B7H6 antibodies and uses thereof

Abstract

The present invention discloses a B7H6 antibody and its use. The antibody comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3. The antibody of the present invention can bind to B7H6, promote the binding of B7H6 to NK cell activating receptor NKp30, and have anti-cancer function.

Description

[Technical Field]

[0001] The present invention relates to the field of antibodies, and in particular to antibodies or antigen-binding fragments thereof capable of binding to B7H6 and uses thereof. [Background technology]

[0002] NK cells are essential innate lymphocytes in the body, playing important roles in antiviral and antitumor activities. NK cells express various activating receptors, including NKp30 and NKG2D, on their surface, which recognize and bind to ligands expressed on the surface of tumor cells. These ligands are encoded by the NK cells' own genes and are expressed on the surface of cells under conditions such as malignant transformation (tumor).

[0003] NKp30 is an important NK cell activating receptor that transmits activation signals after binding to its ligand, promoting tumor killing by NK cells. B7H6 is the only known NKp30 ligand expressed as a membrane protein. B7-H6 is highly expressed in various tumor tissues, such as melanoma and liver cancer, but not in normal tissues, demonstrating tumor-specific expression.

[0004] Cytokines such as TGF-β in the tumor microenvironment downregulate the expression of NKp30 on the surface of NK cells, which results in NK cells being unable to accurately identify and kill tumors using the NKp30-B7H6 interaction, leading to tumor immune escape. Therefore, it is necessary to provide therapeutic antibody drugs that target B7H6. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention overcomes the drawbacks of the prior art and aims to provide a B7H6 antibody that can bind to human and monkey B7H6 and promote the binding of B7H6 to NKp30, and uses thereof. [Means for solving the problem]

[0006] The antibody according to the present invention promotes the binding of B7H6 to NKp30, thereby promoting the anti-cancer function of NK cells.

[0007] In one aspect, the invention provides an antibody or antigen-binding fragment thereof capable of binding to B7H6, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3; the heavy chain CDR1 comprises at least one selected from the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 7, and conservatively modified amino acid sequences thereof; the heavy chain CDR2 comprises at least one selected from the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 8, and conservatively modified amino acid sequences thereof; the heavy chain CDR3 comprises at least one selected from the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 9, and conservatively modified amino acid sequences thereof; the light chain CDR1 comprises at least one selected from the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 10, and conservatively modified amino acid sequences thereof; the light chain CDR2 comprises at least one selected from the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 11, and conservatively modified amino acid sequences thereof; The light chain CDR3 comprises at least one selected from the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 12, and conservatively modified amino acid sequences thereof.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof comprises: 1) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:2, a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:3, a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:6, or 2) A heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:7, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:8, a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:9, a light chain CDR1 having the amino acid sequence shown in SEQ ID NO:10, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:11, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:12.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises an amino acid sequence that is 80% or greater identical to at least one of the amino acid sequences set forth in SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and conservatively modified forms thereof, and / or (ii) the light chain variable region comprises an amino acid sequence that is 80% or greater identical to at least one of the amino acid sequences set forth in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and conservatively modified forms thereof.

[0010] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a heavy chain variable region selected from (i), and the light chain variable region comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a light chain variable region selected from (ii).

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises: 1) the heavy chain variable region, e.g., the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region, e.g., the amino acid sequence shown in SEQ ID NO: 14; 2) the heavy chain variable region, e.g., the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region, e.g., the amino acid sequence shown in SEQ ID NO: 16; 3) the heavy chain variable region, e.g., the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region, e.g., the amino acid sequence shown in SEQ ID NO:18; or 4) The heavy chain variable region, for example, the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region, for example, the amino acid sequence shown in SEQ ID NO:20.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises: 1) the heavy chain, e.g., the amino acid sequence shown in SEQ ID NO:26, and the light chain, e.g., the amino acid sequence shown in SEQ ID NO:27; 2) the heavy chain, e.g., the amino acid sequence shown in SEQ ID NO: 28, and the light chain, e.g., the amino acid sequence shown in SEQ ID NO: 29; 3) the heavy chain, e.g., the amino acid sequence shown in SEQ ID NO:30, and the light chain, e.g., the amino acid sequence shown in SEQ ID NO:31; or 4) The heavy chain, for example, the amino acid sequence shown in SEQ ID NO:32, and the light chain, for example, the amino acid sequence shown in SEQ ID NO:33.

[0013] In some embodiments, the isolated antibody is an IgG1, IgG2, or IgG4.

[0014] In some embodiments, the antibody is a monoclonal antibody, a murine antibody, a chimeric antibody, a humanized antibody, a human-modified antibody, a human antibody, an Fv, a single-chain antibody (scFv), Fab, Fab', Fab'-SH, or F(ab')2.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof is capable of binding to the amino acid sequence set forth in SEQ ID NO:21.

[0016] In another aspect, the invention provides an immunoconjugate, the immunoconjugate comprising a therapeutic agent and an antibody or antigen-binding fragment thereof as described above conjugated to the therapeutic agent.

[0017] In another aspect, the present invention provides a composition, said composition comprising the above-described antibody or antigen-binding fragment thereof, and / or the above-described immunoconjugate, and a pharmaceutically acceptable vector.

[0018] In another aspect, the invention provides a kit for detecting B7H6 in a sample, the kit comprising the above-described antibody or antigen-binding fragment thereof.

[0019] In another aspect, the present invention provides the use of the above-described antibody or antigen-binding fragment thereof in the preparation of a reagent for detecting B7H6 in a sample.

[0020] In another aspect, the present invention provides the use of the above-described antibody or antigen-binding fragment thereof in the preparation of a medicament for preventing and / or treating a B7H6-mediated disease, wherein the B7H6-mediated disease is cancer or an infectious disease.

[0021] Optionally, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0022] In another aspect, the invention provides a nucleic acid comprising a nucleic acid encoding the above-described antibody or antigen-binding fragment thereof.

[0023] In another aspect, the present invention provides a recombinant vector or a transformant comprising the above-mentioned nucleic acid.

[0024] In one aspect, the present invention provides a recombinant cell, wherein the recombinant cell carries the above-described nucleic acid, expression vector, or transformant, or expresses the above-described antibody or antigen-binding fragment thereof.

[0025] In another aspect, the present invention provides a drug comprising the above-described antibody or antigen-binding fragment thereof, immunoconjugate, composition, drug, nucleic acid, recombinant vector, transformant, or recombinant cell.

[0026] In another aspect, the present invention provides use of the above-described antibody or antigen-binding fragment thereof, immunoconjugate, composition, drug, nucleic acid, recombinant vector, transformant, or recombinant cell in the treatment and / or prevention of B7H6-mediated diseases. As described above, the antibody or antigen-binding fragment thereof can effectively bind to B7H6, and further promote the interaction between B7H6 and NKp30, enhancing the anti-cancer function of NK cells. Therefore, the antibody or antigen-binding fragment thereof, and a substance obtained by expressing the antibody or antigen-binding fragment thereof under appropriate conditions, can all effectively prevent and / or treat B7H6-mediated diseases.

[0027] In another aspect, the present invention provides a method for treating and / or preventing a B7H6-mediated disease. According to an embodiment of the present invention, the method comprises administering to a subject at least one of the following: 1) the antibody or antigen-binding fragment thereof described above; 2) the immunoconjugate described above; 3) the composition described above; 4) the drug described above; 5) the nucleic acid described above; 6) the recombinant vector or transformant described above; and 7) the recombinant cell described above. As described above, the antibody or antigen-binding fragment thereof can effectively bind to B7H6 and further promote the interaction between B7H6 and NKp30, enhancing the anti-cancer function of NK cells. Therefore, the antibody or antigen-binding fragment thereof, as well as substances obtained by expressing the antibody or antigen-binding fragment thereof under appropriate conditions, or substances containing the antibody or antigen-binding fragment thereof, can all effectively prevent and / or treat a B7H6-mediated disease. According to an embodiment of the present invention, a B7H6-mediated disease can be effectively prevented and / or treated.

[0028] In another aspect, the present invention provides a method for diagnosing whether a subject has a B7H6-mediated disease. According to an embodiment of the present invention, the method includes detecting B7H6 in a sample to be detected using at least one of 1) the above-described antibody or antigen-binding fragment thereof, 2) the above-described nucleic acid, 3) the above-described recombinant vector or transformant, and 4) the above-described recombinant cell, and determining the content of B7H6 in the sample to be detected based on the B7H6 detection result. As described above, the antibody or antigen-binding fragment of an embodiment of the present invention can effectively bind to B7H6, and therefore can be used to detect B7H6. B7H6 mediates various diseases, and therefore, whether a subject has a B7H6-mediated disease can be determined based on the level of B7H6 contained in a biological sample from the subject. Furthermore, the above substances can be used to monitor the content of B7H6 in a sample to be detected from a subject, i.e., the above substances can also be used to stage disease in subjects with B7H6-mediated diseases and to evaluate the prognosis of B7H6-mediated diseases.

[0029] In another aspect, the present invention provides use of at least one of 1) the above-described antibody or antigen-binding fragment thereof, 2) the above-described nucleic acid, 3) the above-described recombinant vector or transformant, and 4) the above-described recombinant cell in diagnosing whether a subject has a B7H6-mediated disease. As described above, the antibodies or antigen-binding fragments of embodiments of the present invention can effectively bind to B7H6, and therefore can be used to detect B7H6, which mediates various diseases. Therefore, whether a subject has a B7H6-mediated disease can be determined based on the level of B7H6 contained in a biological sample from the subject.

[0030] By the above technical means, the present invention provides an anti-B7H6 antibody or antigen-binding fragment thereof capable of binding to human and monkey B7H6. The antibody or antigen-binding fragment thereof has at least one of the following properties: the ability to bind to human and monkey B7H6; the ability to promote the interaction between B7H6 and NKp30; and the ability to promote the anti-cancer function of NK cells.

[0031] Other features and advantages of the present invention are described in detail in the detailed description that follows.

[0032] Additional aspects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. [Brief explanation of the drawings]

[0033] The above and / or additional aspects and advantages of the present invention will become more apparent and understandable in the following description of the preferred embodiments, when taken in conjunction with the following drawings, in which: The drawings are intended to provide a further understanding of the invention, form a part of this specification, and, together with the following embodiments, are used to explain the invention but are not intended to be limiting thereof. [Figure 1] FIG. 1 shows the results of an ELISA for binding human B7H6 to a mouse-derived B7H6 antibody according to a specific embodiment of the present invention. [Figure 2] FIG. 1 shows the results of binding of HL60-B7H6 cells to a mouse-derived B7H6 antibody according to a specific embodiment of the present invention. [Figure 3] FIG. 1 shows the results of an ELISA for binding human B7H6 to humanized B7H6 antibodies according to specific embodiments of the present invention. [Figure 4] FIG. 1 shows the results of binding of HL60-B7H6 cells to a humanized B7H6 antibody according to a specific embodiment of the present invention. [Figure 5] FIG. 1 shows the results of binding of CHO-K1-cyno B7H6 cells to a humanized B7H6 antibody according to a specific embodiment of the present invention. [Figure 6] FIG. 1 shows ELISA results for binding monkey B7H6 to humanized B7H6 antibodies according to specific embodiments of the present invention. [Figure 7] FIG. 1 shows the results of the B7H6 antibody according to a specific embodiment of the present invention promoting the binding of the receptor NKp30. [Figure 8] FIG. 1 shows the results of B7H6 antibody-mediated complement-dependent cytotoxicity according to a specific embodiment of the present invention. [Figure 9] FIG. 1 shows the results of the B7H6 antibody according to a specific embodiment of the present invention promoting resistance to human acute myeloid leukemia U-937 tumors in nude mice. DETAILED DESCRIPTION OF THE INVENTION

[0034] The following detailed description of the preferred embodiments of the present invention will be given, examples of which are shown in the accompanying drawings. The preferred embodiments described below with reference to the accompanying drawings are merely illustrative and are intended to explain the present invention, and should not be construed as limiting the present invention.

[0035] It should be noted that the terms "first" and "second" are for explanatory purposes only and cannot be considered to indicate or imply relative importance or the number of technical features. Therefore, a feature defined as "first" or "second" can expressly or imply that it includes one or more of the feature, and in the description of the present invention, the concept of "plurality" is two or more than two, unless otherwise clearly and specifically limited.

[0036] Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described herein are used only to illustrate and explain the present invention, and are not intended to limit the present invention.

[0037] The range endpoints and any values ​​disclosed herein are not limited to the exact ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and the individual end values, and the individual end values ​​can be combined with each other to obtain one or more new numerical ranges that are to be considered specifically disclosed herein.

[0038] To facilitate understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs. Abbreviations for amino acid residues are the standard three-letter and / or one-letter codes for one of the 20 common L-amino acids used in the art.

[0039] In the present invention, unless otherwise specified, the term antigen-binding fragment used is "antibody fragment", and "antibody fragment" generally refers to an antigen-binding antibody fragment, which may include an intact portion of an antibody, generally the antigen-binding region or variable region, and examples of antibody fragments include Fab, Fab', F(ab')2, Fv or scFv, diabodies, linear antibodies, single-chain antibody molecules, etc.

[0040] The term "complementarity determining region" or "CDR" or "CDR sequence" refers to the amino acid sequences in an antibody that are responsible for antigen binding, e.g., generally around amino acid residues 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and around amino acid residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or a "hypervariable ring" (e.g., amino acid residues from about 26-32 (LI), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987))

[0041] The term "conservatively modified amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence, including amino acid substitution, addition, or deletion. Modifications can be introduced into the antibodies of the present invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. In a conservative amino acid substitution system, an amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of the antibodies of the invention may be substituted with other amino acid residues from the same side chain family, and the retained function of the altered antibody may be tested using the functional assay methods described herein. Preferably, conservative modifications do not exceed one or two in number.

[0042] In the context of peptides, the term "substantial homology" means that two peptides, or their specific sequences, when optimally aligned and compared (with appropriate insertion or deletion of nucleotides), have at least about 80% of the amino acids identical, generally at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the amino acids identical.

[0043] The percent identity between two sequences varies depending on the number of identical positions shared by the sequences when the sequences are optimally matched (i.e., % homology = number of identical positions / total number of positions × 100). The optimal matching system is determined by considering the number of empty digits that need to be introduced to achieve optimal matching of the two sequences and the length of each empty digit. Sequence matching and determination of percent identity between two sequences can be performed using a mathematical algorithm, as described in the non-limiting examples below.

[0044] Those skilled in the art can obtain variations in the sequence of the antibody or functional fragment thereof by substituting, adding, and / or deleting one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequence of the present invention, provided that the activity of the antibody is not substantially affected (retaining at least 95% activity). All such variations are considered to be within the scope of protection of the present invention. For example, amino acids with similar properties are substituted in the variable region. The sequence of the variant of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the reference sequence. Sequence identity according to the present invention can be determined using sequence analysis software, for example, using the computer program BLAST with default parameters, particularly BLASTP or TBLASTN. All amino acid sequences described in the present invention are shown from the N-terminus to the C-terminus.

[0045] As described above, the antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies), may contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), or may be modified to affect function. The present invention includes anti-B7H6 antibodies with modified glycosylation patterns. In some applications, modifications to remove undesired glycosylation sites may be useful, or the absence of fucosia moieties on the oligosaccharide chains may result in antibodies that enhance antibody-dependent cellular cytotoxicity (ADCC) function, for example. In other applications, galactosylation modifications can be performed to alter complement-dependent cytotoxicity (CDC).

[0046] The term "functional fragment" as used herein refers specifically to antibody fragments such as Fv, scFv (sc refers to single chain), Fab, F(ab')2, Fab', scFv-Fc fragments, or diabodies, or any fragment whose half-life can be increased by chemical modification or incorporation into liposomes. The chemical modification is, for example, the addition of a poly(alkoxy)diol such as polyethylene glycol ("PEGylation, PEGylation") (referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG pegylated fragments) ("PEG" refers to polyethylene glycol), and the fragment retains B7H6-binding activity. Preferably, the functional fragment consists of or includes a subsequence of the heavy or light chain variable region of the antibody from which it was derived. The subsequence is sufficient to retain binding specificity and sufficient affinity similar to that of the antibody from which it was derived, preferably at least 1 / 100 of the affinity of the antibody from which it was derived for B7H6. In more preferred embodiments, it is at least equal to 1 / 10. Such functional fragments include at least 5 amino acids, with 10, 15, 25, 50 and 100 contiguous amino acids of the antibody sequence from which they are derived being preferred.

[0047] As described above, the present invention provides an antibody or antigen-binding fragment thereof capable of binding to B7H6, wherein the antibody is characterized by comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3.

[0048] According to the present invention, the antibody is capable of binding to B7H6, particularly the amino acid sequence shown in SEQ ID NO:21.

[0049] In a preferred embodiment of the present invention, an antibody can be humanized to further enhance its bioacceptability. That is, the antibody is a chimeric or humanized antibody. The term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody from one species (e.g., mouse) with the constant region of an antibody from another species (e.g., human). The term "humanized antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the non-CDR (Fv framework region (FR)) amino acid sequences of the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with the constant and variable regions of an antibody from another species (e.g., human). In other words, if the constant region of one antibody is humanized, it is called a chimeric antibody, and if the non-CDR amino acid sequences of the constant and variable regions are fully humanized, it is called a humanized antibody. Humanization methods can be performed with reference to conventional antibody engineering techniques, but these will not be repeated here.

[0050] The sequence of the heavy chain variable region of the humanized antibody according to the present invention is shown in SEQ ID NO:17 and the sequence of the light chain variable region is shown in SEQ ID NO:18, or the sequence of the heavy chain variable region is shown in SEQ ID NO:19 and the sequence of the light chain variable region is shown in SEQ ID NO:20.

[0051] The immunoconjugates according to the invention comprise a therapeutic agent and an antibody or antigen-binding fragment thereof as described above conjugated to the therapeutic agent. The means for conjugating the antibody or antigen-binding fragment thereof to the therapeutic agent can be conventional means.

[0052] The compositions according to the present invention comprise the above-described antibody or antigen-binding fragment thereof, and / or the above-described immunoconjugate, and a pharmaceutically acceptable vector. In some embodiments, the compositions comprise a combination of components separated in time and / or space, as long as they function together to achieve the objectives of the present invention. For example, the components contained in the composition may be administered to a subject as a whole or separately. When the components contained in the composition are administered to a subject separately, the components may be administered to the subject simultaneously or sequentially.

[0053] The term "pharmaceutically acceptable" indicates that the composition can be administered to a subject without causing adverse physiological reactions that would prevent its administration. For example, a "pharmaceutically acceptable vector" refers to a vector that is generally safe, non-toxic, and useful for preparing a desired pharmaceutical composition. Preferred examples of such vectors or diluents include, but are not limited to, water, saline, Ringer's solution, glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used.

[0054] The compositions of the present invention can also be administered in combination with each other or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Thus, the compositions may also contain a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the present invention may also be combined with a second therapeutic agent. Exemplary second therapeutic agents include, but are not limited to, other agents that inhibit B7H6 activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with B7H6 upstream or downstream signaling.

[0055] Generally, the antibody or antigen-binding fragment thereof is administered in an effective amount, i.e., an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that causes prevention or alleviation of symptoms associated with the disease being treated, such as a B7H6-associated disease. The effective amount of the composition administered to a subject will depend on the type and severity of the disease, as well as the severity and type of disease, which will depend on individual characteristics such as general health, age, sex, weight, and tolerance to drugs; however, one of skill in the art can determine the appropriate dosage based on these and other factors.

[0056] A kit for detecting B7H6 in a sample according to the present invention contains the above-described antibody or an antigen-binding fragment thereof. The sample may be tissue from a patient with a B7H6-mediated disease (particularly a patient with transplant rejection, an autoimmune disease, an infectious disease, or cancer, more preferably a patient with at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer). The kit may also contain conventional reagents for detecting B7H6, such as a coating solution.

[0057] The present invention further provides use of the above-described antibody or antigen-binding fragment thereof in preparing a reagent for detecting B7H6 in a sample. As described above, the sample may be tissue from a patient with a B7H6-mediated disease, and further description is omitted here. The antibody or antigen-binding fragment thereof of the present invention has good affinity for B7H6 and can efficiently detect B7H6 in a sample.

[0058] The present invention further provides use of the antibody or antigen-binding fragment thereof, immunoconjugate, composition, nucleic acid, recombinant vector, transformant, or recombinant cell in the preparation of a medicament for preventing and / or treating a B7H6-mediated disease. Preferably, the B7H6-mediated disease is transplant rejection, an autoimmune disease, an infectious disease, or cancer. More preferably, the cancer is a B7H6-expressing cancer. Even more preferably, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer. More preferably, the infectious disease includes, but is not limited to, HIV virus infection and / or hepatitis B virus infection.

[0059] The present invention relates to a drug comprising the above-described antibody or antigen-binding fragment thereof, immunoconjugate, composition, nucleic acid, recombinant vector, transformant, or recombinant cell.

[0060] According to some specific embodiments of the present invention, the drug may include at least one of the following additional technical features:

[0061] According to some specific embodiments of the present invention, the drug comprises a pharmaceutically acceptable vector and an effective amount of the antibody or antigen-binding fragment thereof, immunoconjugate, composition, nucleic acid, recombinant vector, transformant, or recombinant cell.

[0062] The present invention further relates to methods for preventing and / or treating B7H6-mediated diseases (as described above). The methods comprise administering to a patient an effective amount of at least one of the antibodies or antigen-binding fragments thereof, immunoconjugates, compositions, recombinant cells, nucleic acids, and drugs of the present invention. The administration route may be oral, intranasal, intradermal, subcutaneous, intramuscular, intravenous, or intraperitoneal. As described above, the antibodies or antigen-binding fragments thereof can effectively bind to B7H6 and further promote the interaction between B7H6 and NKp30, thereby enhancing the anti-cancer function of NK cells. Therefore, the antibodies or antigen-binding fragments thereof, as well as substances obtained by expressing the antibodies or antigen-binding fragments thereof under appropriate conditions, or substances containing the antibodies or antigen-binding fragments thereof, can all effectively prevent and / or treat B7H6-mediated diseases. According to embodiments of the present invention, B7H6-mediated diseases can be effectively prevented and / or treated.

[0063] According to some specific embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0064] According to a specific embodiment of the present invention, the B7H6-mediated disease is cancer or an infectious disease.

[0065] According to a specific embodiment of the present invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0066] A "patient" or "subject" according to the present invention generally refers to a mammal, such as a primate and / or rodent, particularly a human or mouse.

[0067] Those skilled in the art can obtain an antibody or antigen-binding fragment thereof described in the present invention by cloning a DNA molecule encoding the antibody or antigen-binding fragment thereof into a vector (particularly an expression vector), transforming the vector into a host cell, and inducing expression of the antibody or antigen-binding fragment thereof. Therefore, the present invention further provides nucleic acids encoding the above-mentioned antibody or antigen-binding fragment thereof (isolated), as well as recombinant vectors and transformants containing the nucleic acids. The nucleic acid is preferably an expression cassette obtained by genetic engineering means.

[0068] The recombinant vector may refer to a clone vector or an expression vector. The recombinant vector can be obtained by operatively linking the nucleic acid with a commercially available vector (e.g., a plasmid or a viral vector). Commonly used plasmids include pSeTag2, PEE14, and pMH3.

[0069] The expression vector of the present invention may contain DNA sequences for the heavy chain variable region, light chain variable region, and / or constant region of the encoded antibody. However, two expression vectors may be constructed separately. One contains the heavy chain variable region and constant region, and the other contains the light chain variable region and constant region, and these vectors are co-transfected into mammalian cells. In one preferred embodiment, the expression vector further contains a promoter, a DNA sequence encoding a secretory signal peptide, and at least one antibiotic gene for screening.

[0070] The host cell described in the present invention may be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. The prokaryotic host cell may be Escherichia coli, Bacillus subtilis, Streptomyces, Proteus mirabilis, or the like. The eukaryotic host cell may be a fungus such as Pasteurella pichia, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, an insect cell such as an armyworm, a plant cell such as tobacco, or a mammalian cell such as a BHK cell, a CHO cell, a COS cell, or a myeloma cell. In some embodiments, the host cell described in the present invention is preferably a mammalian cell, more preferably a BHK cell, a CHO cell, an NSO cell, or a COS cell.

[0071] The present invention relates to recombinant cells carrying the above-described nucleic acid, recombinant vector, or transformant, or antibody or antigen-binding fragment. The recombinant cells can be obtained by transfecting or transforming the above-described expression vector. According to an embodiment of the present invention, the recombinant cells can effectively express the above-described antibody under appropriate conditions, and the antibody or antigen-binding fragment thereof can effectively bind to B7H6. Furthermore, the antibody or antigen-binding fragment has a good effect in preventing and / or treating B7H6-mediated diseases.

[0072] The recombinant cell described in the present invention is not particularly limited and may be a prokaryotic cell, a eukaryotic cell, or a bacteriophage. The prokaryotic cell may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, for example. The eukaryotic cell may be a fungus such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, an insect cell such as an armyworm, a plant cell such as tobacco, or a mammalian cell such as a BHK cell, a CHO cell, a COS cell, or a myeloma cell. In some embodiments, the recombinant cell described in the present invention is preferably a mammalian cell, including a BHK cell, a CHO cell, an NSO cell, or a COS cell, but not including an animal germ cell, a fertilized egg, or an embryonic stem cell.

[0073] The "appropriate conditions" referred to in the specification of the present application refer to conditions suitable for the expression of the antibody or antigen-binding fragment thereof. It will be readily understood by those skilled in the art that suitable conditions for the expression of an antibody or antigen-binding fragment include, but are not limited to, an appropriate transformation or transfection method, appropriate transformation or transfection conditions, a healthy host cell state, an appropriate host cell density, an appropriate cell culture environment, and an appropriate cell culture time. The "appropriate conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibody or antigen-binding fragment thereof depending on the specific environment of their laboratory.

[0074] The present invention further relates to the use of the above-described antibodies or antigen-binding fragments thereof, immunoconjugates, compositions, drugs, nucleic acids, recombinant vectors, transformants, or recombinant cells in the treatment and / or prevention of B7H6-mediated diseases. As described above, the antibodies or antigen-binding fragments thereof can effectively bind to B7H6, promote the interaction between B7H6 and NKp30, and enhance the anti-cancer function of NK cells. Therefore, the antibodies or antigen-binding fragments thereof, and substances obtained by expressing the antibodies or antigen-binding fragments thereof under appropriate conditions, can all effectively prevent and / or treat B7H6-mediated diseases.

[0075] According to a specific embodiment of the present invention, the above-mentioned use in the treatment and / or prevention of B7H6-mediated diseases may further comprise at least one of the following additional technical features:

[0076] According to a specific embodiment of the present invention, the B7H6-mediated disease is cancer or an infectious disease.

[0077] According to a specific embodiment of the present invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0078] In another aspect, the present invention provides a method for diagnosing whether a subject has a B7H6-mediated disease. According to a specific embodiment of the present invention, the method includes detecting B7H6 in a sample to be detected using at least one of 1) the above-described antibody or antigen-binding fragment thereof, 2) the above-described nucleic acid, 3) the above-described recombinant vector or transformant, and 4) the above-described recombinant cell, and determining the content of B7H6 in the sample to be detected based on the B7H6 detection result. As described above, the antibody or antigen-binding fragment of the present invention can effectively bind to B7H6, and therefore can be used to detect B7H6. B7H6 mediates various diseases, and therefore, whether a subject has a B7H6-mediated disease can be determined based on the level of B7H6 contained in a biological sample from the subject. Furthermore, the above substances can be used to monitor the content of B7H6 in a sample to be detected from a subject, i.e., the above substances can also be used to stage the disease in subjects with B7H6-mediated diseases and to evaluate the prognosis of B7H6-mediated diseases.

[0079] According to a specific embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0080] According to a specific embodiment of the present invention, the content of B7H6 in the test sample is equal to or greater than the minimum threshold for the disease, indicating that the test sample is derived from a patient with a B7H6-mediated disease. The minimum threshold value can be determined by differential comparison analysis and verification of the B7H6 content in the test sample from a number of individuals with a B7H6-mediated disease and a number of healthy individuals.

[0081] According to a specific embodiment of the present invention, the B7H6-mediated disease is cancer or an infectious disease.

[0082] According to a specific embodiment of the present invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0083] According to a specific embodiment of the present invention, the detection target sample includes at least one of tissue, cell, blood, serum, sweat, feces, and urine.

[0084] In another aspect, the present invention provides use of at least one of 1) the above-described antibody or antigen-binding fragment thereof, 2) the above-described nucleic acid, 3) the above-described recombinant vector or transformant, and 4) the above-described recombinant cell in diagnosing whether a subject has a B7H6-mediated disease. As described above, the antibodies or antigen-binding fragments of embodiments of the present invention can effectively bind to B7H6, and therefore can be used to detect B7H6, which mediates various diseases. Therefore, whether a subject has a B7H6-mediated disease can be determined based on the level of B7H6 contained in a biological sample from the subject.

[0085] According to a specific embodiment of the present invention, the above use further includes at least one of the following additional technical features:

[0086] According to a specific embodiment of the present invention, the B7H6-mediated disease is cancer or an infectious disease.

[0087] According to a specific embodiment of the present invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0088] A description of the sequences described in this invention is provided in Table 1.

[0089] [Table 1 (01)] [Table 1 (02)] [Table 1(03)] [Table 1 (04)] [Table 1 (05)] [Table 1 (06)]

[0090] Nucleic acids encoding the heavy and / or light chains of the antibodies of the present invention are within the scope of the present invention, and based on the amino acid sequences of the heavy and / or light chains, those skilled in the art can easily obtain the corresponding nucleic acid sequences, which are shown in Table 2.

[0091] [Table 2(01)] [Table 2(02)] [Table 2(03)] [Table 2(04)] [Table 2 (05)] [Table 2(06)] [Table 2(07)] [Table 2(08)] [Table 2 (09)] [Table 2 (10)] [Table 2(11)] [Table 2(12)] [Table 2(13)] [Table 2(14)] [Table 2(15)]

[0092] The present invention will be described in detail below with reference to examples. In the examples or detection examples, experimental methods for which no specific conditions are given are carried out according to ordinary conditions.

[0093] The present invention will be explained with reference to the following examples. The examples described below are for the purpose of illustrating the present invention, and the limitations of the present invention cannot be understood. If specific techniques or conditions are not shown in the examples, they should be carried out according to the techniques or conditions described in the literature in the relevant field or according to the product specifications. Reagents or instruments used are conventional products that are commercially available unless the manufacturer is specified.

[0094] Example 1 Preparation of antibodies

[0095] A mouse-derived monoclonal antibody against human B7H6 was produced and purified. Balb / c mice (9 weeks old, purchased from Shanghai SLAC, weighing approximately 20 g) were immunized with the purified recombinant B7H6 extracellular domain Fc fusion protein (B7H6-Fc) (recombinant B7H6 extracellular domain Fc fusion protein, amino acid sequence shown in SEQ ID NO: 22) as an antigen.

[0096] Immunized mice were immunized three times with purified antigen and complete Freund's adjuvant, and immune responses were detected after tail vein bleeding. Mice with anti-human B7H6 immunoglobulins were identified by screening the sera using ELISA and flow cytometry. Splenocytes from mice with the highest anti-B7H6 immunoglobulin levels were fused with mouse myeloma cells SP2 / 0 (ATCC No. CRL-1581). The fused hybridoma cells were screened for antibodies to obtain mouse monoclonal antibodies.

[0097] Candidate hybridoma cells were cultured until the total number reached 106, and then centrifuged at 800 rpm for 10 minutes to recover the cells. Total RNA was extracted using a Trizol kit (Invitrogen). A cDNA library (Invitrogen) was synthesized by reverse transcription using the total RNA as a template, and the corresponding variable region nucleic acid sequences of the hybridoma cells were amplified by PCR using the cDNA as a template. The primer sequences used in the PCR amplification reaction were complementary to the first frame region or signal peptide region and constant region of the antibody variable region (Larrick, JW, et al., (1990) Scand. J. Immunol., 32, 121-128 and Coloma, JJ, et al., (1991) BioTechniques, 11, 152-156). A 50 μl reaction mixture was prepared by adding 2 μL of cDNA, 5 μL of 10× PCR buffer, 2 μL (5 μmol) of upstream and downstream primers, 2 μL of dNTPs, 1 μL of Taq enzyme (Takara, Ex Taq), and 38 μL of HO. The mixture was pre-denatured at 95°C for 5 minutes, followed by temperature cycling and PCR amplification. The reaction conditions were as follows: 30 s denaturation at 94°C, 45 s annealing at 58°C, 50 s extension at 72°C, for a total of 32 cycles, and 7 min extension at 72°C. After sequencing the amplification product, the sequences of the heavy and light chain variable regions of the mouse monoclonal antibody (including amino acid and nucleic acid sequences) were obtained.

[0098] Example 2: ELISA binding experiment of mouse-derived B7H6 antibody

[0099] The ELISA experiment was conducted to detect the binding properties of the B7H6 antibody. The B7H6 extracellular domain Fc fusion protein (B7H6-Fc) was coated onto a 96-well plate, and the binding properties of the antibody to B7H6 were determined based on the signal intensity after addition of the antibody.

[0100] B7H6-Fc fusion protein (amino acid sequence shown in SEQ ID NO:22) was diluted to 1 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL per well and incubated overnight at 4°C. The PBS buffer in the 96-well plate was then aspirated, and the well plate was washed six times with PBST (pH 7.2 PBS containing 0.1% Tween 20). 200 μL per well of PBS / 10% BSA was then added and incubated at 37°C for 2 hours for blocking. The blocking agent was then removed, and the well plate was washed six times with PBST. 100 μL per well of the target B7H6 antibody, diluted to the appropriate concentration with PBST / 0.05% BSA, was added and incubated at 37°C for 1 hour. The reaction mixture was removed, and the well plate was washed six times with PBST. Then, 100 μL / well of HRP (horseradish peroxidase)-conjugated anti-mouse secondary antibody was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 h. After washing the well plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added, and the plate was incubated at room temperature for 3 min. The reaction was stopped by adding 80 μL / well of 4 M sulfuric acid. The absorbance was read at 450 nm using a microplate reader. The results (Figure 1) demonstrate that the mouse-derived antibodies 12F5 (SEQ ID NO:26 and SEQ ID NO:27) and 12G4 (SEQ ID NO:28 and SEQ ID NO:29) of the present invention can bind to B7H6.

[0101] Example 3: Flow cytometry binding experiments of mouse-derived B7H6 antibody

[0102] Flow cytometry experiments were used to detect the binding properties of the B7H6 antibody. B7H6 protein (HL60-B7H6, SEQ ID NO: 21; SEQ ID NO: 54) was overexpressed in HL60 cells (ATCC No. CCL-240) using the expression vector pLVX-EF1a-B7H6-IRES-ZsGreen. The signal intensity after addition of the antibody was used to determine the binding properties of the antibody to B7H6.

[0103] HEK293T cells were cultured at 5 x 10 5 Seed cells / well into a 6-well plate and culture overnight in DMEM medium without the double antibody. Before transfection, discard the medium and add 1 mL of fresh DMEM medium without the double antibody. pLVX-EF1a-B7H6-IRES-ZsGreen (the coding sequence (SEQ ID NO: 54) of the B7H6 protein (SEQ ID NO: 21) inserted between the EcoRI and BamHI restriction enzyme sites of the pLVX-EF1a-IRES-ZsGreen1 vector), pMD2G, and psPAX2 vectors (3 μg in total) were added to 200 μl of serum-free DMEM medium in a 2:1:1 ratio, followed by the addition of 12 μg of polyetherimide (PEI, Polysciences Co., Ltd.). After uniform mixing, the mixture was left to stand for 16 minutes, and then the entire liquid was added to a 6-well plate seeded with HEK293T cells. After 6 hours of incubation, the medium was discarded and fresh complete DMEM medium was added and incubated. After 48 hours of transfection, the cell culture supernatant was collected and passed through a 0.45 μm filter (Millipore) to obtain the viral supernatant. The viral supernatant was diluted to 1 x 10 4 Add all of this to a 6-well plate containing HL60 cells, add polybrene (Sigma) to a final concentration of 4 μg / ml, and culture for 12 hours. Then, discard the supernatant and add fresh complete RPMI 1640 medium. The resulting cells are HL60-B7H6 cells.

[0104] 2 x 10 HL60-B7H6 cells in PBS 6Dilute to 1 mL / mL and add 100 μL / tube to 1.5 mL EP tubes. Add 10 μL / tube of goat serum and block at 4°C for 30 minutes. Add 1 μg / tube of B7H6 antibody and incubate at 4°C for 30 minutes. Add 1 mL of PBS to the EP tube and centrifuge at 3500 rpm for 5 minutes at 4°C. Discard the supernatant and wash again with PBS. After centrifugation, discard the supernatant and resuspend the cells in 100 μL / tube of PBS. Add 0.1 μL / tube of Alexa-647-labeled goat anti-mouse secondary antibody (Invitrogen) and incubate at 4°C for 30 minutes in the dark. Wash twice with PBS, centrifuge, and discard the supernatant. The cells were resuspended in 200 μL / tube of PBS and detected using flow cytometry, the results of which are shown in Figure 2 and further demonstrate that the murine-derived antibodies 12F5 (SEQ ID NO:26 and SEQ ID NO:27), 12G4 (SEQ ID NO:28 and SEQ ID NO:29) of the present invention can bind to B7H6.

[0105] Example 4: Humanization experiment of mouse antibody

[0106] The humanization template that best matches the non-CDR regions of the B7H6 antibody is selected based on the light chain variable region and heavy chain variable region sequences. The CDR regions of the mouse-derived antibody are grafted onto the selected humanization template, replacing the CDR regions of the human-derived template to obtain a humanized antibody. Based on the three-dimensional structure of the mouse-derived antibody, revert mutations are then performed on buried residues, residues that directly interact with the CDR regions, and residues that have a significant impact on the three-dimensional structure of the VL and VH to obtain a humanized antibody. The sequence of the heavy chain variable region of the humanized B7H6 antibody 12F5 is shown in SEQ ID NO: 17, and the sequence of the light chain variable region is shown in SEQ ID NO: 18. Alternatively, the sequence of the heavy chain variable region of the humanized B7H6 antibody 12G4 is shown in SEQ ID NO: 19, and the sequence of the light chain variable region is shown in SEQ ID NO: 20.

[0107] Example 5: ELISA binding experiments of humanized B7H6 antibodies

[0108] ELISA experiments were used to detect the binding properties of the humanized B7H6 antibody. B7H6 extracellular domain Fc fusion protein (B7H6-Fc) was coated onto a 96-well plate, and the binding properties of the antibody to B7H6 were determined using the signal intensity after antibody addition.

[0109] B7H6-Fc fusion protein (amino acid sequence shown in SEQ ID NO:22) was diluted to 1 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL per well and incubated at 4°C overnight. The PBS buffer in the 96-well plate was then aspirated, and the well plate was washed six times with PBST (pH 7.2 PBS containing 0.1% Tween 20). 200 μL per well of PBS / 10% BSA was then added and incubated at 37°C for 2 hours for blocking. The blocking agent was then removed, and the well plate was washed six times with PBST. 100 μL per well of the target B7H6 antibody, diluted to the appropriate concentration in PBST / 0.05% BSA, was added and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the well plate six times with PBST, 100 μL / well of HRP (horseradish peroxidase)-conjugated mouse anti-human IgG (Fab-specific) secondary antibody (Sigma) was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the well plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added, and the plate was incubated at room temperature for 3 minutes. 80 μL / well of 4M sulfuric acid was added to stop the reaction. The absorbance was read at 450 nm using a microplate reader. The results (Figure 3) demonstrate that the humanized antibodies h12F5 (SEQ ID NO:30 and SEQ ID NO:31) and h12G4 (SEQ ID NO:32 and SEQ ID NO:33) of the present invention can bind to B7H6.

[0110] Example 6: Flow cytometry binding experiments of humanized B7H6 antibodies

[0111] Flow cytometry experiments were used to detect the binding properties of the B7H6 antibody. B7H6 protein (HL60-B7H6) (e.g., as shown in Example 3) was overexpressed in HL60 cells (ATCC No. CCL-240), and the binding properties of the antibody to B7H6 were determined using the signal intensity after addition of the antibody.

[0112] 2 x 10 HL60-B7H6 cells in PBS 6 Dilute to 1 mL / mL and add 100 μL / tube to 1.5 mL EP tubes. Add 10 μL / tube of mouse serum to each tube and block for 30 min at 4°C. Add 1 μg / tube of B7H6 antibody and incubate for 30 min at 4°C. Add 1 mL of PBS to the EP tube and centrifuge at 3500 rpm for 5 min at 4°C. Discard the supernatant and wash again with PBS. After centrifugation, discard the supernatant and resuspend the cells in 100 μL / tube of PBS. Add 1 μL / tube of Alexa-647-labeled mouse anti-human IgG-Fc secondary antibody (Biolegend) to each tube and incubate for 30 min in the dark at 4°C. Wash twice with PBS, centrifuge, and discard the supernatant. The cells were resuspended in 200 μL / tube of PBS and detected using flow cytometry, the results of which are shown in Figure 4 and further demonstrate that the humanized antibodies of the present invention, h12F5 (SEQ ID NO:30 and SEQ ID NO:31), h12G4 (SEQ ID NO:32 and SEQ ID NO:33), can bind to B7H6.

[0113] Example 7: Flow cytometry binding experiments of humanized B7H6 antibodies

[0114] Flow cytometry experiments were used to detect the binding properties of the humanized B7H6 antibody. CHO-K1 cells (ATCC No. CCL-240) were overexpressed with monkey B7H6 protein (CHO-K1-cyno-B7H6), and the signal intensity after antibody addition was used to determine the binding properties of the antibody to B7H6.

[0115] 5 × 10 HEK293T cells 5Seed cells / well into a 6-well plate and culture overnight in DMEM medium without double antibodies. Discard the medium before transfection and add 1 mL of fresh DMEM medium without double antibodies. pLVX-EF1a-cynoB7H6-IRES-ZsGreen (a coding sequence (SEQ ID NO: 57) of the monkey B7H6 protein (SEQ ID NO: 24) inserted between the EcoRI and BamHI restriction enzyme sites of the pLVX-EF1a-IRES-ZsGreen1 vector), pMD2G, and psPAX2 vector (3 μg in total) were added to 200 μL of serum-free DMEM medium in a 2:1:1 ratio, and 12 μg of polyetherimide (PEI, Polysciences Co., Ltd.) was added. After uniform mixing, the mixture was left to stand for 16 minutes, and then the entire liquid was added to a 6-well plate seeded with HEK293T cells. After 6 hours of incubation, the medium was discarded and fresh complete DMEM medium was added and incubated. After 48 hours of transfection, the cell culture supernatant was collected and passed through a 0.45 μm filter (Millipore) to obtain the viral supernatant. All viral supernatants were diluted to 1 × 10 4 Add polybrene (Sigma) to a final concentration of 4 μg / ml to a 6-well plate containing CHO-K1 cells and culture for 12 hours. Then, discard the supernatant and add fresh complete RPMI 1640 medium. The resulting cells are CHO-K1-cyno B7H6 cells.

[0116] CHO-K1-cynoB7H6 cells were diluted to 2 x 106 / mL with PBS and added to 1.5 mL EP tubes at a volume of 100 μL / tube. 10 μL / tube of mouse serum was added and blocked at 4°C for 30 minutes. 1 μg / tube of humanized B7H6 antibody was added and incubated at 4°C for 30 minutes. 1 mL of PBS was added to the EP tube and centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was discarded and the cells were washed again with PBS. After centrifugation, the supernatant was discarded and the cells were resuspended in 100 μL / tube of PBS. 1 μL / tube of Alexa-647-labeled mouse anti-human IgG-Fc secondary antibody (Biolegend) was added and incubated in the dark at 4°C for 30 minutes. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL / tube of PBS and detected using flow cytometry, and the results, as shown in FIG. 5, further demonstrate that the antibodies h12F5, h12G4 of the present invention can bind to monkey B7H6.

[0117] Example 8: ELISA binding experiments of humanized B7H6 antibodies

[0118] ELISA experiments were used to detect the binding properties of the humanized B7H6 antibody. The monkey B7H6 extracellular domain Fc fusion protein (cynoB7H6-Fc) was coated onto a 96-well plate, and the binding properties of the antibody to B7H6 were determined using the signal intensity after antibody addition.

[0119] The cynoB7H6-Fc fusion protein (amino acid sequence shown in SEQ ID NO:23) was diluted to 1 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL per well and incubated at 4°C overnight. The PBS buffer in the 96-well plate was then aspirated, and the well plate was washed six times with PBST (pH 7.2 PBS containing 0.1% Tween 20). 200 μL per well of PBS / 10% BSA was then added and incubated at 37°C for 2 hours for blocking. The blocking agent was then removed, and the well plate was washed six times with PBST. 100 μL per well of the target B7H6 antibody, diluted to the appropriate concentration in PBST / 0.05% BSA, was added and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the well plate six times with PBST, 100 μL / well of HRP (horseradish peroxidase)-labeled mouse anti-human IgG (Fab-specific) secondary antibody (Sigma) was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the well plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes. 80 μL / well of 4M sulfuric acid was added to stop the reaction. The absorbance was read at 450 nm using a microplate reader. The results (Figure 6) demonstrate that the humanized antibody h12G4 of the present invention can bind to monkey B7H6.

[0120] Example 9: In vitro binding affinity and kinetics experiments

[0121] The Biacore method is a known detection method for objectively detecting affinity and binding kinetics between proteins, and the affinity and binding kinetics are characterized by analyzing the B7H6 antibody of the present invention using a Biacore T200.

[0122] Using standard amino acid coupling techniques, B7H6 extracellular Fc fusion protein (B7H6-Fc) was covalently coupled to a CM5 (GE) chip. A gradient series of B7H6 antibodies diluted in PBS was injected in each cycle, followed by regeneration with 10 mM NaOH solution. Antigen-antibody binding kinetics were monitored for 3 minutes and dissociation kinetics for 10 minutes. The data were analyzed using GE's BIAevaluation software with a 1:1 (Langmuir) binding model. The KD values ​​for murine 12F5 and murine 12G4, determined by this method, are 2.2 nM and 0.15 nM, respectively.

[0123] Example 10: Screening of antibodies for their ability to promote B7H6 binding to NKp30

[0124] B7H6 antibody promotes the signal transduction pathway of B7H6 and its receptor NKp30 by binding to the extracellular domain of B7H6, and flow cytometry experiments are used to detect the enhanced binding of B7H6 antibody to the receptor NKp30.

[0125] 2 × 10 HL60-B7H6 cells (same as above) in PBS 6Dilute to 1 / ml and add 100 μL / tube to 1.5 mL EP tubes. Add 10 μL / tube of mouse serum and block for 30 min at 4°C. Add 4 μg / tube of NKp30 extracellular domain Fc fusion protein (NKp30-Fc, SEQ ID NO:25, expressed in this laboratory) and incubate for 30 min at 4°C. Add B7H6 antibody and incubate for 30 min at 4°C. Add 1 mL of PBS to the EP tube and centrifuge at 3500 rpm for 5 min at 4°C. Discard the supernatant and wash again with PBS. After centrifugation, discard the supernatant and resuspend the cells in PBS in 100 μL / tube. Add 1 μL / tube of 647-labeled mouse anti-human secondary antibody (Biolegend) and incubate for 30 min in the dark at 4°C. Wash twice with PBS, centrifuge, and discard the supernatant. The cells were resuspended in 200 μL / tube of PBS and detected using flow cytometry. The results, shown in Figure 7, demonstrate that the mouse-derived 12F5 antibody of the present invention can promote the binding of B7H6 to the receptor NKp30.

[0126] Example 11: Complement-dependent cytotoxicity detection

[0127] Complement-dependent cytotoxicity experiments are used to detect the effect of B7H6 antibody in killing tumor cells, and the tumor cells used are HL60-B7H6 cells.

[0128] (1) Tumor cells are resuspended in serum-free RPMI-1640 medium and counted. 4 × 10 cells were counted using complete RPMI-1640 medium. 5 Dilute the diluted cells to 100 μL / mL. Add the diluted cells in a volume of 25 μL / well to a 96-well round-bottom plate. (2) Dilute B7H6 antibody or control mouse IgG in serum-free RPMI-1640 medium, and add the diluted B7H6 antibody or mouse IgG to a 96-well round-bottom plate in a volume of 25 μL / well. (3) Dissolve rabbit complement (Cedarlane) in 1 mL of ultrapure water, add 1 mL of serum-free RPMI-1640 medium, mix thoroughly, and use as the working solution. Add 40 μL of the working solution to a 96-well round-bottom plate. Incubate at 37°C for 30 minutes. (4) Add complete RPMI-1640 medium to a 96-well round-bottom plate at a volume of 110 μL / well, and add 7-AAD (BD) to the 96-well round-bottom plate at a volume of 1 μL / well, and mix evenly. Transfer all liquid from the wells to a flow cytometry tube and detect using flow cytometry. The results, as shown in Figure 8, show that the B7H6 mouse-derived 12F5 antibody of the present invention has complement-dependent cytotoxicity.

[0129] Example 12: Enhancement of anti-cancer activity in mice by B7H6 antibody

[0130] In vivo efficacy experiments were performed to detect the anti-tumor activity of B7H6 antibody in nude mice, using acute myeloid leukemia U-937 cells (ATCC No. CRL-1953.2).

[0131] (1) Right ventral subcutaneous tumors in nude mice, 1.5 × 10 cells per mouse 6 There are U-937 cells, and the mice are randomly grouped. (2) On days 5, 7, 9, 11, and 13, inject the antibody intraperitoneally into the mice, administering 1 mg per mouse. (3) Tumor volume is measured every two days.

[0132] The results, as shown in FIG. 9, demonstrate that the mouse-derived antibody 12F5 of the present invention has anti-cancer function.

[0133] As can be seen from the above experimental results, the antibodies obtained in the present invention can bind to human and monkey B7H6, promote the interaction between B7H6 and NKp30, and promote anti-cancer activity in mice.

[0134] Although the preferred embodiments of the present invention have been described in detail above, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications of the technical solutions of the present invention may be implemented, including combinations of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as the contents disclosed in the present invention, and all fall within the scope of protection of the present invention.

[0135] In the description herein, when a description includes terms such as "one embodiment," "some embodiments," "exemplary," "particular examples," or "some examples," it is intended that the specific feature, structure, material, or advantage described using the embodiment or example is included in at least one embodiment or example of the present invention. In the description herein, the use of exemplary terms does not necessarily refer to the same embodiment or example. It should be noted that the specific features, structures, materials, or advantages described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, unless mutually inconsistent, those skilled in the art may blend or combine different embodiments or examples and features of different embodiments or examples described herein.

[0136] Although the embodiments of the present invention have been shown and described above, the above embodiments are merely illustrative and do not limit the scope of the present invention, and those skilled in the art can change, modify, substitute, and alter the above embodiments within the scope of the present invention.

[0137] This application claims priority from a Chinese patent application with application number 202111485380.9, filed with the China Patent Office on December 7, 2021, the entire contents of which are incorporated herein by reference.

Claims

1. An antibody or antigen-binding fragment thereof capable of binding to B7H6, said antibody or antigen-binding fragment thereof comprising: 1) a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6; or 2) The antibody or antigen-binding fragment thereof, comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 7, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8, a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 9, a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 10, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 11, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

12.

2. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and (i) the heavy chain variable region comprises an amino acid sequence having 90% or more identity to at least one of the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, and / or (ii) the light chain variable region comprises an amino acid sequence having 90% or more identity to at least one of the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO:

20.

3. 4. The antibody or antigen-binding fragment thereof of claim 3, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a heavy chain variable region selected from (i), and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a light chain variable region selected from (ii).

4. The antibody or antigen-binding fragment thereof 1) the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 14; 2) the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 16; 3) the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 17 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 18; or 4) The antibody or antigen-binding fragment thereof according to claim 1, comprising the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 19 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:

20.

5. The antibody or antigen-binding fragment thereof 1) the amino acid sequence of the heavy chain shown in SEQ ID NO: 26 and the amino acid sequence of the light chain shown in SEQ ID NO: 27; 2) the heavy chain amino acid sequence shown in SEQ ID NO: 28 and the light chain amino acid sequence shown in SEQ ID NO: 29; 3) the heavy chain amino acid sequence shown in SEQ ID NO: 30 and the light chain amino acid sequence shown in SEQ ID NO: 31; or 4) The antibody or antigen-binding fragment thereof according to claim 1, comprising the heavy chain amino acid sequence shown in SEQ ID NO: 32 and the light chain amino acid sequence shown in SEQ ID NO:

33.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the isolated antibody is IgG1, IgG2, or IgG4.

7. The antibody may be a monoclonal antibody, a mouse-derived antibody, a chimeric antibody, a humanized antibody, a human-modified antibody, an Fv, a single-chain antibody (scFv), an Fab, an Fab', an Fab'-SH, or an F(ab') ２ The antibody or antigen-binding fragment thereof of claim 1,

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is capable of binding to the amino acid sequence shown in SEQ ID NO:

21.

9. 10. An immunoconjugate comprising a therapeutic agent and the antibody or antigen-binding fragment thereof of claim 1 conjugated to the therapeutic agent.

10. A composition comprising the antibody or antigen-binding fragment thereof of claim 1 and a pharmaceutically acceptable vector.

11. A kit for detecting B7H6 in a sample, the kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a reagent for detecting B7H6 in a sample.

13. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 8 in the preparation of a medicament for preventing and / or treating a B7H6-mediated disease, wherein the B7H6-mediated disease is cancer or an infectious disease.

14. 14. The use of claim 13, wherein the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

15. A nucleic acid comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof of claim 1.

16. A recombinant vector or transformant comprising the nucleic acid of claim 15.

17. A recombinant cell carrying the antibody or antigen-binding fragment thereof of claim 1.

18. A drug comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the immune complex according to claim 9, the composition according to claim 10, the nucleic acid according to claim 15, the recombinant vector or transformant according to claim 16, or the recombinant cell according to claim 17.

19. A therapeutic agent for treating and / or preventing a B7H6-mediated disease, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, an immune complex according to claim 9, a composition according to claim 10, a nucleic acid according to claim 15, a recombinant vector or transformant according to claim 16, or a recombinant cell according to claim 17.

20. The therapeutic agent of claim 19, wherein the B7H6-mediated disease is cancer or an infectious disease.

21. The therapeutic agent of claim 20, wherein the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

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