B7H6 variants with enhanced binding to NKp30

Abstract

The present invention relates to B7-H6 variants with enhanced binding affinity to NKp30. The B7-H6 variant of the present invention has significantly increased binding affinity to NKp30, which is an activating receptor for natural killer cells (NK cells), compared to the wild type, thereby increasing the activation of natural killer cells. Additionally, because it is significantly smaller than an antibody, it facilitates penetration into the tumor microenvironment and is easy to produce. Therefore, it can be usefully used for the treatment of cancer or infectious diseases, either alone or in combination with various immunotherapies.

Description

Technology Field

[0001] The present invention relates to B7-H6 variants with improved binding strength to NKp30. Background Technology

[0002] Drugs for cancer treatment are broadly classified into small molecule and large molecule drugs; compared to small molecule drugs, which lack specificity and carry relatively significant side effects, large molecule drugs, which possess specificity, are gaining prominence as therapeutic agents. Cancer cells express immune checkpoint proteins on their surface—which are utilized by normal cells to suppress immune cell activation—to evade the killing mechanisms of immune cells; consequently, research on immune checkpoint inhibitor proteins has been actively conducted recently as a method for treating cancer. Although the use of immune checkpoint inhibitors is increasing explosively due to reports of superior therapeutic effects compared to conventional anticancer drugs across various cancer types and fewer side effects in patients who respond to them, more than half of patients still do not respond to clinically approved immune checkpoint inhibitors, and resistance is being observed in some early responders, leading to cancer progression after treatment. Since the causes of resistance to immune checkpoint inhibitors vary depending on individual patient tumor immunological characteristics, new biomarkers capable of selecting patients suitable for treatment are needed for effective treatment and cost reduction. Additionally, effective immunostimulatory therapies are urgently needed, as many preclinical and clinical studies have reported that combination therapies using immunostimulatory drugs improve patient response rates. However, antibodies have the disadvantage of being macromolecular proteins with a molecular weight of 150,000, making it difficult for them to penetrate into tumor tissue. Consequently, for more effective treatment, there is a growing need for protein therapies that target new immune checkpoint proteins. These therapies are much smaller than antibodies, facilitate easy penetration into tumor tissue, and overcome low response rates.

[0003] Immune cells express immune checkpoint proteins on their surface that are used to inhibit and activate themselves. Among various immune checkpoint receptors, it has been confirmed that B7-H6 binds to NKp30, an activating receptor expressed on the surface of NK cells (Natural Killer Cells), thereby activating the NK cells. NK cells are innate immune cells that detect and eliminate cancer cells and virus-infected cells even without specific antigens, regulate immune and inflammatory responses by inducing the activation of other immune cells, and suppress the proliferation, recurrence, and metastasis of cancer cells. However, there has been a problem in utilizing these cells because the wild-type B7-H6 that activates them binds to NKp30 with a very low affinity (equilibrium dissociation constant = ~1.0 μM). Therefore, in order to overcome the low response rate of existing immune checkpoint inhibitors and to induce effective immune checkpoint activation of NK cells that recognize and eliminate cancer cells and virus-infected cells even without specific antigens, it is very necessary to discover a B7-H6 variant that increases binding affinity with the activating receptor NKp30.

[0004] Meanwhile, cell therapy refers to pharmaceuticals that use living cells to induce regeneration and restore damaged or diseased cells or tissues. It is manufactured by manipulating autologous, allogeneic, or xenogeneic cells through physical, chemical, or biological methods, such as culturing, proliferating, or selecting them in vitro. Among these, immunomodulatory cell therapy refers to pharmaceuticals used to treat diseases by activating the body's immune response using immune cells such as dendritic cells, natural killer cells, and T cells. Currently, immunomodulatory cell therapies are primarily being developed for cancer treatment. Because they achieve therapeutic effects by directly administering immune cells to patients to activate immune function, they possess therapeutic mechanisms and efficacy distinct from conventional cancer treatments such as surgery, anticancer drugs, or radiation therapy. Consequently, this field is expected to constitute a major segment of future biopharmaceuticals. Depending on the type of immunomodulatory cell therapy, the physical and chemical characteristics of the antigens introduced into the cells differ; furthermore, when foreign genes are introduced into immune cells via viral vectors, the product exhibits characteristics of both cell therapy and gene therapy. Immunomodulatory cell therapy involves activating various immune cells, such as peripheral blood mononuclear cells (PBMCs), T cells, and natural killer (NK) cells, isolated from the patient via apheresis using various antibodies and cytokines, then proliferating them in vitro and re-injecting them into the patient, or re-injecting immune cells into which genes such as TCRs (T-Cell Receptors) or CARs (Chimeric Antigen Receptors) have been introduced. In particular, immunotherapy using natural killer cells, which can be mass-produced and frozen, is being researched. Natural killer cells are lymphoid cells that account for approximately 15% of peripheral blood lymphocytes and play an important role in innate immune responses.Natural killer (NK) cells eliminate tumor cells by activating dendritic cells and inducing cytotoxic T lymphocytes (CTLs) to respond specifically to tumors. NK cells directly kill malignant tumors such as sarcomas, myelomas, carcinomas, lymphomas, and leukemia. However, most NK cells present in the bodies of healthy individuals exist in an inactive state, and activated NK cells are required to eliminate tumors. Furthermore, NK cells in cancer patients exhibit functional defects due to the immune evasion mechanisms of cancer cells. Therefore, activating NK cells is crucial for utilizing them as a therapeutic agent. Additionally, since the number of NK cells in the body is limited, the development of technology to mass-produce and freeze NK cells from the blood of healthy individuals or patients is essential. In vitro expansion methods are utilized to proliferate natural killer cells in large quantities, and research is being conducted on mass culture methods for natural killer cells using peripheral blood lymphocytes (PBMCs), cord blood (CB), or human-induced pluripotent stem cells as raw materials. For the in vitro expansion culture of natural killer cells, PBMCs, CD3. - Cells, CD3-CD56 + Cell, CD56 +Cells are used as source cells, and cytokines such as IL-2, IL-12, IL-15, and IL-21, as well as LPS (Goodier et al., J. Immunol. 165(1):139-147, 2000) and CD3-stimulating OKT-3 antibodies (Condiotti et al., Experimental Hematol. 29(1):104-113, 2001) are used as natural killer cell proliferation factors, but they have a proliferation rate that makes it difficult to commercialize natural killer cells as therapeutic agents. The problem to be solved

[0005] The objective of the present invention is to provide a B7-H6 variant with increased binding affinity to natural killer cells.

[0006] In addition, the objective of the present invention is to provide a natural killer cell activator.

[0007] In addition, the object of the present invention is to provide a bispecific or multispecific antibody.

[0008] In addition, the objective of the present invention is to provide a pharmaceutical composition for the treatment or prevention of cancer.

[0009] In addition, the objective of the present invention is to provide a method for the in vitro proliferation of activated natural killer cells. means of solving the problem

[0010] To achieve the above objective, the present invention provides a B7-H6 variant with increased binding affinity to NKp30 (Natural cytotoxicity triggering receptor 3).

[0011] In addition, the present invention provides a natural killer cell activator comprising the above B7-H6 variant.

[0012] In addition, the present invention provides a bispecific or multispecific antibody comprising the B7-H6 variant and a portion that binds to a target antigen.

[0013] In addition, the present invention provides a pharmaceutical composition for the treatment or prevention of cancer comprising the above-mentioned B7-H6 variant, or a bispecific or multispecific antibody.

[0014] In addition, the present invention provides a method for the in vitro proliferation of activated natural killer cells. Effects of the invention

[0015] The B7-H6 variant of the present invention has a significantly increased binding affinity to NKp30, an activating receptor of natural killer cells (NK cells), compared to the wild type, which can increase the activation of natural killer cells. Since it is significantly smaller than an antibody, it facilitates penetration into the tumor microenvironment and is easy to produce, so it can be usefully used for the treatment of cancer or infectious diseases, either alone or in combination with various immunotherapies. Brief explanation of the drawing

[0016] Figure 1 shows an SDS-PAGE gel image of the NKp30-streptavidin protein expression vector and the purified protein. Figure 2 is a figure showing the results of flow cytometry analysis for selecting the B7-H6 display method. Figure 3 is a figure showing the results of amino acid sequence analysis of the constructed initial library. Figure 4 shows the results of verification of the concentration of clones with enhanced NKp30 binding affinity according to the screening process using a flow cytometer. Figure 5 shows the results of the analysis of the binding affinity of B7-H6 variants with NKp30 obtained through a flow cytometer. Figure 6 shows an SDS-PAGE gel image of purified B7-H6 variant-Fc fusion proteins. Figure 7 shows the results of analyzing the NKp30 binding affinity of B7-H6 variant-Fc fusion proteins using ELISA. Figure 8 shows the amino acid sequence analysis results of the constructed B7-H6 focused library. Figure 9 shows the results of verifying the amplification of B7-H6 variant clones with high NKp30 affinity following screening using a flow cytometer. Figure 10 shows the results of the analysis of the binding affinity of B7-H6 variants with NKp30 using a flow cytometer. Figure 11 shows an SDS-PAGE gel image of purified B7-H6 variant-Fc fusion proteins. Figure 12 is a figure showing the results of analyzing the NKp30 binding affinity of the B7-H6 variant-Fc fusion proteins of the present invention by ELISA. Specific details for implementing the invention

[0017] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0018] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0019] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0020] Throughout this specification, not only are conventional one- and three-character codes for naturally occurring amino acids used, but generally accepted three-character codes for other amino acids, such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc., are also used. Additionally, amino acids referred to by abbreviations in this invention are described according to the IUPAC-IUB nomenclature as follows:

[0021] Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y, and Valine: V.

[0023] In one aspect, the present invention relates to a B7-H6 variant or a fragment thereof having increased binding affinity to NKp30 (Natural cytotoxicity triggering receptor 3), wherein one or more amino acids selected from the group consisting of the 31st, 32nd, 37th, 40th, 51st, 53rd, 57th, 60th, 67th, 86th, 101st, 102nd, 108th, 114th, 129th, 136th, 142nd, and 143rd amino acids in the amino acid sequence of wild type B7-H6 (B7 homolog 6, NCR3LG1) are substituted with a sequence different from that of the wild type amino acids.

[0024] In one embodiment, the amino acid of wild-type B7-H6 may include the amino acid sequence of SEQ ID NO. 1, and the amino acid position may be based on the amino acid sequence of SEQ ID NO. 1.

[0025] In one embodiment, the B7-H6 variant of the present invention may comprise one or more amino acid substitutions selected from the group consisting of M31I, A32T, I37T, I37F, L40Q, F51I, F51S, F51L, F51Y, F51T, F51H, F51Q, F51K, F51R, S53G, N57D, S60I, S60Y, S60T, S60H, S60L, W67R, Q86L, K101E, S102C, S102R, R108M, L114M, L129M, Q136R, S142N, and P143S.

[0026] In one embodiment, the B7-H6 variant of the present invention may be a variant comprising an amino acid variation of the extracellular domain (ectodomain) region (SEQ ID NO. 2), which is the region of the entire amino acid of wild-type B7-H6 that is exposed outside the cell.

[0027] In one embodiment, the B7-H6 variant of the present invention may be a B5 comprising amino acid substitutions F51S and S60I, and may include an extracellular domain region (SEQ ID NO. 3) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 4.

[0028] In one embodiment, the B7-H6 variant of the present invention may be a B6 comprising an amino acid substitution N57D, and may include an extracellular domain region (SEQ ID NO. 5) comprising said amino acid substitution, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 6.

[0029] In one embodiment, the B7-H6 variant of the present invention may be a B7 comprising amino acid substitutions K101E and S102R, and may include an extracellular domain region (SEQ ID NO. 7) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 8.

[0030] In one embodiment, the B7-H6 variant of the present invention may be a B8 comprising amino acid substitutions A32T and S60I, and may include an extracellular domain region (SEQ ID NO. 9) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 10.

[0031] In one embodiment, the B7-H6 variant of the present invention may be a B9 comprising amino acid substitutions A32T, L40Q and S60I, and may include an extracellular domain region (SEQ ID NO. 11) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 12.

[0032] In one embodiment, the B7-H6 variant of the present invention may be a B14 comprising amino acid substitutions F51L and S60I, and may include an extracellular domain region (SEQ ID NO. 13) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 14.

[0033] In one embodiment, the B7-H6 variant of the present invention may be a B16 comprising an amino acid substitution K101E, and may include an extracellular domain region (SEQ ID NO. 15) comprising said amino acid substitution, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 16.

[0034] In one embodiment, the B7-H6 variant of the present invention may be a B19 comprising amino acid substitutions A32T, W67R, Q86L, K101E and L129M, and may include an extracellular domain region (SEQ ID NO. 17) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 18.

[0035] In one embodiment, the B7-H6 variant of the present invention may be a B23 comprising amino acid substitutions S60I, L114M and S142N, and may include an extracellular domain region (SEQ ID NO. 19) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 20.

[0036] In one embodiment, the B7-H6 variant of the present invention may be a B29 comprising amino acid substitutions M31I, A32T, F51I, S60I, S102C, and R108M, and may include an extracellular domain region (SEQ ID NO. 21) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 22.

[0037] In one embodiment, the B7-H6 variant of the present invention may be a B35 comprising amino acid substitutions A32T, S60I, K101E and P143S, and may include an extracellular domain region (SEQ ID NO. 23) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 24.

[0038] In one embodiment, the B7-H6 variant of the present invention may be a B40 comprising amino acid substitutions I37T, Q86L, and K101E, and may include an extracellular domain region (SEQ ID NO. 25) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 26.

[0039] In one embodiment, the B7-H6 variant of the present invention may be a B41 comprising amino acid substitutions A32T, S53G, S60I, and Q136R, and may include an extracellular domain region (SEQ ID NO. 27) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 28.

[0040] In one embodiment, the B7-H6 variant of the present invention may be a B47 comprising amino acid substitutions F51Y and S60I, and may include an extracellular domain region (SEQ ID NO. 29) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 30.

[0041] In one embodiment, the B7-H6 variant of the present invention may be a B52 comprising amino acid substitutions S60I and K101E, and may include an extracellular domain region (SEQ ID NO. 31) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 32.

[0042] In one embodiment, the B7-H6 variant of the present invention may be a B53 comprising amino acid substitutions A32T and K101E, and may include an extracellular domain region (SEQ ID NO. 33) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 34.

[0043] In one embodiment, the B7-H6 variant of the present invention may be a B54 comprising amino acid substitutions A32T, S60I, and K101E, and may include an extracellular domain region (SEQ ID NO. 35) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 36.

[0044] In one embodiment, the B7-H6 variant of the present invention may be a BF2 comprising amino acid substitutions F51H and S60I, and may include an extracellular domain region (SEQ ID NO. 37) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 38.

[0045] In one embodiment, the B7-H6 variant of the present invention may be a BF3 comprising amino acid substitutions F51I and S60Y, and may include an extracellular domain region (SEQ ID NO. 39) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 40.

[0046] In one embodiment, the B7-H6 variant of the present invention may be a BF5 comprising amino acid substitutions I37F, F51L, and S60T, and may include an extracellular domain region (SEQ ID NO. 41) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 42.

[0047] In one embodiment, the B7-H6 variant of the present invention may be a BF8 comprising amino acid substitutions F51T and S60T, and may include an extracellular domain region (SEQ ID NO. 43) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 44.

[0048] In one embodiment, the B7-H6 variant of the present invention may be a BF11 comprising amino acid substitutions F51L and S60H, and may include an extracellular domain region (SEQ ID NO. 45) comprising said amino acid substitution, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 46.

[0049] In one embodiment, the B7-H6 variant of the present invention may be a BF19 comprising amino acid substitutions F51T and S60Y, and may include an extracellular domain region (SEQ ID NO. 47) comprising said amino acid substitution, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 48.

[0050] In one embodiment, the B7-H6 variant of the present invention may be a BF25 comprising amino acid substitutions F51Q and S60H, and may include an extracellular domain region (SEQ No. 49) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ No. 50.

[0051] In one embodiment, the B7-H6 variant of the present invention may be a BF39 comprising amino acid substitutions F51K and S60L, and may include an extracellular domain site (SEQ ID NO. 51) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 52.

[0052] In one embodiment, the B7-H6 variant of the present invention may be a BF46 comprising amino acid substitutions F51R and S60T, and may include an extracellular domain region (SEQ ID NO. 53) comprising said amino acid substitutions, which may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO. 54.

[0053] In the present invention, the amino acid substitution location is described based on the total amino acids of wild-type B7-H6 (Sequence No. 1), but since the actual mutation location is located in the extracellular domain region (Sequence No. 2), the location described based on the total amino acids of wild-type B7-H6 may differ from the location in the extracellular domain region containing the amino acid substitution. For example, the F51S amino acid substitution of the B5 variant is F27I based on the amino acids of Sequence No. 3.

[0054] The B7-H6 variant of the present invention refers to a wild-type B7-H6 protein (or peptide) in which some amino acid sequences are substituted. As used in the present invention, the term "variant" refers to a corresponding amino acid sequence containing at least one amino acid difference (substitution, insertion, or deletion) when compared to a reference substance. In certain embodiments, the "variant" has high amino acid sequence homology and / or conservative amino acid substitutions, deletions, and / or insertions when compared to a reference sequence. Additionally, as used in the present invention, the term "B7-H6 variant" refers to a B7-H6 variant protein mutated at one or more amino acids to regulate its binding activity with NKp30.

[0055] Specifically, the B7-H6 variant of the present invention may be produced by a standard synthesis method, a recombinant expression system, or any other method in the art. Accordingly, the peptides according to the present invention may be synthesized by a number of methods, including, for example, a method comprising the following:

[0056] (a) a method for synthesizing a peptide stepwise or by fragment assembly by means of a solid-phase or liquid-phase method, and isolating and purifying the final peptide product; or

[0057] (b) a method of expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from a host cell culture; or

[0058] (c) a method for performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or

[0059] A method for obtaining a peptide fragment by any combination of (a), (b) and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

[0060] As a more specific example, the B7-H6 variant of the present invention can be produced by manufacturing a gene encoding the B7-H6 variant of the present invention through genetic engineering, transforming it into a host cell, and then expressing it.

[0061] In one aspect, the present invention relates to a nucleic acid molecule encoding a B7-H6 variant of the present invention, a vector containing the same, and a host cell containing said vector.

[0062] As used in the present invention, the term “nucleic acid molecule” refers to a deoxyribonucleotide or ribonucleotide existing in a single-stranded or double-stranded form, and includes natural nucleic acid analogs unless otherwise specifically noted (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0063] The term "vector" as used in the present invention refers to any nucleic acid comprising a competent nucleotide sequence that is inserted into a host cell, recombines with the host cell genome, is inserted therein, or spontaneously replicates as an episome. Examples of such vectors include linear nucleic acids, plasmids, phagesmids, cosmids, RNA vectors, viral vectors, etc.

[0064] The term "host cell" as used in the present invention refers to a eukaryotic or prokaryotic cell into which one or more DNA or vectors are introduced, and should be understood to refer not only to a specific target cell but also to its offspring or potential offspring. Although the offspring are not identical to the parent cell in fact because modifications may occur in subsequent generations due to mutations or environmental influences, they are still included within the scope of the term as used in the present invention.

[0065] In one aspect, the present invention relates to a natural killer cell (NK cell) activator comprising a B7-H6 variant of the present invention or a fragment thereof.

[0066] In one embodiment, the fragment may be an ectodomain site of the B7-H6 variant of the present invention.

[0067] In one aspect, the present invention relates to a composition for detecting natural killer cells comprising the B7-H6 variant of the present invention.

[0068] In one embodiment, the composition can detect and quantify NKp30 protein expressed on the surface of natural killer cells.

[0069] In one embodiment, the B7-H6 variant may be labeled with one selected from the group consisting of a chromogenic enzyme, a radioisotope, a chromopore, a luminescent substance, and a fluorescent substance, and the fluorescent substance may be a Cy (cyanine) series, Rhodamine series, Alexa series, BODIPY series, or ROX series fluorescent substance, and may be Nile Red, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), cyanine, fluorescein, Rhodamine, coumarine, or Alexa.

[0070] In one aspect, the present invention relates to a bispecific or multispecific antibody comprising a portion that binds to a target antigen and a B7-H6 variant of the present invention.

[0071] In one embodiment, the bispecific antibody may be a bispecific NK cell engager.

[0072] In one embodiment, the portion binding to the target antigen may include an antibody or a fragment having immunological activity thereof, and the fragment having immunological activity may be any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv, Fv, single-strand antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody and diabody, and it is more preferable that it be scFv or Fab.

[0073] In one embodiment, the target antigen is 17-1A antigen, GD3 ganglioside R24, EGFRvⅢ, PSMA, PSCA, HLA-DR, EpCAM, MUC1 core protein, atypically glycosylated MUC1, fibronectin isoform containing an ED-B domain, HER2 / neu, carcinoma-embryonic antigen (CEA), gastrin-releasing peptide (GRP) receptor antigen, mucin antigen, epidermal growth factor receptor (EGF-R), HER3, HER4, MAGE antigen, SART antigen, MUC1 antigen, c-erb-2 antigen, TAG 72, carbonic anhydrase IX, alpha-fetoprotein, antigen specific to A3 and A33 antibodies, Ba 733, BrE3-antigen, CA125, CDl, CD1a, CD3, CD5, CDl5, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, Colon-specific antigen-p (CSAp), CSAp, EGP-1, EGP-2, Ep-CAM, FIt-1, Flt-3, Folate receptor, HLA-DR, Human chorionic gonadotropin (HCG) and its subunits, Hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, Insulin-growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, Macrophage inhibitor factor (MIF), MAGE, MUCl, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigens specific to PAM-4 antibodies, placental growth factor, p53, prostatic acid phosphatase, PSA, RS5, SLOO, TAC, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF,ED-B may be one or more selected from the group consisting of fibronectin, angiogenesis markers, oncogene markers, or oncogene products, and the oncogene may be an apoptosis-related gene, a transcription factor gene, a metastasis-related gene, angiogenesis-related gene, or a tyrosine-kinase gene.

[0074] In one embodiment, the apoptosis-related gene is ABL1, AKT1, AKT2, BARD1, BAX, BCL11B, BCL2, BCL2A1, BCL2L1, BCL2L12, BCL3, BCL6, BIRC2, BIRC3, BIRC5, BRAF, CARD11, CAV1, CBL, CDC25A, CDKN1A, CFLAR, CNR2, CTNNB1, CUL4A, DAXX, DDIT3, E2F1, E2F3, E2F5, ESPL1, FOXO1, HDAC1, HSPA5, IGF1R, IGF2, JUN, JUNB, JUND, MALT1, MAP3K7, MCL1, MDM2, MDM4, MYB, MYC, NFKB2, NPM1, NTRK1, PAK1, It may be PAX3, PML, PRKCA, PRKCE, PTK2B, RAF1, RHOA, TGFB1, TNFRSF1B, TP73, TRAF6, YWHAG, YWHAQ, or YWHAZ;상기 전사인자 유전자는 AR, ARID3A, ASCL1, ATF1, ATF3, BCL11A, BCL11B, BCL3, BCL6, CDC5L, CDX2, CREB1, CUX1, DDIT3, DLX5, E2F1, E2F3, E2F5, ELF4, ELK1, ELK3, EN2, ERG, ETS1, ETS2, ETV1, ETV3, ETV4, ETV6, FEV, FEZF1, FLI1, FOS, FOSL1, FOXA1, FOXG1, FOXM1, FOXO1, FOXP1, FOXQ1, GATA1, GATA6, GFI1, GFI1B, GLI1, GLI2, GLI3, HES6, HHEX, HLF, HMGA1, HMGA2, HOXA1, HOXA9, HOXD13, HOXD9, ID1, ID2, IKZF1, IRF2, IRF4, JUN, JUNB, JUND, KAT6A, KDM2A, KDM5B, KLF2, KLF4, KLF5, KLF6, KLF8, KMT2A, LEF1, LHX1, LMX1B, MAF, MAFA, MAFB, MBD1, MECOM, MEF2C, MEIS1, MITF, MYB, MYC, MYCL, MYCN, NANOG, NCOA3, NFIB, NFKB2, NKX2-1, OTX2, PATZ1, PAX2, PAX3, PAX4, PAX8, PBX1, PBX2, PITX2, PLAG1,PLAGL2, PPARG, PPP1R13L, PRDM10, PRDM13, PRDM14, PRDM15, PRDM16, PRDM6, PRDM8, PRDM9, RARA, REL, RERE, RUNX1, RUNX3, SALL4, SATB1, SFPQ, SIX1, SNAI1, SOX2, SOX4, SPI1, SREBF1, STAT3, TAF1, TAL1, TAL2, TBX2, TBX3, TCF3, TFCP2, TFE3, THRA, TLX1, TP63, TP73, TWIST1, WT1, YBX1, YY1, ZBTB16, ZBTB7A, ZIC2, ZNF217 또는 ZNF268일 수 있고;The above metastasis-related genes may be AKT1, AKT2, AR, CBL, CDH1, CRK, CSF1, CTNNB1, CTTN, CXCR4, EGFR, FGFR1, FLT3, FYN, GLI1, ILK, ITGA3, JAK2, MET, PDGFRB, PLXNB1, PRKCI, PTCH1, PTPN11, RAC1, RHOA, RHOC, ROCK1, SMO, SNAI1, SRC, TCF3, or WT1; and the above angiogenesis-related genes may be BRAF, CAV1, CTGF, EGFR, ERBB2, ETS1, FGF4, FGF6, FGFR1, FGFR3, FGFR4, ID1, NRAS, PDGFB, PDGFRA, PDGFRB, or SPARC; The above tyrosine-kinase gene may be ABL1, ABL2, ALK, AXL, BLK, EGFR, EPHA2, ERBB2, ERBB3, ERBB4, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT3, FYN, ITK, JAK1, JAK2, KIT, LCK, MERTK, MET, MST1R, NTRK1, NTRK3, PDGFRA, PDGFRB, PTK2B, PTK7, RET, ROS1, SRC, SYK, TEC, or YES1.

[0075] 일 구현예에서, 종양유전자는 SEPTIN9, ACOD1, ACTN4, ADAM28, ADAM9, ADGRF1, ADRBK2, AFF1, AFF3, AGAP2, AGFG1, AGRN, AHCYL1, AHI1, AIMP2, AKAP13, AKAP9, AKIRIN2, AKTIP, ALDH1A1, ALL1, ANIB1, ANP32C, ANP32D, AQP1, ARAF, ARHGEF1, ARHGEF2, ARHGEF5, ASPSCR1, AURKA, BAALC, BAIAP2L1, BANP, BCAR4, BCKDHB, BCL9, BCL9L, BCR, BMI1, BMP7, BOC, BRD4, BRF2, CABIN1, CAMK1D, CAPG, CBFB, CBLB, CBLL1, CBX7, CBX8, CCDC28A, CCDC6, CCNB1, CCNB2, CCND1, CCNE1, CCNL1, CD24, CDC25C, CDC6, CDH17, CDK1, CDK14, CDK4, CDK5R2, CDK6, CDK8, CDKN1B, CDKN3, CDON, CEACAM6, CENPW, CHD1L, CHIC1, CHL1, CKS1B, CMC4, CNTN2, COPS3, COPS5, CRKL, CRLF2, CROT, CRTC1, CRYAB, CSF1R, CSF3, CSF3R, CSNK2A1, CSNK2A2, CT45A1, CTBP2, CTNND2, CTSZ, CUL7, CXCL1, CXCL2, CXCL3, CYGB, CYP24A1, DCD, DCUN1D1DDB2, DDHD2, DDX6, DEK, DIS3, DNPH1, DPPA2, DPPA4, DSG3, DUSP12, DUSP26, ECHS1, ECT2, EEF1A1, EEF1A2, EEF1D, EIF3E, EIF3I, EIF4E, EIF5A2, ELAVL1, ELL, EML4, EMSY, ENTPD5, EPCAM, EPS8, ERAS, ERGIC1, ERVW-1, EVI2A, EVI5, EWSR1, EZH2, FAM189B, FAM72A,FAM83D, FASN, FDPS, FGF10, FGF3, FGF5, FGF8, FR1OP, FHL2, FIP1L1, FNDC3B, FRAT1, FUBP1, FUS, FZD2, GAB2, GAEC1, GALNT10, GALR2, GLO1, GMNN, GNA12, GNA13, GNAI2, GNAQ, GNAS, GOLPH3, GOPC, GPAT4, GPM6A, GPM6B, GPR132, GREM1, GRM1, GSK3A, GSM1, H19, HAS1, HAX1, HDGFRP2, HMGN5, HNRNPA1, HOTAIR, HOTTIP, HOXA-AS2, HRAS, HSPA1A, HSPA4, HSPB1, HULC, IDH1, IFNG, IGF2BP1, IKBKE, IL7R, INPPL1, INTS1, INTS2, INTS3, INTS4, INTS5, INTS7, INTS8, IRS2, IST1, JUP, KDM4C, KIAA0101, KIAA1524, KIF14, KRAS, KSR2, LAMTOR5, LAPTM4B, LCN2, LDHB, LETMD1, LIN28A, LIN28B, LMO1, LMO2, LMO3, LMO4, LSM1, LUADT1, MACC1, MACROD1, MAGEA11, MALAT1, MAML2, MAP3K8, MAPRE1, MAS1, MCC, MCF2, MCF2L, MCTS1, MEFV, MFHAS1, MFNG, MIEN1, MINA, MKL2, MLANA, MLLT1, MLLT11, MLLT3, MLLT4, MMP12, MMS22L, MN1, MNAT1, MOS, MPL, MPST, MRAS, MRE11A, MSI1, MTCP1, MTDH, MTOR, MUC1, MUC4, MUM1, MYD88, NAAA, NANOGP8, NBPF12, NCOA4, NEAT1, NECTIN4, NEDD4, NEDD9, NET1, NINL, NME1, NOTCH1, NOTCH4, NOV, NSD1, NUAK2, NUP214, NUP98, NUTM1, OLR1,PA2G4, PADI2, PAK7, PARK7, PARM1, PBK, PCAT1, PCAT5, PDGFA, PDZK1IP1, PELP1, PFN1P3, PIGU, PIK3CA, PIK3R1, PIM1, PIM2, PIM3, PIRD,PLKPLM1,PICWIL1, PPP1R10, PPP1R14A, PPP2R1A, PRAME, PRDM12, PRMT5, PSIP1, PSMD10, PTCH2, PTMA, PTP4A1, PTP4A2, PTP4A3, PTTG1, PTTG1IP, PTTG2, R21 8AB, PVTAB, RAB8A, RALGDS, RAP1A, RASSF1, RBM14, RBM15, RBM3, RBMY1A1, RFC3, RGL4, RGR, RHO, RING1, RINT1, RIT1, RNF43, RPL23, RRAS, RRAS2, RSF14, S100T, S1X1T S100A8, SAG, SART3, SBSN, SEA, SEC62, SERTAD1, SERTAD2, SERTAD3, SET, SETBP1, SETDB1, SGK1, SIRT1, SIRT6, SKI, SKIL, SKP2, SLC12A5, SLCNO2, SCG9, SMR1,B, SNORA80E, SPAG9, SPATA4, SPRY2, SQSTM1, SRSF1, SRSF2, SRSF3, SRSF6, SS18, SSX1, SSX2, SSX2B, STIL, STMN1, STRA6, STYK1, SUZTA101CSTD, SSW TAF15, TALDO1, TAZ, TBC1D1, TBC1D15, TBC1D3, TBC1D3C, TBC1D7, TCL1A, TCL1B, TCL6, TCP1, TFG, TGM3, TINCR, TKTL1, TLE1, TMEMPR140,2, TMPRSSPD2, TMPOP2 TRE17, TREH, TRIB1, TRIB2,TRIM28, TRIM32, TRIM8, TRIO, TRIP6, TSPAN1, TSPY1, TXN, TYMS, TYRP1, UBE2C, UBE3C, UCA1, UCHL1, UHRF1, URI1, USP22, USP4, USP6, VAV1, VAV2, VAV3, VIM, WAPL, WHSC1, WHSC1L1, WISP1, It may be WNT1, WNT10A, WNT10B, WNT2, WNT3, WNT5A, WWTR1, XCL1,

[0076] In one embodiment, the target antigen may be a cell surface antigen or an autoantigen, and the cell surface antigen may be one or more selected from the group consisting of CEA, ED-B fibronectin, CD20, CD22, CD19, EGFR, IGF1R, VEFGR1 / Flt-1, VEGFR2 / KDR, VEGRF3 / Flt-4, HER2 / neu, CD30, CD33, CD3, CD16, CD64, CD89, CD2, adenovirus fibrous nop, PfMSP-1, HN / NDV, EpCAM / 17-1A, hTR, IL-2R / Tac, CAl9-9, MUCl, HLA class II, GD2, G250, TAG-72, PSMA, CEACAM6, HMWMAA, CD40, M13 envelope protein and GPIIb / IIIa.

[0077] In one embodiment, the fragment having immunological activity of the present invention may be any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single-chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody, and diabody.

[0078] The above antibody is in the form of a whole antibody as well as includes functional fragments of the antibody molecule. The whole antibody has a structure comprising two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. A functional fragment of the antibody molecule refers to a fragment possessing antigen-binding function, and examples of antibody fragments include: (i) a Fab fragment consisting of the variable region (VL) of the light chain and the variable region (VH) of the heavy chain, and the constant region (CL) of the light chain and the first constant region (CH1) of the heavy chain; (ii) an Fd fragment consisting of the VH and CH1 domains; and (iii) an Fv fragment consisting of the VL and VH domains of a monoclonal antibody. (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)]; (v) a separated CDR region; (vi) a divalent fragment F(ab')2 fragment containing two linked Fab fragments; (vii) a single-stranded Fv molecule (scFv) linked by a peptide linker that links the VH domain and VL domain to form an antigen-binding site; (viii) a bispecific single-stranded Fv dimer (PCT / US92 / 09965); and (ix) a diabody WO94 / 13804, a multivalent or multispecific fragment produced by gene fusion.

[0079] The antibody of the present invention or the fragment having immunological activity thereof may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fragments having immunological activity thereof. The antibody may be produced recombinantly or synthetically.

[0080] Animal-derived antibodies, produced by immunizing animals with a desired antigen, can generally cause immune rejection when administered to humans for therapeutic purposes; chimeric antibodies were developed to suppress this rejection. Chimeric antibodies are created by using genetic engineering methods to replace the constant region of animal-derived antibodies—which causes anti-isotype reactions—with the constant region of human antibodies. While chimeric antibodies show significant improvement in anti-isotype reactions compared to animal-derived antibodies, they still carry potential side effects related to anti-idiotypic reactions because animal-derived amino acids remain in the variable region. Humanized antibodies were developed to address these side effects. They are produced by transplanting the Complementarity Determining Regions (CDRs), which play a crucial role in antigen binding within the variable region of chimeric antibodies, into a human antibody framework.

[0081] In CDR grafting technology for producing humanized antibodies, the most critical aspect is selecting an optimized human antibody capable of best accepting the CDR site of an animal-derived antibody; to achieve this, techniques such as antibody databases, crystal structure analysis, and molecular modeling are utilized. However, even when the CDR site of an animal-derived antibody is grafted onto an optimized human antibody scaffold, there are a significant number of cases where antigenic binding affinity is not preserved due to the presence of amino acids on the animal antibody scaffold that affect antigen binding. Therefore, the application of additional antibody engineering techniques to restore antigenic binding affinity is essential.

[0082] The above antibody or the fragment having immunological activity thereof may be isolated from a living organism (not present in the living organism) or non-naturally occurring, for example, may be produced synthetically or recombinantly.

[0083] In the present invention, the term "antibody" refers to a substance produced within the immune system by stimulation of an antigen; its type is not particularly limited and can be obtained naturally or unnaturally (e.g., synthetically or recombinantly). Antibodies are advantageous for mass expression and production because they are very stable and have a long half-life, both in vitro and in vivo. Furthermore, antibodies have a very high avidity because they inherently possess a dimer structure. A complete antibody has a structure comprising two full-length light chains and two full-length heavy chains, with each light chain connected to a heavy chain by a disulfide bond. The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0084] In the present invention, the term "heavy chain" refers to a variable region domain V comprising an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen. H and 3 invariant domains C H1 , C H2 and C H3 It is interpreted to include both the full-length heavy chain containing the hinge and its fragments. Additionally, the term "light chain" refers to a variable region domain V containing an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen.L and invariant domain C L It is interpreted to mean that it includes all of the battlefield light chains and fragments thereof.

[0085] In the present invention, the term "variable region" or "variable domain" refers to a part of an antibody molecule that performs the function of specifically binding to an antigen and exhibits many variations in sequence, and the variable region contains complementary determining regions CDR1, CDR2, and CDR3. Between the CDRs, there exists a framework region (FR) that serves to support the CDR loop. The "complementary determining region" is a loop-shaped region involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this region changes.

[0086] The term "scFv (single chain fragment variable)" as used in the present invention refers to a single-chain antibody produced by expressing only the variable region of an antibody through genetic recombination, and refers to a single-chain antibody in which the VH region and the VL region of the antibody are connected by a short peptide chain. Unless otherwise specified or understood from the context, the term "scFv" is intended to include scFv fragments, including antigen-binding fragments. This is obvious to a person skilled in the art.

[0087] In the present invention, the term "complementarity determining region (CDR)" refers to the amino acid sequence of a hypervariable region of the heavy chain and light chain of an immunoglobulin. The heavy chain and light chain may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide major contact residues for the antibody to bind to an antigen or an epitope.

[0088] In the present invention, the terms "specifically binding" or "specifically recognizing" have the same meaning as commonly known to those skilled in the art, and refer to an antigen and an antibody specifically interacting to produce an immunological reaction.

[0089] In the present invention, the term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure thereof, meaning a part of a polypeptide comprising a portion capable of binding to an antigen. For example, it may be scFv, (scFv) 2, scFv-Fc, Fab, Fab', or F(ab') 2, but is not limited thereto. Among the antigen-binding fragments, Fab comprises a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region of the heavy chain (C H1 It has a structure containing ) and possesses one antigen-binding site. Fab' is heavy chain C H1It differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the domain. F(ab')2 antibodies are generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Recombinant techniques for generating Fv fragments from minimal antibody fragments that possess only a heavy chain variable region and a light chain variable region are widely known in the art. Two-chain Fv has the heavy chain variable region and the light chain variable region connected by non-covalent bonds, while single-chain Fv generally has the heavy chain variable region and the single chain variable region connected by covalent bonds or directly at the C-terminus via a peptide linker, thereby forming a dimer-like structure similar to that of two-chain Fv. The linker may be a peptide linker composed of any 1 to 100 or 2 to 50 amino acids, and suitable sequences are known in the art. The above antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restricting the whole antibody with papain and F(ab')2 fragment can be obtained by cleaving it with pepsin), and can be produced through genetic recombination technology.

[0090] In the present invention, the term "hinge region" refers to a region included in the heavy chain of an antibody, C H1 and C H2 It refers to a region that exists between regions and functions to provide flexibility to the antigen binding site within the antibody. For example, the hinge may be derived from a human antibody, specifically, from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3, or IgG4.

[0091] In one aspect, the present invention relates to an isolated nucleic acid molecule encoding a B7-H6 variant or a fragment thereof, or a bispecific or multispecific antibody, a vector comprising the same, and a host cell transformed therefrom.

[0092] The nucleic acid molecules of the present invention may be isolated or recombinant and include DNA and RNA in single-stranded and double-stranded forms, as well as corresponding complementary sequences. In the case of nucleic acids isolated from natural sources, isolated nucleic acids are nucleic acids separated from surrounding genetic sequences present in the genome of the individual from which the nucleic acid was isolated. In the case of nucleic acids synthesized enzymatically or chemically from a template, such as PCR products, cDNA molecules, or oligonucleotides, the nucleic acids produced from these procedures may be understood as isolated nucleic acid molecules. Isolated nucleic acid molecules represent nucleic acid molecules in the form of separate fragments or as components of larger nucleic acid constructs. Nucleic acids are operably linked when arranged in a functional relationship with other nucleic acid sequences. For example, the DNA of a presequence or secretion leader is operably linked to the polypeptide DNA when the polypeptide is expressed as a preprotein, which is the form prior to secretion; a promoter or enhancer is operably linked to the coding sequence when it influences the transcription of the polypeptide sequence; or a ribosome binding site is operably linked to the coding sequence when positioned to facilitate translation. Generally, operably linked means that the DNA sequences to be linked are located adjacently, and in the case of a secretion leader, this means they exist adjacently within the same reading frame. However, enhancers do not need to be located adjacently. Linkage is achieved by ligation at a convenient restriction enzyme site. If such a site is not present, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

[0093] The isolated nucleic acid molecule encoding the antibody of the present invention, the fragment having immunological activity thereof, or the bispecific or multispecific antibody of the present invention may undergo various modifications to the coding region within a range that does not alter the amino acid sequence of the antibody expressed from the coding region, due to codon degeneracy or considering the codons preferred by the organism to express said antibody; and may also undergo various modifications or alterations to parts excluding the coding region within a range that does not affect gene expression, and such modified genes are also included within the scope of the present invention, as will be well understood by those skilled in the art. That is, as long as the nucleic acid molecule of the present invention codes for a protein having equivalent activity, one or more nucleic acid bases may be modified by substitution, deletion, insertion, or a combination thereof, and these are also included within the scope of the present invention. The sequence of such nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

[0094] An isolated nucleic acid molecule encoding the antibody of the present invention or a fragment having immunological activity thereof, or the bispecific or multispecific antibody of the present invention, according to the present invention, may be inserted into an expression vector for protein expression. The expression vector typically comprises a protein in which the antibody of the present invention or a fragment having immunological activity thereof, or any fusion partner, and / or additional elements are operably linked, i.e., functionally related. Under appropriate conditions, the antibody of the present invention or a fragment having immunological activity thereof, or the bispecific or multispecific antibody of the present invention, may be produced by a method of inducing protein expression by culturing a host cell transformed with nucleic acid, preferably, an expression vector containing the isolated nucleic acid molecule encoding the antibody of the present invention or a fragment having immunological activity thereof, or the bispecific or multispecific antibody of the present invention. Various suitable host cells, including mammalian cells, bacteria, insect cells, and yeast, may be used, but are not limited thereto. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used. Preferably, Escherichia coli, which has low production costs and high industrial utility value, can be produced as a host cell.

[0095] The vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Suitable vectors may include signal sequences or leader sequences for membrane targeting or secretion in addition to expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, and may be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequences may include, but are not limited to, PhoA signal sequences, OmpA signal sequences, etc. when the host is Escherichia sp., α-amylase signal sequences, subtilisin signal sequences, etc. when the host is Bacillus sp., MFα signal sequences, SUC2 signal sequences, etc. when the host is yeast, and insulin signal sequences, α-interferon signal sequences, antibody molecule signal sequences, etc. when the host is an animal cell. Additionally, the vector may include a selection marker for selecting a host cell containing the vector, and if it is a replicable expression vector, it includes a replication origin.

[0096] In the present invention, the term “vector” refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. The nucleic acid sequence may be exogenous or heterologous. Examples of vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages). Those skilled in the art may construct vectors using standard recombinant techniques (Maniatis, et al., *Molecular Cloning*, *A Laboratory Manual*, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., *Current Protocols in Molecular Biology*, John, Wiley & Sons, Inc, NY, 1994, etc.).

[0097] In one embodiment, when producing the vector, expression control sequences such as a promoter, terminator, and enhancer, sequences for membrane targeting or secretion, etc., can be appropriately selected and combined in various ways according to the purpose, depending on the type of host cell to be used to produce the antibody.

[0098] In the present invention, the term "expression vector" means a vector comprising a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. The expression vector may include various regulatory sequences. Along with regulatory sequences that regulate transcription and translation, the vector and the expression vector may also include nucleic acid sequences that provide other functions.

[0099] In the present invention, the term "host cell" refers to any transgenic organism, including eukaryotes and prokaryotes, capable of replicating said vector or expressing a gene encoded by said vector. The host cell may be transfected or transformed by said vector, which refers to the process in which an exogenous nucleic acid molecule is delivered or introduced into the host cell.

[0100] In one embodiment, the host cell may be a bacterium or an animal cell, the animal cell line may be a CHO cell, an HEK cell, or an NSO cell, and the bacterium may be E. coli.

[0101] In one aspect, the present invention relates to a pharmaceutical composition for the treatment or prevention of cancer, comprising a B7-H6 variant or a fragment thereof, or a bispecific or multispecific antibody.

[0102] In one embodiment, the cancer may be any one selected from the group consisting of brain tumor, melanoma, multiple myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, pro-anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma.

[0103] In the present invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, spread, and recurrence of a disease or illness by administering a composition according to the present invention.

[0104] As used in this invention, the term "treatment" refers to any act of improving or beneficially altering the symptoms of a disease or condition and its resulting complications through the administration of a composition according to this invention. A person skilled in the art to which this invention pertains would be able to determine the precise criteria for diseases to which the composition of this invention is effective, and to assess the degree of improvement, enhancement, and treatment, by referring to materials provided by organizations such as the Korean Medical Association.

[0105] In the present invention, the term "therapeutically effective amount" used in combination with the active ingredient refers to an amount effective for preventing or treating a disease or disorder, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, and the patient's condition. Therefore, when used in humans, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These matters to be considered when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0106] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level may be determined based on factors including the patient's health status, the type of disease or condition, the severity of the disease or condition, the drug's activity, sensitivity to the drug, the method of administration, the time of administration, the route of administration and elimination rate, the duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without adverse effects, and this can be easily determined by a person skilled in the art.

[0107] The pharmaceutical composition of the present invention may include a carrier, a diluent, an excipient, or a combination of two or more of these commonly used in biological preparations. As used in the present invention, the term "pharmaceutical acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and may be used, for example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into primary formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the field or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0108] In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, topical preparations, suppositories, sterile injectable solutions, and sprays, and an oral or injectable formulation is more preferred.

[0109] As used in the present invention, the term "administration" means providing a specific substance to an individual or patient by any appropriate method. Depending on the intended method, it may be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally as an injectable formulation) or orally. The dosage varies depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. Liquid formulations for oral administration of the composition of the present invention include suspensions, liquid formulations, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, etc. The pharmaceutical composition of the present invention may also be administered by any device capable of delivering the active substance to target cells. Preferred modes of administration and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip infusions, etc. Injectables can be prepared using aqueous solvents such as physiological saline solution and Ringer's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleate), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), non-aqueous solvents, and may include pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0110] The term “individual” used in the present invention means any animal including monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, bats, camels, rats, rabbits, or guinea pigs, including humans, on which the disease or illness has developed or may develop, and the term “specimen” may be droplets, sputum, whole blood, plasma, serum, urine, or saliva separated therefrom.

[0111] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0112] In one aspect, the present invention relates to a method for in vitro proliferation of activated natural killer cells, comprising the steps of: isolating natural killer cells; and culturing said isolated natural killer cells in the presence of a B7-H6 variant of the present invention or a fragment thereof.

[0114] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0116] Example 1. Cloning of tetrameric human NKp30 for B7-H6 variant search

[0117] To search for B7-H6 variants with enhanced binding affinity to NKp30, we induced binding avidity effects through the tetramerization of the NKp30 protein. Specifically, streptavidin was expressed at the C-terminus of NKp30 to induce tetramerization, and glycine and serine linkers were inserted between NKp30 and streptavidin to ensure the mobility of each protein. The NKp30 and streptavidin genes were amplified separately using primers and Vent polymerase (New England Biolab), followed by assembly PCR using Vent polymerase. To the synthesized genes Bss HII and Xba The gene was treated with restriction enzymes using I (New England Biolab). The restriction enzyme-treated NKp30-Streptavidin gene was ligated into the pMAZ vector, an animal cell vector treated with the same restriction enzymes. After transforming the ligated plasmid into E. coli Jude1, a single clone was obtained and sequenced to confirm that NKp30-Streptavidin was successfully inserted into the pMAZ vector (Fig. 1).

[0119] Example 2. Expression, Purification, and Labeling of Tetrameric Human NKp30

[0120] 2x10 Expi293F cells 6After subculturing 300 ml at a density of cells / ml for one day, PEI (Polyehylenimine, Polyscience, 23966) and the NKp30-streptavidin-His tag expression vector prepared in Example 1 were mixed in a 1:4 ratio with 30 ml of Freestyle 293 expression medium (Gibco. 12338-018), incubated at room temperature for 20 minutes, and then transfected into the Expi293F animal cells. The cells were cultured in a CO2 shaking incubator at 37 ℃, 125 rpm, and 8% CO2 for 7 days, after which the supernatant was collected by centrifugation. Subsequently, the mixture was equilibrated using 25x PBS and filtered through a 0.2 μm filter (Merck Millipore) using a bottle top filter. 1 ml of Ni-NTA resin was added to the filtered culture medium and stirred at 4°C for 16 hours. The resin was then passed through a column to be recovered and washed with 10 ml of PBS. The washed resin was sequentially washed with 10 ml each of 10 mM imidazole buffer and 20 mM imidazole buffer, followed by elution with 4 ml of 250 mM imidazole buffer. After exchanging the buffer with PBS using centrifugal filter units 3K (Merck Millipore), the purified NKp30-streptavidin tetramer protein was fluorescently labeled using the Alexa-488 labeling kit.

[0122] Example 3. Selection of Screening Method

[0123] 3-1. B7-H6 Cloning for Selection of Yeast Surface Display Method

[0124] To ensure efficient screening, we decided to determine yeast surface anchoring motifs and cloned B7-H6 to compare a system anchoring the N-terminus of B7-H6 to Aga2 (pCTCON-Aga2-B7-H6-FLAG) and a system anchoring the C-terminus to Aga2 (pCTCON-B7-H6-Aga2-FLAG). Specifically, we synthesized the wild-type B7-H6 gene and amplified the gene using designed primers and Vent Polymerase (New England Biolab). The amplified wild-type B7-H6 gene Sfi Restriction enzyme treatment was performed using I (New England Biolab), and the restriction enzyme-treated gene was ligated into the same restriction enzyme-treated pCTCON vector. After transgenerating the ligated plasmid into Jude1 E. coli, a single clone was obtained and sequenced, confirming that two types of plasmids, pCTCON-Aga2-B7-H6_WT-FLAG and pCTCON-B7-H6_WT-Aga2-FLAG, were successfully cloned.

[0126] 3-2. Selection of Display Method through Bonding Strength Verification

[0127] The display method was selected by verifying the binding affinity between B7-H6 expressed on the yeast surface and the probe NKp30-streptavidin using a flow cytometer. Specifically, the two plasmids prepared in Example 3-1 were each transduced into the AWY101 (Trp-) strain and cultured for 16 hours at 30°C and 225 rpm in 5 ml of SDCAA medium (20 g / L Glucose, 6.7 g / L Yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L Na2HPO4, and 8.56 g / L NaH2PO4) supplemented with 50 μg / ml Kanamycin and 40 μg / ml Chloramphenicol. The cultured cells (5 x 10⁶) 7 After obtaining the sample by centrifugation (2,500 g, 5 min, 4°C), it was incubated for 24 hours at 20°C and 225 rpm in 5 ml of SGCAA medium (20 g / L Galactose, 6.7 g / L Yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L Na2HPO4, and 8.56 g / L NaH2PO4) supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol. After incubation, 2 x 10 7 Cells were centrifuged (14,000g, 30 seconds, 4℃) and collected in e-tubes. 1 ml of PBSB (0.1% BSA in PBS) was added to each e-tube containing collected cells to resuspend them, and after collecting the cells again by centrifugation (14,000g, 30 seconds, 4℃), 0.5 ml of PBSB was added to resuspend them to obtain 4 x 10 7cells / ml were prepared. 25 μl of cells were transferred to a new e-tube, and 25 μl of PBSB, NKp30-streptavidin-Alexa488 (200 nM), and Anti-FLAG-iFluor647 (1000:1) probes were added, respectively. The cells were incubated at room temperature for 30 minutes to label them with fluorescent probes. Subsequently, the cells were centrifuged (14,000g, 30 seconds, 4℃), the supernatant was discarded, and the cells were resuspended in 200 μl of PBSB for washing. The samples were then prepared by centrifuging again (14,000g, 1 minute, 4℃) and resuspending in 200 μl of PBSB. The binding affinity of wild-type B7-H6 and NKp30 was indirectly analyzed by measuring the fluorescence signal values ​​of the prepared samples using a FACSLyric (BD Biosciences) instrument.

[0128] As a result of the analysis, it was confirmed through the signal of wild-type B7-H6 that fluorescently labeled tetrameric NKp30 was active when using tetrameric NKp30. In addition, although both were well expressed, it was confirmed that the binding affinity to NKp30 was significantly increased in B7-H6 displayed in yeast with the C-terminal anchored to Aga2 compared to wild-type B7-H6 displayed in yeast with the N-terminal anchored to Aga2, so it was decided to proceed with screening in the form with the C-terminal anchored (Fig. 2).

[0130] Example 4. Creation of a large B7-H6 error-prone library for using ultrafast screening techniques

[0131] To rapidly screen B7-H6 variants with increased binding affinity to NKp30, both sides are based on pCTCON-B7-H6_WT-Aga2-FLAG to allow random mutations to be introduced into all regions of B7-H6. Sfi A primer containing the I site was designed. The designed primer and TaqDNA was primarily amplified using the error-prone PCR technique with polymerase (TAKARA), dNTPs (Invitrogen), MgCl2, and MnCl2 (SIGMA). The amplified gene was secondary amplified using vent polymerase and prepared (24 μg), and the corresponding vector is Sfi It was prepared by treatment with restriction enzyme I (8 μg). The two prepared genes were transduced into the AWY101 strain to construct a library via homologous recombination. The constructed library was 5.1 x 10⁶ 7 It was of size, and through sequence analysis, it was confirmed to have an error-rate of 0.92% based on DNA (average 2.3 mutations / total 360 bp) and 1.92% based on amino acids (average 3.3 mutations / total 120 amino acids) (Fig. 3).

[0133] Example 5. B7-H6 variant screening

[0134] The initial library prepared by transformation into AWY101 in Example 4 above was cultured for 16 hours at 30°C and 225 rpm in 500 ml of SDCAA medium (20 g / L Glucose, 6.7 g / L Yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L Na2HPO4, and 8.56 g / L NaH2PO4) supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol, and to remove dead cells, OD in 100 ml of SDCAA medium 450 After inoculation at 0.7, the cells were cultured for an additional 16 hours at 30°C and 225 rpm. Subsequently, the cultured cells were placed in 100 ml of SGCAA medium with an OD level. 450 Inoculated at =0.7 and cultured at 20°C and 225 rpm for 2 days, after induction, 1x10 8The cells were centrifuged (14,000g, 30 seconds, 4℃) and collected in an e-tube. The cells were resuspended in 1 ml of PBSB (0.1% BSA in PBS) in the e-tube and collected again by centrifugation (14,000g, 30 seconds, 4℃). Then, the cells were labeled by resuspending them in 1 ml of PBSB containing Anti-FLAG-iFluor647 (1000:1) and NKp30-streptavidin-Alexa488 (200 nM) probes and incubating at room temperature for 1 hour. Subsequently, the sample was prepared by centrifugation (14,000g, 30 seconds, 4℃), discarding the supernatant, resuspending with 1 ml of PBSB for washing, and centrifugation again (14,000g, 1 min, 4℃) followed by resuspending with 1 ml of PBSB. The fluorescence signal values ​​of the prepared library samples were measured using an S3 sorter (Bio-Rad) to recover yeasts with high binding affinity to NKp30, and the recovered yeasts were cultured in 20 ml of SDCAA at 30℃ and 225 rpm. The cultured cells were harvested the next day and incubated in 100 ml of SGCAA at 20℃ and 225 rpm for 2-3 days, after which the next round was performed. The screening process described above was repeated a total of four times while gradually reducing the probe concentration.

[0136] Example 6. Confirmation of amplification of B7-H6 variants with increased binding affinity to NKp30

[0137] The libraries of the initial, Round 1, Round 2, Round 3, and Round 4 of Example 5 were separately inoculated into 100 ml of SDCAA medium (20 g / L Glucose, 6.7 g / L Yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L Na2HPO4, 8.56 g / L NaH2PO4) supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol, and cultured at 30°C and 225 rpm for 16 hours. To remove dead cells, the cultured cells were placed in 100 ml of SDCAA medium, each containing OD 450 After inoculating with =0.7, it was incubated at 30°C and 225 rpm for 16 hours, and then OD was added to 100 ml of SGCAA medium. 450 Inoculated at 0.7 and cultured at 20°C and 225 rpm for 2 days. After induction, the libraries were 2x10 7 Centrifuge each sample (14,000g, 30 seconds, 4℃) with an amount corresponding to cells and collect in e-tubes. Add 1 ml of PBSB (0.1% BSA in PBS) to each e-tube to resuspend, then centrifuge again (14,000g, 30 seconds, 4℃) to collect the cells, add 0.5 ml of PBSB to resuspend, and collect 4 x 10 7cells / ml were prepared. 25 μl of cells were transferred to a new e-tube, and 25 μl of PBSB, NKp30-streptavidin-Alexa488 (100 nM), and Anti-FLAG-iFluor647 (1000:1) probes were added, respectively. The cells were incubated at room temperature for 30 minutes to label them with fluorescent probes. Subsequently, the cells were centrifuged (14,000g, 30 seconds, 4℃), the supernatant was discarded, and the cells were resuspended in 200 μl of PBSB for washing. The samples were prepared by centrifuging again (14,000g, 1 minute, 4℃) and resuspending in 200 μl of PBSB. The fluorescence signal values ​​of the prepared samples were measured using a FACSLyric (BD Biosciences) instrument to indirectly analyze the binding affinity of each library to NKp30. As a result, it was confirmed that variants with improved binding affinity to NKp30 were amplified as the screening rounds increased (Fig. 4).

[0139] Example 7. Amino acid sequence analysis of B7-H6 variants and securing B7-H6 variants with increased binding affinity to NKp30

[0140] After obtaining DNA from a 4-round library using Zymoprep Yeast Plasmid Miniprep kits (Zymo Research), sequence analysis was performed on 50 colonies obtained by transforming them into Jude1 E. coli. As a result, 26 variants were selected, and to analyze their binding affinity with NKp30, the plasmids of the 26 variants were each transformed into the AWY101(Trp-) strain. To confirm the binding affinity of the variants to NKp30 using a flow cytometer, wild-type B7-H6 and 26 variants were cultured for 16 hours at 30°C and 225 rpm in 5 ml of SDCAA medium (20 g / L Glucose, 6.7 g / L Yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L Na2HPO4, 8.56 g / L NaH2PO4) supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol, and 5 x 10⁶ cultured cells 7 After obtaining the sample by centrifugation (2,500 g, 5 min, 4°C), it was incubated for 24 hours at 20°C and 225 rpm in 5 ml of SGCAA medium (20 g / L Galactose, 6.7 g / L Yeast nitrogen base without amino acids, 5 g / L casamino acids, 5.4 g / L Na2HPO4, 8.56 g / L NaH2PO4) supplemented with 50 μg / ml kanamycin and 40 μg / ml chloramphenicol. After incubation, 2x10 7 Cells were centrifuged (14,000g, 30 seconds, 4℃) and collected in e-tubes. 1 ml of PBSB (0.1% BSA in PBS) was added to each e-tube containing collected cells to resuspend them, and after centrifuging (14,000g, 30 seconds, 4℃) to collect the cells again, 0.5 ml of PBSB was added and resuspended to obtain 4 x 10 7cells / ml were prepared. After transferring 25 μl of cells to a new e-tube, 25 μl of PBSB, NKp30-streptavidin-Alexa488 (100 nM), and Anti-FLAG-iFluor647 (1000:1) probe were added, respectively, and the cells were incubated at room temperature for 30 minutes to label the cells with fluorescent probes. Afterward, the cells were centrifuged (14,000g, 30 seconds, 4℃), the supernatant was discarded, and the cells were resuspended in 200 μl of PBSB and washed. The sample was prepared by centrifuging again (14,000g, 1 minute, 4℃) and resuspending in 200 μl of PBSB. The expression levels and binding affinities of each variant with NKp30 were indirectly analyzed by measuring the fluorescence signal values ​​of the samples prepared using FACSLyric (BD Biosciences) equipment, and a total of 17 variants (B5, B6, B7, B8, B9, B14, B16, B19, B23, B29, B35, B40, B41, B47, B52, B53, and B54) with improved binding affinity with NKp30 were selected (Fig. 5 and Table 1).

[0141] B7-H6 variant B7-H6 mutation site and substituted amino acid B5 F51S / S60I B6 N57D B7 K101E / S102R B8 A32T / S60I B9 A32T / L40Q / S60I B14 F51L / S60I B16 K101E B19 A32T / W67R / Q86L / K101E / L129M B23 S60I / L114M / S142N B29 M31I / A32T / F51I / S60I / S102C / R108M B35 A32T / S60I / K101E / P143S B40 I37T / Q86L / K101E B41 A32T / S53G / S60I / Q136R B47 F51Y / S60I B52 S60I / K101E B53 A32T / K101E B54 A32T / S60I / K101E

[0143] Example 8. Expression and purification of B7-H6 variants with increased binding affinity to NKp30

[0144] To construct animal cell expression vectors for B7-H6 variants with increased binding affinity to NKp30, wild-type B7-H6 and B7-H6 variants were expressed with the Fc domain of an IgG antibody to induce dimer formation as a control group; at this time, a GS linker composed of glycine and serine was inserted between the Fc and the B7-H6 variants to ensure the fluidity of each protein. Specifically, the genes and Fc domains of wild-type B7-H6 and three variants (B5, B7, and B14) selected from the 17 B7-H6 variants selected in Example 7 were amplified using designed primers and Vent Polymerase (New England Biolab), and then Assembly PCR was performed on the amplified genes, Bss HII and Xba I was treated with restriction enzyme (New England Biolab). The genes of the restriction enzyme-treated B7-H6 variants were ligated into the pMAZ vector, an animal cell vector treated with the same restriction enzyme. The ligated plasmid E. coliAfter transformation into Jude1, a single clone was obtained and sequenced, confirming that the genes of the variants were successfully inserted into the vector. The constructed vector for B7-H6 variant-Fc fusion protein expression was transfected into Expi293F animal cells and cultured in a CO2 incubator at 37°C, 125 rpm, and 8% CO2 for 7 days; after centrifugation, only the supernatant was separated. Subsequently, the mixture was equilibrated using 25x PBS and filtered using a 0.2 μm syringe filter (Sartorius, S6634). 0.15 ml of Protein A resin was added to the filtered culture medium and stirred at room temperature for 1 hour to recover the resin, which was then washed with PBS. Afterward, the solution was eluted with 100 mM glycine buffer (pH 2.7) and neutralized with 1 M Tris-HCl (pH 8.0), and then the buffer was exchanged with 1× PBS (pH 7.4) using centrifugal filter units 10K (Merck Millipore). The expression of the wild-type B7-H6-Fc fusion protein expressed and purified in this manner, and the B7-H6 variant-Fc fusion proteins containing the three variants (B5, B7, and B14) discovered in the present invention, were confirmed by SDS-PAGE (Fig. 6).

[0146] Example 9. Verification of NKp30 binding strength of B7-H6 variants

[0147] ELISA was performed to analyze the NKp30 binding affinity of the B7-H6 variant-Fc fusion proteins purified in Example 8 above. Specifically, 50 μl of B7-H6 variant-Fc fusion proteins diluted to 4 μg / ml in 0.05 M Na2CO3 pH 9.6 were dispensed into Flat Bottom Polystyrene High Bind 96-well microplates (costar), immobilized at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk (Biopure) (in PBS, pH 7.4) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST (pH 7.4), 50 μl of NKp30-GST serially diluted with 1% Skim Milk (Biopure) (in PBS, pH 7.4) was dispensed into each well and reacted at room temperature for 1 hour. After washing four times, 50 μl of anti-GST-HRP conjugate was added and reacted at room temperature for 1 hour, followed by four washes. Subsequently, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to induce color development, followed by the addition of 50 μl of 2 M H2SO4 to terminate the reaction, after which the samples were analyzed using an Epoch Microplate Spectrophotometer (BioTek).

[0148] As a result, the three selected variants (B5, B7, and B14) showed significantly higher binding affinity than wild-type B7-H6 (Fig. 7).

[0150] Example 10. Additional screening of B7-H6 variants

[0151] 10-1. Construction of a Large B7-H6 Focused Library for Ultrafast Screening Techniques

[0152] A library was constructed to discover additional novel B7-H6 variants with enhanced NKp30 binding by introducing mutations at two amino acid positions in three variants (B5, B7, and B14), excluding mutations consistent with the wild-type B7-H6. A yeast surface display library was constructed using the same method as in Example 4. The constructed library was 1.5 × 10⁶ 7 The size was, and sequence analysis confirmed that mutations were introduced in two places (Fig. 8).

[0154] 10-2. B7-H6 Variant Screening

[0155] Yeasts having a high binding affinity to NKp30 were recovered by measuring the fluorescence signal value of the library sample in the same manner as in Example 5 above, and the screening process was performed once.

[0157] 10-3. Confirmation of enrichment of B7-H6 variants with increased binding affinity to NKp30

[0158] The binding affinity of the library with NKp30 was indirectly analyzed using the same method as in Example 6 above. As a result, it was confirmed that variants with improved binding affinity with NKp30 were amplified after a single screening (Fig. 9).

[0160] Example 11. Amino acid sequence analysis and selection of additional B7-H6 variants

[0161] The expression levels of the variants and their binding affinity with NKp30 were indirectly analyzed by measuring fluorescence signal values ​​in the same manner as in Example 7 above. Through this, a total of nine variants (BF2, BF3, BF5, BF8, BF11, BF19, BF25, BF39, and BF46) with enhanced binding affinity with NKp30 were selected (Table 2 and Fig. 10).

[0162] B7-H6 variant B7-H6 mutation site and substituted amino acid B5 F51S / S60I B6 N57D B7 K101E / S102R B8 A32T / S60I B9 A32T / L40Q / S60I B14 F51L / S60I B16 K101E B19 A32T / W67R / Q86L / K101E / L129M B23 S60I / L114M / S142N B29 M31I / A32T / F51I / S60I / S102C / R108M B35 A32T / S60I / K101E / P143S B40 I37T / Q86L / K101E B41 A32T / S53G / S60I / Q136R B47 F51Y / S60I B52 S60I / K101E B53 A32T / K101E B54 A32T / S60I / K101E BF2 F51H / S60I BF3 F51I / S60Y BF5 I37F / F51L / S60T BF8 F51T / S60T BF11 F51L / S60H BF19 F51T / S60Y BF25 F51Q / S60H BF39 F51K / S60L BF46 F51R / S60T

[0164] Example 12. Preparation, expression, and purification of animal cell expression vectors for additionally obtained B7-H6 variants

[0165] Using the 9 variants (BF2, BF3, BF5, BF8, BF11, BF19, BF25, BF39, and BF46) selected in Example 11 above, animal cell expression vectorals were prepared, expressed, and purified in the same manner as in Example 8 above, and it was confirmed by SDS-PAGE that wild-type B7-H6 and all 9 additionally discovered variants were well expressed (Fig. 11).

[0167] Example 13. Verification of NKp30 binding strength of B7-H6 variants of the present invention

[0168] As a result of conducting ELISA on the three initially discovered variants (B5, B7, and B14) and the three additionally discovered variants with the highest NKp30 binding affinity (BF2, BF8, and BF19) in the same manner as in Example 9, all variants discovered in the present invention showed significantly higher binding affinity than wild-type B7-H6 (Fig. 12).

Claims

Claim 1 A B7-H6 variant with increased binding affinity to NKp30 (Natural cytotoxicity triggering receptor 3), comprising, in the amino acid sequence of wild type B7-H6 (B7 homolog 6, NCR3LG1), amino acid substitutions of F51S and S60I; amino acid substitutions of K101E and S102R; amino acid substitutions of F51L and S60I; amino acid substitutions of F51H and S60I; amino acid substitutions of F51T and S60T; or amino acid substitutions of F51T and S60Y. Claim 2 In claim 1, the amino acid of the wild-type B7-H6 comprises the amino acid sequence of SEQ ID NO. 1, and is a B7-H6 variant with increased binding affinity with NKp30. Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 A natural killer cell (NK cell) activator comprising the B7-H6 variant of claim 1. Claim 29 A bispecific or multispecific antibody comprising the B7-H6 variant of claim 1 and a portion binding to a target antigen. Claim 30 In paragraph 29, the target antigens are 17-1A antigen, GD3 ganglioside R24, EGFRvⅢ, PSMA, PSCA, HLA-DR, EpCAM, MUC1 core protein, atypically glycosylated MUC1, fibronectin isoform containing an ED-B domain, HER2 / neu, carcinoma-embryonic antigen (CEA), gastrin-releasing peptide (GRP) receptor antigen, mucin antigen, epidermal growth factor receptor (EGF-R), HER3, HER4, MAGE antigen, SART antigen, MUC1 antigen, c-erb-2 antigen, TAG 72, carbonic anhydrase IX, alpha-fetoprotein, antigens specific to A3 and A33 antibodies, Ba 733, BrE3-antigen, CA125, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, Colon-specific antigen-p (CSAp), CSAp, EGP-1, EGP-2, Ep-CAM, FIt-1, Flt-3, Folate receptor, HLA-DR, Human chorionic gonadotropin (HCG) and its subunits, Hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, Insulin-growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, Macrophage inhibitor factor (MIF), MAGE, MUCl, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigens specific to PAM-4 antibodies, placental growth factor, p53, prostatic acid phosphatase, PSA, RS5, SLOO, TAC, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF,A bispecific or multispecific antibody selected from the group consisting of ED-B fibronectin, angiogenesis markers, oncogene markers, or oncogene products. Claim 31 A pharmaceutical composition for the treatment or prevention of cancer, comprising the B7-H6 variant of claim 1 or the bispecific or multispecific antibody of claim 29. Claim 32 A method for the in vitro proliferation of activated natural killer cells, comprising: i) a step of isolating natural killer cells; and ii) a step of culturing said isolated natural killer cells in the presence of the B7-H6 variant of claim 1 or a fragment thereof.

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