ, α, and ε, respectively.

[0029] 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. The heavy chain constant region includes CH1, H (hinge), CH2, and CH3 domains. Each domain consists of two β-sheets, between which an intramolecular disulfide bond is linked.

[0030] An antibody containing a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 is referred to herein as "antibody hAb-1".

[0031] In one embodiment, the antibody of the present invention may be a monoclonal antibody. In another embodiment, the antibody of the present invention may be a chimeric antibody, a humanized antibody, or a human antibody.

[0032] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0033] As used herein, the term "humanized antibody" refers to an antibody in which the CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted into human framework sequences. The framework sequences may be further engineered, for example, by back mutations.

[0034] As used herein, the term "human antibody" refers to an antibody in which both the framework and CDR regions contain variable regions derived from human immunoglobulin sequences. The constant region of the antibody is also derived from human immunoglobulin sequences.

[0035] As used herein, the term "antigen-binding fragment" or "antibody fragment" refers to antigen-binding fragments and analogs of antibodies that typically include 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 antibody scFv, single-domain antibodies, bispecific antibodies (dAb), or linear antibodies.

[0036] In particular, the Fab fragment refers to a monovalent fragment composed of the VL, VH, CL, and CH1 domains.

[0037] The Fab’ fragment differs from the Fab fragment in that several residues have been added from the antibody hinge region to the carboxyl terminus of the CH1 domain that contains at least one cysteine.

[0038] Fab’-SH refers to a Fab’ in which the cysteine residue of the constant domain has a free thiol group.

[0039] The F(ab’)2 antibody fragment is produced as a pair of Fab’ fragments via the hinge cysteine between the Fab’ fragments.

[0040] Fv is the smallest antibody fragment that contains a complete antigen-recognition site and -binding site. This fragment consists of a dimer of one heavy-chain variable region and one light-chain variable region in a tight non-covalent association. From the folding of these two regions, the antibody is provided with amino acid residues for antigen binding, and six hypervariable loops (three loops each from the heavy and light chains) that confer antigen-binding specificity are generated. However, even a single variable region has the ability to recognize and bind to an antigen, but with a lower affinity than the entire binding site.

[0041] A single-chain antibody scFv is an antibody fragment that contains VH and VL antibody domains linked in a single polypeptide chain. Preferably, the scFv polypeptide further includes 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 an scFv antibody fragment, an antigen-binding fragment scFv, an scFv antibody, an antibody scFv, or simply scFv.

[0042] Bispecific antibodies are prepared by constructing scFv fragments using short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain rather than intra-chain pairing of the V domains is achieved, resulting in a small antibody fragment that is a bivalent fragment, i.e., a fragment having two antigen-binding sites. A bispecific bispecific antibody is a heterodimer consisting of two "crossover" scFv fragments in which the VH and VL domains of two antibodies are present on different polypeptide chains.

[0043] The anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention can specifically bind to the CNTN4 protein, preferably the human or mouse CNTN4 protein.

[0044] As used herein, the term "specifically binds to" or "specific for" refers 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 biomolecules. For example, an antibody that specifically binds to a particular target (e.g., an epitope) is an antibody that binds to this target with higher affinity, binding ability, more readily, and / or for a longer period than it binds to other targets.

[0045] As used herein, the term "specifically binds to human CNTN4 protein" refers to an antibody that binds to human CNTN4 protein with a dissociation constant (Kd) of 1×10 -7 M or less, or preferably 5×10 -8 M or less. Accordingly, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention can bind to human CNTN4 protein with a dissociation constant (Kd) of 1×10 -7 M or less, preferably 5×10 -8 M or less.

[0046] As used herein, the term "Kd" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, and the constant has the unit 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 (SPR), preferably using a biosensor system such as a Biacore® system.

[0047] In one embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention is monospecific and specifically binds to a single epitope, i.e., the CNTN4 protein.

[0048] 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. The multispecific antibody molecule includes a plurality of variable regions, and each variable region has a binding specificity for a different epitope. In one embodiment, the first variable region of the bispecific antibody molecule has a first binding specificity for a first epitope, such as the CNTN4 protein, and the second variable region has a second binding specificity for a second epitope, such as a target protein other than the CNTN4 protein (including, but not limited to, CTLA-4, PD-1, or PD-L1). In a specific 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 trispecific antibody including a first binding specificity for CNTN4 and second and third binding specificities for at least two of CTLA-4, PD-1, or PD-L1. The multispecific antibody molecule of the present invention may be prepared using standard molecular biological techniques known to those skilled in the art (e.g., recombinant DNA and protein expression techniques).

[0049] 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" can be represented by the formula M-[L-D] n wherein M represents an antibody molecule, i.e., the anti-CNTN4 antibody or 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 from about 1 to about 20. The drug included in the ADC can be appropriately selected according to therapeutic or diagnostic uses as 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 agent (e.g., a chemotherapeutic agent), a prodrug-converting enzyme, a radioisotope or compound, or a toxin. The drugs and linkers that can be included in the ADC and the method for preparing the ADC may follow methods known in the art.

[0050] The anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention exhibits excellent competitive binding ability to CNTN-4 protein even in the presence of the CNTN-4 receptor. Therefore, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention can bind to CNTN-4 protein substantially equally specifically even when the CNTN-4 receptor is present in the body.

[0051] In another embodiment, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention binds to CNTN4 protein (e.g., human or mouse CNTN4 protein) with an EC50 of 5 nM or less, preferably 3 nM or less, more preferably 2 nM, and even more preferably 1 nM or less as determined by an ELISA assay.

[0052] As used herein, the term "EC50" is a term related to in vitro or in vivo assays using an antibody and refers to the concentration of the antibody that induces a 50% of the maximum response, i.e., an intermediate response between the maximum response and the baseline.

[0053] Nucleic acid molecule and vector Another aspect of the present invention relates to a nucleic acid molecule encoding the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention.

[0054] The nucleic acid can exist in whole cells, in cell lysates, or in a particularly purified or substantially pure form. The nucleic acid is "isolated" or "substantially pure" when separated and purified from other cell components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques including alkaline / SDS treatment, CsCl band formation, column chromatography, agarose gel electrophoresis, and other techniques well known in the art.

[0055] The nucleic acid of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0056] In one embodiment, the nucleic acid molecule of the present invention encodes the light chain region, the heavy chain region, or both the light chain and heavy chain regions of the anti-CNTN4 antibody of the present invention or an antigen-binding fragment thereof, preferably encodes the light chain variable region, the heavy chain variable region, or both the light chain and heavy chain variable regions. In one embodiment, the nucleic acid molecule of the present invention encodes a light chain variable region containing SEQ ID NO: 1 and / or encodes a heavy chain variable region containing SEQ ID NO: 2, or encodes the antibody hAb-1.

[0057] When a DNA fragment encoding the VL and / or VH region is obtained, such a DNA fragment may be further manipulated by standard recombinant DNA techniques, for example, the variable region gene may be converted into a full-length antibody chain gene, a Fab fragment gene or a scFv gene. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another protein, for example, an antibody constant region, or another DNA fragment encoding a flexible linker. As used herein, the term "operably linked" means that two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0058] Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably binding the DNA encoding VH to another DNA molecule encoding the heavy chain constant regions (CH1, CH2 and CH3). The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region.

[0059] For the Fab fragment heavy chain gene, the DNA encoding VH may be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0060] To generate the scFv gene, DNA fragments encoding VL and VH are operably linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser)3, such that the VL and VH sequences can be expressed as adjacent single-chain proteins in which the VL and VH regions are linked by the flexible linker.

[0061] 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 systems, any recombinant expression system, including, for example, yeast, insect, or mammalian systems, can be used. For example, manipulations of nucleic acids, such as subcloning into an expression vector, labeling probes, sequencing, and hybridization, may be performed as known in the art.

[0062] Accordingly, the present invention provides a recombinant expression vector containing a nucleic acid molecule.

[0063] As used herein, the term "vector" refers to a DNA molecule capable of self-replication in prokaryotic and / or eukaryotic cells and is used interchangeably with recombinant vector, cloning vector, or expression vector, which is commonly used as a carrier for delivering a gene or DNA fragment into a cell or the like. 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 specific conditions / substances such as antibacterial degrading enzymes, a promoter capable of transcribing a gene in eukaryotic or prokaryotic cells, and a translatable sequence.

[0064] One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, to which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).

[0065] Preparation of antibody or antigen-binding fragment thereof The antibody or antigen-binding fragment thereof of the present invention may be prepared according to conventionally known methods.

[0066] In one embodiment, to express the antibody or antigen-binding fragment thereof, a recombinant expression vector containing nucleic acid molecules encoding the partial or full-length light and heavy chains is transfected into a host cell. As methods of transfection, for example, but not limited to, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc., techniques generally used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells may be used.

[0067] The host cell for the expression of the antibody or antigen-binding fragment thereof of the present invention may be a prokaryotic cell or a eukaryotic cell, preferably a eukaryotic cell, particularly a mammalian cell.

[0068] Examples of mammalian host cells include, but are not limited to, human fetal kidney cells (e.g., 293 cells or 293 cells subcloned for growth in suspension culture), Expi293F™ cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), dog kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC5 cells, FS4 cells, human hepatocellular carcinoma cell lines (e.g., Hep G2), and myeloma cells (e.g., NS0 and Sp2 / 0 cells).

[0069] The transformed host cells can be cultured in a suitable medium to produce the polypeptide of the heavy chain, light chain, or antigen-binding fragment thereof of the antibody according to the present invention from the recombinant expression vector introduced into the host cells. The composition of the medium for culturing the host cells, the culture conditions, the culture time, etc. can be appropriately selected according to the methods commonly used in the art. For example, commercially available media such as Ham's F10 (Sigma-Aldrich Co., St. Louis, MO), Minimum Essential Medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich Co.) can be used, but are not limited thereto. Hormones, growth factors, salts, buffers, nucleotides, antibiotics, trace elements, etc. may be further added to the medium as necessary.

[0070] Antibodies or their antigen-binding fragments produced in host cells can be obtained by processes such as purification. The method of obtaining can be appropriately selected considering the characteristics of the polypeptide of the antibody or its antigen-binding fragments produced in the host cell, the characteristics of the host cell, the expression method, or whether the polypeptide is targeted. For example, antibodies or their antigen-binding fragments secreted into the culture medium can be recovered by obtaining the medium in which the host cells were cultured and centrifuging the medium to remove impurities. Further, the obtained antibody can be further subjected to steps of removing further impurities and concentrating the antibody by methods such as chromatography, filtration through a filter or the like, or dialysis, and a two-step purification method, for example, a first purification by protein A affinity chromatography and then a second purification by cation exchange chromatography can be used.

[0071] Use and method The antibody or its antigen-binding fragment of the present invention restores an immune response inhibited by the binding of the CNTN4 protein. The CNTN4 protein inhibits the proliferation of T cells, particularly CD4+ T cells and CD8+ T cells. The antibody or its antigen-binding fragment of the present invention may be used to treat diseases related to immunosuppression by specifically binding to the CNTN4 protein and thereby increasing T cell activity, particularly the activity of CD4+ T cells or CD8+ T cells.

[0072] In one embodiment, the antibody or its antigen-binding fragment of the present invention increases T cell activity.

[0073] As used herein, the term "T cell" means a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on its cell surface. Helper T cells (also called TH cells or CD4+ T cells) and subtypes (including TH1, TH2, TH3, TH17, TH9, and TFH cells), cytotoxic T cells (also called TC cells, CD8+ T cells, cytotoxic T lymphocytes, T killer cells, killer T cells), memory T cells and subtypes (central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells)), regulatory T cells (also called Treg cells or suppressor T cells) and subtypes (including CD4+FOXP3+ Treg cells, CD4+FOXP3- Treg cells, Tr1 cells, Th3 cells, and Treg17 cells), natural killer T cells (also known as NKT cells), mucosa-associated invariant T cells (MAIT), and gamma delta T cells (γδT cells) (including Vγ9 / Vδ2 T cells) are included, but not limited to these, and there are several subsets of T cells. In the present invention, preferably, the T cell is a CD4+ T cell or a CD8+ T cell.

[0074] As used herein, the term "T cell activation" means a cellular process in which mature T cells expressing antigen-specific T cell receptors on their surface recognize their cognate antigens, and in response, the mature T cells enter the cell cycle, secrete cytokines or lytic enzymes, and initiate the execution of the effector functions of T cells.

[0075] Accordingly, the antibody or antigen-binding fragment thereof of the present invention can 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 can increase the proliferation of T cells inhibited by CNTN4. The antibody or antigen-binding fragment thereof of the present invention has an excellent ability to neutralize CNTN4.

[0076] Accordingly, the present invention relates to the induction of 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 the use of an anti-CNTN4 antibody or an antigen-binding fragment thereof for inducing or enhancing T cell activation.

[0077] 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.

[0078] In another embodiment, the present invention relates to the prevention, remission, or treatment of immunosuppression-related diseases by using an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0079] In one embodiment, the present invention provides a method for preventing, remitting, or treating an immunosuppression-related disease, the method comprising administering to a subject an effective amount of an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0080] In another embodiment, the present invention provides the use of an anti-CNTN4 antibody or an antigen-binding fragment thereof for preventing, remitting, or treating an immunosuppression-related disease.

[0081] In another embodiment, the present invention provides a pharmaceutical composition for the prevention, amelioration, or treatment of an immunosuppression-related disease, the pharmaceutical composition comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0082] As used herein, the term "subject" is intended to include both humans and non-human animals. Non-human animals include mammals and non-mammals, including all vertebrates, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, with non-human primates, sheep, dogs, cats, cows, and horses being preferred. Preferred subjects are humans in need of activation or enhancement of an immune response.

[0083] Preferably, the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention blocks the binding of the CNTN4 protein, thereby activating T cells in cancer patients and / or enhancing the immune response against cancer cells, and thus can inhibit the growth of cancer cells in vivo, and therefore can be effectively used to prevent, remit, or treat cancer.

[0084] Preferred cancers whose growth can be inhibited when the antibody of the present invention is used include cancers that are normally responsive to immunotherapy. For example, cancers in the present invention include, but are not limited to, gastric cancer, pancreatic cancer, endometrial cancer, liver cancer, gallbladder cancer, prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), melanoma (e.g., metastatic malignant melanoma or cutaneous and ocular malignant melanoma), kidney cancer (e.g., clear cell carcinoma), breast cancer (e.g., invasive breast cancer, non-invasive breast cancer), colorectal cancer, rectal cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). Specific examples of cancers may include gastric cancer, pancreatic cancer, endometrial cancer, liver cancer, gallbladder cancer, prostate cancer, or melanoma. Furthermore, subjects to be treated in the present invention include refractory or recurrent malignant tumors whose growth can be inhibited when the antibody of the present invention is used.

[0085] Examples of other cancers that can be treated using the method of the present invention include bone cancer, skin cancer, cancers of the head and neck, uterine cancer, ovarian cancer, rectal cancer, cancers of the anal region, testicular cancer, uterine cancer, cancer of the fallopian tubes, cervical cancer, vaginal cancer, cancer of the vulva, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic or acute leukemia including chronic lymphocytic leukemia, solid tumors in childhood, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal cord tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T cell lymphoma, or combinations of these cancers.

[0086] The antitumor activity of the anti-CNTN4 antibody or antigen-binding fragment thereof of the present invention can be tested in vivo. For this purpose, syngeneic tumor models known in the art, such as the CT26 tumor model for colorectal cancer, the EMT6 tumor model for breast cancer, or the LLC-1 tumor model for lung cancer, can be used.

[0087] In another embodiment, the cancer may be a cancer that expresses CNTN4.

[0088] In another embodiment, the cancer may be refractory or resistant to conventional immune checkpoint inhibitors (e.g., resistant to PD-1 pathway inhibitors, PD-1 pathway inhibitors, or CTLA-4 pathway inhibitors).

[0089] The antibody or antigen-binding fragment of the present invention may be used alone or in combination with other anti-cancer therapies. Other anti-cancer therapies include, for example, standard cancer therapies (e.g., chemotherapy, radiation therapy, or surgery), or other anti-cancer agents, such as cytotoxic, cytostatic, anti-angiogenic or metabolic antagonist drugs, tumor-targeting agents, immune stimulants or immunomodulators, or antibodies conjugated to cytotoxic, cytostatic, or other toxic drugs, immune checkpoint inhibitors, and the like.

[0090] 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. Immune checkpoint inhibitors may be, for example, anti-CTLA-4 antibodies (e.g., ipilimumab), anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab), or anti-PD-L1 antibodies (e.g., atezolizumab, avelumab, durvalumab). Chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic / antineoplastic antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens and selective estrogen receptor modulators (SERMs), antiprogestins, 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 associated with abnormal cell proliferation or tumor growth. Specific examples of the 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 docetaxel. 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.

[0091] When the antibody or antigen-binding fragment of the present invention is used in combination with other anti-cancer agents, they may be administered separately or in the form of a combination product in which a plurality of active ingredients are present in a single pharmaceutical formulation. When they are administered as separate formulations, the two formulations may be administered sequentially or simultaneously. In simultaneous administration, they are administered together to the patient. In sequential administration, they may be administered at short time intervals, for example, these formulations may be administered to the patient within a period of 12 hours or less, or 6 hours or less.

[0092] In one embodiment, the present invention provides a method for preventing, remitting or treating an immunosuppression-related disease, such as cancer, the method comprising administering to a subject an effective amount of an anti-CNTN4 antibody or an antigen-binding fragment thereof in combination with a further anti-cancer agent.

[0093] Embodiments include not only simultaneously administering to a patient in need thereof a single composition containing an anti-CNTN4 antibody or antigen-binding fragment together with a further anti-cancer agent, but also simultaneously or sequentially administering to a patient in need thereof compositions containing the anti-CNTN4 antibody or antigen-binding fragment and a further anti-cancer agent separately.

[0094] In another embodiment, the present invention provides the use of an anti-CNTN4 antibody or an antigen-binding fragment thereof for use in combination with a further anti-cancer agent for preventing, remitting or treating an immunosuppression-related disease, such as cancer.

[0095] In another embodiment, the present invention provides a pharmaceutical composition or combination for preventing, remitting, or treating immunosuppression-related diseases, such as cancer, the composition or combination comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof, and a further anti-cancer agent. The pharmaceutical composition or combination herein comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof, and a further anti-cancer agent includes not only the case where the two components are physically 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. Accordingly, the present invention provides a kit for preventing, remitting, or treating immunosuppression-related diseases, such as cancer, the kit comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof and a further anti-cancer agent.

[0096] The further 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 and nivolumab), or an anti-PD-L1 antibody (e.g., atezolizumab, avelumab, and durvalumab).

[0097] Another preferred further anti-cancer agent may include a chemotherapeutic agent, such as gemcitabine, vinorelbine, etoposide (VP-16), a platinum analog, such as cisplatin or carboplatin, a taxoid, such as paclitaxel, albumin-bound paclitaxel, or docetaxel.

[0098] Another preferred further anti-cancer therapy used together with the antibody or an antigen-binding fragment thereof of the present invention may include radiotherapy.

[0099] The present invention also provides a method for detecting the presence of CNTN4 protein in a sample or measuring the amount of anti-CNTN4 antibody by using an anti-CNTN4 antibody or an antigen-binding fragment thereof as an active ingredient. The method includes contacting the antibody or its antigen-binding fragment with a sample and a control sample under conditions where the antibody or its antigen-binding fragment can bind to the CNTN4 protein to form a complex. Then, it is detected whether a complex is formed, and the difference in the degree of complex formation between the sample compared with the control sample is evidence that the antigen in human blood is present in the sample (e.g., blood).

[0100] Accordingly, the present invention provides a composition for diagnosing cancer, the composition comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof.

[0101] Pharmaceutical composition The present invention provides a pharmaceutical composition comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof. The composition may contain an inert ingredient, i.e., a pharmaceutically acceptable excipient (see, e.g., Handbook of Pharmaceutical Excipients, etc.). The composition of the therapeutic and diagnostic formulations may be prepared, for example, in the form of a lyophilized powder, slurry, aqueous solution or suspension by mixing the formulation with a physiologically acceptable carrier, excipient, or stabilizer.

[0102] Suitable routes of administration include parenteral administration, such as intramuscular, intravenous, or subcutaneous administration. Administration of the antibody used in the pharmaceutical composition of the present invention or used to carry out the method of the present invention may be carried out by various conventional methods, such as topical application, or intradermal, subcutaneous, intraperitoneal, parenteral, intraarterial, or intravenous injection. In one embodiment, the antibody of the present invention is administered intravenously or subcutaneously.

[0103] Hereinafter, the present invention will be described in more detail in relation to examples. It should be apparent to those skilled in the art that these examples are only intended to describe the present invention in more detail and that the scope of the present invention is not limited by these examples according to the subject matter of the present invention.

Examples

[0104] Example 1: Confirmation of the ability to inhibit human T cell activity of CNTN4 In this example, the degree to which CNTN4 inhibits the activity of human T cells was confirmed as compared with PD-L1, which is known as an immune checkpoint protein.

[0105] α-CD3 antibody at a concentration of 4 μg / mL and human PD-L1 and human CNTN4 recombinant proteins at concentrations of 50, 100, 150, and 200 nM, respectively, were prepared to a final volume of 50 μL and placed in a 96-well plate. The 96-well plate was incubated at 37 °C for about 16 hours while taking care not to generate bubbles at the bottom of the 96-well plate.

[0106] 50 mL of blood per donor (contained in a heparin sodium-treated blood collection tube) and PBS were mixed at a ratio of 1:1, 15 mL of Ficoll was pre-contained in a 50-mL tube, and 30 mL of the diluted blood was carefully placed on top of the Ficoll so that the Ficoll layer and the blood layer were separated and stacked. Centrifugation (acceleration and deceleration rates were 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 and dissolved thoroughly, and left at room temperature for 5 minutes. After filling 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 then the cells (PBMC) were observed under a microscope and the cell count was calculated.

[0107] 2×10 of PBMC cells obtained in the above experiment8 1,000 μL of MACS buffer was added per cell and then resuspended. The solution was divided into two equal parts, one placed in a CD4 tube and the other in a CD8 tube. 1×10 8 50 μL of the antibody cocktail was resuspended per cell and stored at room temperature for 10 minutes. 1×10 8 100 μL of anti-biotin microbeads was resuspended per cell and stored at room temperature for 10 minutes. On the other hand, 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 passed through each column, and then the buffer passed through each column was discarded. After the reaction was completed, the cells of each group were loaded onto the two LS columns placed in the magnetic field. When the cell solution stopped dropping, 3 mL of MACS buffer was passed through each column. Then, the cells in the solution dropped from each column were observed under a microscope, and the cell count was calculated.

[0108] 18 μL of DMSO was added to 50 μg of CFSE, measured with a pipette to prepare a concentration of 5 mM, and the cells were used. 1 mL of MTM medium was added to the CD4 cells and CD8 cells obtained in the above experiment, CFSE was added to reach a final concentration of 5 μM, resuspended, and then stored in a 37°C water bath for 10 minutes. The solution was centrifuged at 1,800 rpm at 4°C for 5 minutes, and the supernatant was removed. Then, 1 mL of MTM medium was added thereto, resuspended, and then centrifuged again at 1,800 rpm at 4°C for 5 minutes, and the supernatant was removed. Finally, the cells were resuspended in MTM medium to reach 2×10 5 cells / 200 μL and used for the experiment.

[0109] The incubated plates were washed twice with 200 μL of PBS, 200 μL of T cells were added per well, and then 5% CO 2 was stored in an incubator at 37°C for 3 days. After 3 days, T cells were obtained, transferred to a FACS tube, and the degree of differentiated cells stained with CFSE was observed using a FACS instrument. The results are shown in Figure 1.

[0110] As shown in Fig. 1, the PD-L1 and CNTN4 recombinant proteins inhibited the proliferation of CD4 + T cells and CD8 + T cell proliferation was confirmed. This result was commonly observed in two donors. In particular, when comparing the treatment with 200 nM CNTN4 and the treatment with 200 nM PD-L1, the proliferation of T cells in the CNTN4 treatment group was significantly inhibited, indicating that the ability of CNTN4 to inhibit T cell activity may be stronger than that of PD-L1, a conventional immune checkpoint protein.

[0111] This result suggests that when the function of the CNTN4 protein is suppressed, its immune evasion mechanism is blocked, indicating that the CNTN4 recombinant protein can be used as a therapeutic agent for diseases.

[0112] Example 2: Generation of monoclonal antibody against CNTN4 The light and heavy chain regions in Fig. 2 and Table 1 were cloned into an expression vector. The DNA was transiently transfected into Expi293F™ cells and cultured until 50% viability or until day 6 to allow IgG expression. The culture medium was bound to a Protein A affinity column (MabSelect SuRe LX, Cytiva) and eluted to perform the first purification of IgG. Next, to remove aggregates and high molecular weight impurities, the first purified IgG was bound to a cation exchange column (Capto SP ImpRes, Cytiva), and IgG was eluted using a concentration gradient of acetate buffer (pH 5.0) and sodium chloride to perform the second purification. As a result, anti-CNTN4 IgG4-S228P (hereinafter referred to as "hAb-1") with a purity of 95.9% was prepared by SEC-HPLC.

[0113]

Table 1

[0114] Example 3: Confirmation of the binding ability of monoclonal antibody hAb-1 3.1 Confirmation of the binding ability of monoclonal antibody hAb-1 to the antigen protein by ELISA An ELISA test was performed on the monoclonal antibody hAb-1 prepared in Example 2. The ELISA plate was coated with 20 nM of the antigen (human CNTN4-10xHis or mouse CNTN4-10xHis). For antibody binding, the antibody hAb-1 was serially diluted from a maximum of 1 μM to 0.0001 nM and thereby bound to the antigen. The HRP-conjugated anti-human kappa antibody was used as the secondary antibody for antibody detection. Color development was carried out using ABTS, a chromogenic reagent, and the absorbance at 405 nm was measured. The resulting binding ability results are shown in Figure 3 and Table 2.

[0115] [Table 2]

[0116] 3.2 Confirmation of the binding ability of hAb-1 to the antigen protein by surface plasmon resonance (SPR) The SPR test was performed with the monoclonal antibody hAb-1 prepared in Example 2. More specifically, to bind the anti-human IgG Fc (capture antibody) using amine coupling, the CM5 chip was placed, the running buffer was flowed, the chip was activated, and then the capture antibody was bound to the chip. The dextran to which the capture antibody did not bind was inactivated using ethanolamine, and then regeneration was carried out using 3M magnesium chloride to remove analytes other than the capture antibody. hAb-1 prepared at a concentration of 0.05 μg / mL was flowed at a rate of 10 μL / min for 30 seconds for binding. Then, as the antigen, human CNTN4 (400 nM to 0.78 nM) prepared by serial dilution was flowed at a rate of 30 μL / min for 3 minutes for binding, and dissociation was carried out for 5 minutes.

[0117] Next, to remove all analytes bound to the capture antibody, a regeneration solution (3M magnesium chloride) was flowed at a rate of 20 μL / min for 30 seconds to perform regeneration. For each antigen concentration, the binding of hAb-1, the binding of the antigen, dissociation, and regeneration were repeated in order. The results are shown in Table 3.

[0118]

Table 3

[0119] From this, it was confirmed that the antibody of the present invention binds to human CNTN4 protein with high affinity.

[0120] Example 4: Confirmation of whether monoclonal antibody hAb-1 binds competitively to the CNTN4 receptor for CNTN4 An ELISA plate was coated with 20 nM of the antigen human CNTN4-10xHis. For antibody binding, a mixture of antibody hAb-1 diluted to a final concentration of 0 to 1,000 nM and human CNTN4 receptor (amyloid precursor protein, APP) diluted to 0 or 500 nM was allowed to bind to the antigen. HRP-conjugated anti-human kappa antibody was used as the secondary antibody for antibody detection. Color development was performed using ABTS, a chromogenic reagent, and the absorbance at 405 nm was measured.

[0121] The results are shown in Figure 4 and Table 4. Even when the antibody of the present invention was incubated with the CNTN-4 receptor, the degree of binding to the CNTN-4 protein hardly changed.

[0122] This indicates that the antibody of the present invention binds competitively to the CNTN-4 protein in the presence of the CNTN-4 receptor and has excellent binding ability.

[0123]

Table 4

[0124] Therefore, the antibody of the present invention can specifically bind to the CNTN-4 protein even when the CNTN-4 receptor is present in the body, and thus can effectively inhibit the immunosuppressive ability of the CNTN-4 protein.

[0125] Example 5: Test on the CNTN4 neutralizing ability of hAb-1 antibody using human T cells Similar to the experiment regarding the ability of CNTN4 to inhibit T cell activity in Example 1, an α-CD3 antibody at a concentration of 4 μg / mL and a CNTN4 recombinant protein at a concentration of 150 nM were prepared to a final volume of 50 μL, placed in a 96-well plate, and incubated at 37°C for approximately 16 hours. Next, the incubated plate was washed twice with 200 μL of PBS, and hAb-1 antibodies at 0.375, 0.75, 1.5, 3, and 6 μM were each prepared to a final volume of 50 μL in the wells treated with CNTN4 and placed in a 96-well plate. Incubation was carried out at 37°C for approximately 4 hours. The subsequent process was carried out in the same manner as the experiment regarding the ability of CNTN4 to inhibit T cell activity in Example 1.

[0126] As shown in Figure 5, it was confirmed that the CNTN4 recombinant protein inhibited the proliferation of CD4 + T cells and CD8 + T cells. It was observed that the proliferation of T cells inhibited by CNTN4 increased depending on the concentration of the treated hAb-1. This indicates that the antibody hAb-1 of the present invention exhibits an extremely excellent ability to neutralize CNTN4.

[0127] 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.

[0128] Example 6: Confirmation of anti-tumor efficiency (in vivo) in a CT26 syngeneic mouse model of colorectal cancer In this example, it was intended to confirm the anti-tumor efficiency of the anti-CNTN4 antibody of the present invention in tumor-bearing mice, specifically in a CT26 syngeneic mouse model of colorectal cancer.

[0129] 1×10 6 CT26 cells were inoculated into the right flank of BALB / c mice. After cell transplantation, when the tumor size was 75 mm 3 ≤ tumor volume ≤ 150 mm 3Mice were selected and randomly divided into 4 groups of 8 mice each (day 0), and 10, 20, and 40 mg / kg of antibody hAb-1 and 20.4 mg / kg of hIgG4 as a control were intraperitoneally (i.p.) administered to the groups on days 0, 3, 6, and 9, respectively. After the start of administration, the tumor size was measured twice a week, and the difference in size from the control based on the tumor size on day 12 was expressed as TGI (%). Statistical analysis of the data was performed using Graphpad PRISM 8.0, statistical processing was performed using two-way ANOVA, and analysis was performed by a statistical two-sided Student's t-test ( * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. group 1 (hIgG4)).

[0130] The average values of the data values of 8 mice in each experimental group were calculated, and the results are shown in Table 5 and Figure 6. Administration of 10 mg / kg of antibody hAb-1 showed an excellent tumor shrinkage effect. As the dose increased to 20 mg / kg and 40 mg / kg, the tumor shrinkage effect also increased.

[0131]

Table 5

[0132] From this, it was confirmed that the anti-CNTN4 antibody of the present invention exhibits an effect of inhibiting the growth of colorectal cancer tumor cells.

[0133] Example 7: Confirmation of the effectiveness of increased T cell activity (in vivo) in a CT26 syngeneic mouse model of colorectal cancer In this example, it was intended to confirm the effect of the anti-CNTN4 antibody of the present invention on the T cell level in tumor-bearing mice, specifically in a CT26 syngeneic mouse model of colorectal cancer.

[0134] Mice inoculated with CT26 colorectal cancer cells according to the method described in Example 6 were divided into 4 groups of 5 mice per group, and 10, 20, and 40 mg / kg of antibody hAb-1, and 20.4 mg / kg of hIgG4 as a control were intraperitoneally administered to the groups on days 0, 3, and 6, respectively (administered 3 times every 3 days; Q3DX3). On day 7, the spleen was collected to obtain splenocytes. After the spleen was crushed and centrifuged, the cells with erythrocytes removed were used. To detect the indicators related to intracellular immune factors, the isolated cells were divided into two groups, and one group was treated with an immune cell activator to enable the detection of the indicator substance. The isolated cells were incubated with antibodies against CD3+, CD4+, CD45+, and IFNγ, anti-CD3-PerCP / Cy5.5, anti-CD4-PE / Cy7, anti-CD45-APC / Cy7, and anti-IFNγ-FITC, respectively, the cells were washed with buffer, and the fluorescence intensity was measured. In the case of CD3, CD4, and CD45, surface staining was performed for the markers expressed on the cell surface, and in the case of IFNγ, intracellular staining was performed using an intracellular staining method for the markers expressed inside the cells. During staining, the isotype of each fluorescent substance was used to prevent misinterpretation. After the staining was completed, the cells were washed with buffer to remove the remaining fluorescent substances, and then the expression of intracellular and extracellular markers was analyzed using a flow cytometer (FACS CantoII). The statistical analysis of the data was confirmed by the Graphpad PRISM 8.0 program, statistical processing was performed using one-way analysis of variance, and statistical significance was verified by Dunnett's multiple comparison test. ( * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

[0135] The results are shown in Figs. 7 - 10.

[0136] Figures 7 to 9 show, respectively, the percentage of CD4+ T cells relative to CD45+ cells found in the spleen (i.e., CD3+CD4+ / CD45+), the percentage of CD8+ T cells relative to CD45+ cells (i.e., CD3+CD8+ / CD45+), and the percentage of cells secreting IFNγ among CD8+ T cells (i.e., IFNγ / CD8+). From this, it was confirmed that when the antibody hAb-1 was used, the levels of CD4+ T cells and CD8+ T cells were approximately equal to or increased compared to the control (hlgG4), and in particular, the secretion of cytokines by CD8+ T cells was significantly increased compared to the control.

[0137] Figure 10 shows the degree of reduction in tumor volume used in the analysis. In all cases where the antibody hAb-1 was used, the tumor volume was reduced compared to the control.

[0138] From this, it was confirmed that the anti-CNTN4 antibody of the present invention exhibits an effect of inhibiting the growth of tumor cells by increasing T cell activity, particularly CD8+ T cell activity, and increasing cytokine secretion.

[0139] Example 8: Confirmation of antitumor efficiency (in vivo) in an EMT6 syngeneic mouse model of breast cancer In this example, it was intended to confirm the antitumor efficiency of the anti-CNTN4 antibody of the present invention in tumor-bearing mice, specifically, in an EMT6 syngeneic mouse model of breast cancer, which is known as an immunologically cold tumor.

[0140] The experiment was carried out in the same manner as in Example 6, except that 1×10 6 EMT6 breast cancer cells were inoculated into the right flank of BALB / c mice.

[0141] The average values of the data of 8 mice in each experimental group were calculated, and the results are shown in Table 6 and Figure 11. Administration of the antibody hAb-1 at 10 mg / kg showed an excellent tumor reduction effect, and as the dose increased to 20 mg / kg and 40 mg / kg, the tumor reduction effect also increased.

[0142]

Table 6

[0143] From this, it was confirmed that the anti-CNTN4 antibody of the present invention exhibits the effect of inhibiting the proliferation of breast cancer tumor cells.

[0144] Example 9: Confirmation of the effectiveness of increasing T cell activity (in vivo) in an EMT6 syngeneic mouse model of breast cancer In this example, it was intended to confirm the effect of the anti-CNTN4 antibody of the present invention on T cell levels in tumor-bearing mice, specifically in an EMT6 syngeneic mouse model of breast cancer.

[0145] The experiment was carried out in the same manner as in Example 7, except that mice were inoculated with 1×10 6 individual EMT6 breast cancer cells and 4 mice were used per experimental group. Anti-CD4-APC / Cy7, anti-CD8-PerCP / Cy5.5, and anti-IFNγ-FITC antibodies were used for the markers CD4+, CD8+, and IFNγ+ used for detection, respectively.

[0146] The results are shown in FIGS. 12 and 13.

[0147] FIGS. 12 and 13 show the percentages of cells secreting IFNγ among CD4+ T cells and CD8+ T cells found in the spleen (i.e., IFNγ / CD4+ and IFNγ / CD8+), respectively. From this, it was confirmed that when the antibody hAb-1 was used, the secretion of cytokines by CD4+ T cells and CD8+ T cells was significantly increased compared to the control (hlgG4).

[0148] From this, it was confirmed that the anti-CNTN4 antibody of the present invention exhibits the effect of inhibiting the proliferation of tumor cells by increasing T cell activity and increasing cytokine secretion.

[0149] Example 10: Confirmation of the effectiveness of increased T cell activity (in vivo) in an LLC-1 syngeneic mouse model of lung cancer In this example, it was intended to confirm the effect of the anti-CNTN4 antibody of the present invention on T cell levels in tumor-bearing mice, specifically in an LLC-1 syngeneic mouse model of lung cancer.

[0150] The experiment was carried out in the same manner as in Example 7, except that mice were inoculated with 1×10 6 LLC-1 lung cancer cells and 3 to 4 mice were used per experimental group.

[0151] Anti-CD3-PerCP / Cy5 and anti-IFNγ-FITC antibodies were used for CD3+ and IFNγ+ used as detectable markers, respectively.

[0152] The results are shown in Figures 14 and 15.

[0153] Figure 14 shows the percentage of cells secreting IFNγ among T cells dispersed in the spleen (i.e., IFNγ / CD3+). From this, it was confirmed that when the antibody hAb-1 was used, the secretion of cytokines by T cells was significantly increased compared to the control (hlgG4).

[0154] Figure 15 shows the degree of reduction in tumor volume used for the analysis. In all cases where the antibody hAb-1 was used, the tumor volume was reduced compared to the control.

[0155] From this, it was confirmed that the anti-CNTN4 antibody of the present invention exhibits the effect of inhibiting the growth of tumor cells by increasing T cell activity, particularly CD8+ T cell activity, and increasing cytokine secretion.

Claims

1. 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 An anti-CNTN4 antibody or an antigen-binding fragment thereof comprising:

2. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 1 , which specifically binds to human or mouse CNTN4 protein.

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

4. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 1, which increases T cell activity.

5. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 4, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

6. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 1, which increases cytokine secretion.

7. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 6, wherein the cytokine is IL-2, TNFα, IFNγ, or a combination thereof.

8. 2. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 1, 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 claim 1 , wherein the antibody is a monoclonal antibody.

10. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody is a chimeric antibody or a humanized antibody.

11. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody is a multispecific antibody.

12. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody is conjugated to a drug.

13. The anti-CNTN4 antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody is IgG1, IgG2, IgG3, or IgG4.

14. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

15. A recombinant expression vector comprising the nucleic acid molecule of claim 14.

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 13 as an active ingredient.

17. The pharmaceutical composition of claim 16, wherein the cancer is a CNTN4-expressing cancer.

18. 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 13, and a further anti-cancer agent.

19. 19. The pharmaceutical composition of claim 18, wherein the additional anti-cancer agent is an immune checkpoint inhibitor or a chemotherapeutic agent.

20. 20. The pharmaceutical composition of claim 19, 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.

21. 20. The pharmaceutical composition of claim 18, 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.

22. 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 13, used in combination with a further anti-cancer therapy.

23. 23. The pharmaceutical composition of claim 22, 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.

24. A composition for diagnosing cancer, comprising an anti-CNTN4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 13.

25. A method for detecting CNTN4 protein in a sample by using an anti-CNTN4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 13 in vitro as an active ingredient.

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