High-affinity b7h7 antibody or antigen-binding fragment thereof and use thereof

Abstract

The present invention relates to the field of antibodies. Provided are a high-affinity B7H7 antibody or an antigen-binding fragment thereof and the use thereof. The antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (HCDR) and a light chain complementarity determining region (LCDR), wherein the HCDR contains an amino acid sequence selected from at least one of SEQ ID NOs: 1-3 or having at least 80% homology thereto; and the LCDR contains an amino acid sequence selected from at least one of SEQ ID NOs: 4-6 or having at least 80% homology thereto. The antibody or antigen-binding fragment has high specificity and high affinity, and is capable of binding to human and cynomolgus monkey B7H7, specifically blocking the binding of B7H7 to the receptor KIR3DL3 thereof, promoting the killing of tumor cells by immune cells, and effectively treating and / or preventing related diseases mediated by B7H7, such as tumors.

Description

High affinity b7h7 antibodies or antigen binding fragments thereof and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of antibodies, and in particular, relates to high affinity B7H7 antibodies or antigen binding fragments thereof and uses thereof. BACKGROUND

[0002] In recent years, anti-tumor immunotherapy targeting immune checkpoints has achieved great success in clinical application. Very representative multi-humanized or fully human monoclonal antibodies targeting CTLA-4 and PD-1 / PD-L1 have been approved for marketing and applied to the treatment of various tumors. However, the overall response rate of patients is only 30%, and a large number of clinical patients cannot benefit. Therefore, it is necessary to find new immune checkpoint molecules and new combination therapy strategies.

[0003] B7H7 is also known as HHLA2, which belongs to type I transmembrane protein, consisting of three extracellular Ig-like domains, a transmembrane region, and a 49-amino acid long cytoplasmic tail without recognizable motifs. B7H7 belongs to one of the B7 family members, and has about 20%-30% similarity with other members of the B7 family. In normal tissues, B7-H7 is mainly expressed in gastrointestinal tract, bile duct and other tissues, and is not expressed in other normal tissues. However, in many tumor types such as breast cancer, lung cancer, intestinal cancer, pancreatic cancer and the like, B7H7 is in a high expression state, and is negatively correlated with the prognosis of patients.

[0004] It has been reported that B7H7 can bind to its receptor KIR3DL3, thereby inhibiting the effector function of NK cells and T cells. Blocking the binding of B7H7 to KIR3DL3 by blocking antibody can significantly restore the anti-tumor function of NK cells and T cells. Therefore, targeting the B7H7-KIR3DL3 signaling pathway is very promising.

[0005] Currently, there are relatively few clinical studies on B7H7, and only HBM1020 of Hengrui Medicine is in the clinical phase I stage. Therefore, it is urgent to develop a high affinity therapeutic anti-B7H7 antibody. SUMMARY

[0006] The present application is based on the inventors' discovery of the following problems and facts:

[0007] B7-H7 is mainly expressed in gastrointestinal tract, bile duct and other tissues, and is not expressed in other normal tissues. However, in many tumor types such as breast cancer, lung cancer, intestinal cancer, pancreatic cancer and the like, B7H7 is in a high expression state, and is negatively correlated with the prognosis of patients.

[0008] It has been reported that B7H7 can bind to its receptor KIR3DL3, thereby inhibiting the effector function of NK cells and T cells. Blocking the binding of B7H7 to KIR3DL3 by blocking antibodies can significantly restore the anti-tumor function of NK cells and T cells.

[0009] The inventors of the present application obtained high-affinity humanized anti-B7H7 antibodies through affinity maturation. The inventors found that the high-affinity humanized anti-B7H7 antibodies obtained in the present application can specifically target and bind to B7H7, block the binding of B7H7 to its receptor KIR3DL3, promote the killing of tumor cells by immune cells, and thus effectively treat and / or prevent B7H7-mediated related diseases such as tumors.

[0010] Specifically, the present application provides the following technical solutions:

[0011] In the first aspect of the present application, an antibody or an antigen binding fragment thereof is provided. According to embodiments of the present application, it comprises: a heavy chain complementarity determining region (HCDR) and a light chain complementarity determining region (LCDR); wherein the HCDR comprises at least one of the amino acid sequences selected from SEQ ID NO: 1-3 or an amino acid sequence having at least 80% homology thereto; and the LCDR comprises at least one of the amino acid sequences selected from SEQ ID NO: 4-6 or an amino acid sequence having at least 80% homology thereto. In some examples of the present application, the aforementioned antibody or antigen binding fragment has high specificity and high affinity, can bind to human and cynomolgus monkey B7H7, and specifically block the binding of B7H7 to its receptor KIR3DL3, promote the killing of tumor cells by immune cells, and effectively treat and / or prevent B7H7-mediated related diseases such as tumors.

[0012] In the second aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule encodes the antibody or antigen binding fragment thereof of the first aspect of the present application. In some examples of the present application, the aforementioned nucleic acid molecule can be used to prepare a large amount of the aforementioned antibody or antigen binding fragment thereof in vitro.

[0013] In the third aspect of the present application, an expression vector is provided. According to embodiments of the present application, the expression vector carries the nucleic acid molecule of the second aspect of the present application. In some examples of the present application, the expression vector can efficiently express the aforementioned antibody or antigen binding fragment thereof in a suitable host cell.

[0014] In a fourth aspect of the present application, a method for preparing the antibody or antigen-binding fragment thereof of the first aspect is provided. According to embodiments of the present application, the method comprises: introducing the expression vector of the third aspect into a cell; and culturing the cell under conditions suitable for protein expression and secretion, so as to obtain the antibody or antigen-binding fragment thereof. In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof can be efficiently prepared by the aforementioned method.

[0015] In a fifth aspect of the present application, a recombinant cell is provided. According to embodiments of the present application, the recombinant cell carries the antibody or antigen-binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, or the expression vector of the third aspect. The recombinant cell is obtained by transfecting or transforming the expression vector. In some examples of the present application, the recombinant cell can efficiently express the aforementioned antibody or antigen-binding fragment thereof under suitable conditions.

[0016] In a sixth aspect of the present application, an immunoconjugate is provided. According to embodiments of the present application, the immunoconjugate comprises: the antibody or antigen-binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. As previously described, the antibody or antigen-binding fragment thereof can effectively bind to human and cynomolgus monkey B7H7 protein. Therefore, the immunoconjugate comprising the antibody or antigen-binding fragment thereof can also bind to human and cynomolgus monkey B7H7 protein, and the aforementioned immunoconjugate has a good effect in preventing and / or treating B7H7-mediated tumor diseases, such as head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, renal cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia, etc.

[0017] In a seventh aspect of the present application, a composition is provided. According to embodiments of the present application, the composition comprises: the antibody or antigen-binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the immunoconjugate of the sixth aspect. As previously described, the antibody or antigen-binding fragment thereof can effectively bind to human and cynomolgus monkey B7H7 protein. Therefore, the composition comprising the antibody or antigen-binding fragment thereof, such as a food composition, a pharmaceutical composition, etc., can also effectively treat or prevent tumor diseases, such as head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, renal cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia, etc.

[0018] In an eighth aspect, the present application provides use of the antibody or antigen binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, the immunoconjugate of the sixth aspect, or the composition of the seventh aspect in the manufacture of a medicament for treating or preventing a B7H7-mediated disease. As described above, the antibody or antigen binding fragment thereof is capable of binding to human and cynomolgus monkey B7H7, and thus, the medicament comprising the antibody or antigen binding fragment thereof and the like has a significant effect on treating and / or preventing a B7H7-mediated tumor disease (e.g., head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia).

[0019] In a ninth aspect, the present application provides a medicament. According to an embodiment of the present application, the medicament comprises the antibody or antigen binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, the immunoconjugate of the sixth aspect, or the composition of the seventh aspect, and is used for treating or preventing a B7H7-mediated disease. As described above, the antibody or antigen binding fragment thereof is capable of binding to human and cynomolgus monkey B7H7. Thus, the medicament comprising the antibody or antigen binding fragment thereof and the like has a significant effect on treating and / or preventing a B7H7-mediated tumor disease (e.g., head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia).

[0020] In a tenth aspect, the present application provides use of the antibody or antigen binding fragment thereof of the first aspect in the manufacture of a kit for detecting B7H7. As described above, the antibody or antigen binding fragment thereof is capable of binding to human and cynomolgus monkey B7H7, and thus, the kit comprising the antibody or antigen binding fragment thereof can be used to effectively detect B7H7. In some examples of the present application, the aforementioned kit can be used for scientific research, such as qualitative or quantitative detection of B7H7 protein in a biological sample.

[0021] In an eleventh aspect, the present application provides a kit. According to embodiments of the present application, the kit comprises the antibody or antigen binding fragment thereof of the first aspect. As previously described, the antibody or antigen binding fragment thereof is capable of binding to human and cynomolgus B7H7 effectively, thus the kit comprising the antibody or antigen binding fragment thereof can be used to detect B7H7 effectively. In some examples of the present application, the kit can be used for scientific research, such as qualitative or quantitative detection of B7H7 protein in a biological sample, and can also be used to determine the state of an individual, such as determining whether the B7H7 level of the individual is higher or lower than the normal level after obtaining the B7H7 level of the individual.

[0022] Additional aspects and advantages of the present application will be made apparent from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the following embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0024] Figure 1 is an ELISA result schematic diagram of the humanized antibody provided by the embodiments of the present application binding to human and cynomolgus B7H7 protein;

[0025] Figure 2 is a flow cytometry result schematic diagram of the humanized antibody provided by the embodiments of the present application binding to HCC827 cell line overexpressing B7H7;

[0026] Figure 3 is an ELISA result schematic diagram of the humanized antibody provided by the embodiments of the present application blocking the binding of B7H7 protein to KIR3DL3 protein;

[0027] Figure 4 is a flow cytometry result schematic diagram of the humanized antibody provided by the embodiments of the present application blocking the binding of B7H7 protein to CHO-K1-KIR3DL3 cells;

[0028] Figure 5 is a flow cytometry result schematic diagram of the humanized antibody provided by the embodiments of the present application promoting PBMC killing K562 cells overexpressing B7H7. DETAILED DESCRIPTION

[0029] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0030] In the present application, the terms "first", "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, such as two, three, etc., unless otherwise explicitly specified.

[0031] In the present application, the term "antibody" refers to an immunoglobulin, which is a four-chain structure connected by inter-chain disulfide bonds, consisting of two identical heavy chains and two identical light chains. The antigenicity of immunoglobulin heavy chain constant region is different due to different amino acid composition and arrangement order. Immunoglobulins can be divided into five types, namely IgM, IgD, IgG, IgA, and IgE. Immunoglobulins of the same type can also be divided into different subtypes according to the amino acid composition, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin light chains are divided into kappa chains or lambda chains according to the constant region.

[0032] In the present application, the term "antibody affinity maturation" refers to an antibody obtained by mutating one or more CDR sequences of an antibody. In some examples of the present application, an antibody with higher affinity is obtained by affinity maturation of antibody h17H7B8 (application number: 202311708700.1).

[0033] In the present application, the antibody heavy chain variable region can further comprise a heavy chain constant region, which comprises human IgG1, IgG2, IgG3, IgG4, or a variant thereof.

[0034] In the present application, the antibody light chain variable region can further comprise a light chain constant region, which comprises human kappa, lambda chain, or a variant thereof.

[0035] The sequence of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly, which is the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable, which is the constant region. The variable region includes 3 hypervariable regions (HVR) and 4 relatively conserved framework regions (FR). The 3 hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminal to the carboxyl terminal. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0036] In the present application, the term "humanized antibody" refers to an antibody or antibody fragment in which all or part of the CDR regions of a human immunoglobulin (recipient antibody) are replaced by the CDR regions of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. In addition, some of the amino acid residues of the framework regions (FR) of the recipient antibody can also be replaced by the corresponding amino acid residues of the non-human antibody, or by the amino acid residues of other antibodies, in order to further improve or optimize the performance of the antibody.

[0037] In the present application, the term "antigen-binding fragment" refers to Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments, ScFv fragments that bind to human B7H7; which comprise one or more CDR regions of the antibodies described in the present application selected from SEQ ID NO: 1 to SEQ ID NO: 6. Fv fragments contain the variable regions of the heavy and light chains of an antibody, but not the constant regions, and are the smallest antibody fragments that have all of the antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. Two antibody variable regions can also be joined by a different linker to form a single polypeptide chain known as a single-chain antibody or single-chain Fv.

[0038] In the present application, the amino acid sequences of the listed CDRs are shown according to the Kabat definition rule. However, it is well known in the art that the CDRs of an antibody can be defined by various methods in the art, such as the IMGT rule, the Chothia rule, and the like. It is understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or region thereof (e.g., variable region) are understood to encompass the complementary determining regions defined by any of the above known schemes described in the present application. Although the scope of protection claimed in the present application is based on the sequences shown according to the Kabat definition rule, the amino acid sequences corresponding to the CDRs defined according to other CDR definition rules should also be included in the scope of protection of the present application.

[0039] In the present application, the term "amino acid" is represented by the single letter or three letter code, having the following meaning: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gin (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: His (histidine); I: He (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0040] For nucleotides, the terms "homology", "identity" or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding positions] by [the number of nucleotides in the second sequence]. The number of nucleotides in the second sequence is subtracted from the total number of nucleotides in the first sequence, and then multiplied by [100%], wherein each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - relative to the first nucleotide sequence - is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A.

[0041] For polypeptides, the terms "(substantial) homology", "identity" or "similarity" are used to describe or compare the degree of amino acid similarity between two or more polypeptides or specified sequences thereof when optimally aligned and compared (with appropriate insertion or deletion of nucleotides, if needed). The % homology between two sequences varies as the number of identical positions in these sequences (i.e. % homology = number of identical positions / total number of positions x 100) upon optimal alignment of the sequences, wherein optimal alignment is determined taking into account the number of gaps needed to achieve optimal alignment of the two sequences and the length of each gap. Sequence comparison and determination of percent sequence identity between two sequences can be accomplished using a mathematical algorithm, such as described in the non-limiting examples below.

[0042] In the present application, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids can be substituted, added and / or deleted from the sequences of the present application by one of skill in the art without materially affecting the activity of the antibody (retaining at least 95% activity) to obtain variants of the sequences of the antibody or functional fragments thereof. They are all considered to be included in the scope of protection of the present application. Amino acids with similar properties are substituted in the variable region. The variant sequences described in the present application can have at least 80% identity (or homology) to the reference sequences, which means at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence. The sequence identity described in the present application can be measured using sequence analysis software. For example, using the computer program BLAST, especially BLASTP or TBLASTN with default parameters. The amino acid sequences referred to in the present application are shown in the order of N-terminal to C-terminal.

[0043] In the present application, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it is directed against. In certain embodiments, an antibody that specifically binds to (or has specificity for) an antigen means that the antibody binds to the antigen with an affinity (KD) of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less.

[0044] In the present application, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Generally, an antibody binds to an antigen with an affinity (KD) of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less. One of skill in the art can determine KD based on commonly used biological methods.

[0045] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host, which transfers an inserted nucleic acid molecule into and / or between host cells. The vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having a plurality of the above-described functions. The vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0046] In the present application, the term "composition" generally refers to a unit dosage form, and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing into association active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping or confectioning the product.

[0047] In the present application, the term "pharmaceutically acceptable" ingredient is a substance which is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0048] In the present application, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, and the like, suitable for a particular intended dosage form. Except insofar as any conventional excipient is incompatible with the compound of the present application, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this application.

[0049] In the present application, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate, or pharmaceutical composition of the present application can be administered by any common route, so long as it can reach the intended tissue. Various means of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, and the like, but the present application is not limited to these exemplified means of administration. Preferably, the composition of the present application is administered by intravenous injection or subcutaneous injection.

[0050] In the present application, the term "treatment" refers to any action that achieves a desired pharmacological and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure of a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease. "Treatment" as used herein covers any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce the symptoms of, or inhibit the disease in the individual, including, but not limited to, administering a drug comprising a compound described herein to an individual in need thereof.

[0051] In the present application, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity and can be accepted by a human and / or an animal.

[0052] In the present application, the amino acid sequences are shown in Table 1.

[0053] The present application provides an antibody or antigen-binding fragment thereof targeting B7H7, an expression vector, a preparation method, a recombinant cell, an immunoconjugate, a composition, a pharmaceutical use, a drug, a preparation kit use, a kit, a disease treatment method, and a disease treatment use, which will be described in detail respectively.

[0054] Antibody or antigen-binding fragment thereof

[0055] In an aspect of the present application, the present application provides an antibody or antigen-binding fragment thereof, comprising: a heavy chain complementarity determining region (HCDR) and a light chain complementarity determining region (LCDR); wherein the HCDR comprises at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1-3 or an amino acid sequence having at least 80% homology thereto; and the LCDR comprises at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 4-6 or an amino acid sequence having at least 80% homology thereto. In some examples of the present application, the antibody or antigen-binding fragment thereof having the aforementioned HCDR and LCDR amino acid sequences can specifically bind to human and cynomolgus monkey B7H7 proteins with high affinity.

[0056] In some examples of the present application, the aforementioned HCDRs include: an HCDR1, an HCDR2, and an HCDR3, wherein the aforementioned HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1 or having at least 80% homology with SEQ ID NO: 1, the aforementioned HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2 or having at least 80% homology with SEQ ID NO: 2, and the aforementioned HCDR3 comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 3 or having at least 80% homology with SEQ ID NO: 3. Antibodies or antigen-binding fragments thereof based on the aforementioned HCDR sequences have higher binding affinity and binding specificity to B7H7.

[0057] In some preferred examples of the present application, the aforementioned HCDRs include: an HCDR1 as set forth in SEQ ID NO: 1, respectively; an HCDR2 as set forth in SEQ ID NO: 2, respectively; and an HCDR3 as set forth in SEQ ID NO: 3, respectively. Antibodies or antigen-binding fragments thereof having the amino acid sequences as set forth in SEQ ID NO: 1-3 have been experimentally verified to have higher binding affinity and specificity to B7H7.

[0058] In some examples of the present application, the aforementioned LCDRs include: an LCDR1, an LCDR2, and an LCDR3, wherein the aforementioned LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 4 or having at least 80% homology with SEQ ID NO: 4, the aforementioned LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 5 or having at least 80% homology with SEQ ID NO: 5, and the aforementioned LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 6 or having at least 80% homology with SEQ ID NO: 6. Antibodies or antigen-binding fragments thereof based on the aforementioned LCDR sequences have higher binding affinity and specificity to B7H7.

[0059] In some preferred examples of the present application, the aforementioned LCDRs include: an LCDR1 as set forth in SEQ ID NO: 4, respectively; an LCDR2 as set forth in SEQ ID NO: 5, respectively; and an LCDR3 as set forth in SEQ ID NO: 6, respectively. Antibodies or antigen-binding fragments thereof having the amino acid sequences as set forth in SEQ ID NO: 4-6 have been experimentally verified to have higher binding affinity and specificity to B7H7.

[0060] It is noted that one or more of the aforementioned amino acid residues in the HCDRs and / or LCDRs of the B7H7 antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for retained function using the functional assays described herein. Preferably, no more than 1 or 2 conservative modifications are made.

[0061] In some examples of the present application, the aforementioned antibody or antigen binding fragment thereof further comprises: a heavy chain framework region.

[0062] In some examples of the present application, the aforementioned antibody or antigen binding fragment thereof further comprises: a light chain framework region.

[0063] In some examples of the present application, at least a portion of the aforementioned heavy chain framework region and the aforementioned light chain framework region are independently derived from at least one of a rabbit-derived antibody, a murine-derived antibody, a sheep-derived antibody, a goat-derived antibody, a primate-derived antibody, or a mutant thereof.

[0064] In some preferred examples of the present application, the heavy chain variable region of the aforementioned antibody or antigen binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and an amino acid sequence having at least 80% homology with SEQ ID NO: 7. In some more preferred examples of the present application, the amino acid sequence of the heavy chain variable region of the aforementioned antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 7.

[0065] In some preferred examples of the present application, the light chain variable region of the aforementioned antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology with SEQ ID NO: 8. In some more preferred examples of the present application, the amino acid sequence of the light chain variable region of the aforementioned antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 8.

[0066] The aforementioned antibody or antigen binding fragment thereof based on the aforementioned heavy chain variable region and light chain variable region sequence has high binding affinity and specificity to B7H7.

[0067] In some examples of the present application, the aforementioned antibody or antigen binding fragment thereof further comprises: a heavy chain constant region.

[0068] In some examples of the present application, the aforementioned antibody or antigen binding fragment thereof further comprises: a light chain constant region.

[0069] In some examples of the present application, at least a portion of the aforementioned heavy chain constant region and the aforementioned light chain constant region are independently derived from at least one of a rabbit-derived antibody, a murine-derived antibody, a sheep-derived antibody, a goat-derived antibody, a primate-derived antibody, or a mutant thereof.

[0070] In some preferred examples of the present application, at least one of the aforementioned heavy chain constant region and the aforementioned light chain constant region is from a human antibody.

[0071] In some preferred examples of the present application, the aforementioned heavy chain constant region is selected from a human IgG1 constant region.

[0072] In some more preferred examples of the present application, the aforementioned human IgG1 constant region has L234A / L235A mutations.

[0073] It is noted that L234A means that the leucine (Leu, L) at position 234 is mutated to alanine (Ala, A); similarly, L235A means that the leucine (Leu, L) at position 235 is mutated to alanine (Ala, A).

[0074] In some preferred examples of the present application, the aforementioned light chain constant region is selected from a human Ig kappa constant region.

[0075] In some examples of the present application, the heavy chain of the aforementioned antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and an amino acid sequence having at least 80% homology with SEQ ID NO: 9. In some preferred examples of the present application, the amino acid sequence of the heavy chain of the aforementioned antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 9.

[0076] In some examples of the present application, the light chain of the aforementioned antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and an amino acid sequence having at least 80% homology with SEQ ID NO: 10. In some preferred examples of the present application, the amino acid sequence of the light chain of the aforementioned antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 10.

[0077] In some examples of the present application, the aforementioned antibody comprises at least one selected from the group consisting of a polyclonal antibody and a monoclonal antibody; or the aforementioned antigen-binding fragment comprises at least one selected from the group consisting of a F(ab’)2 fragment, a Fab’ fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimal recognition unit. In some preferred examples of the present application, the aforementioned antibody is a monoclonal antibody.

[0078] Nucleic acid molecule

[0079] In another aspect of the present application, the present application provides a nucleic acid molecule, wherein the aforementioned nucleic acid molecule encodes the aforementioned antibody or antigen-binding fragment thereof. Based on the aforementioned nucleic acid molecule, the aforementioned antibody or antigen-binding fragment thereof can be prepared in large quantities in vitro.

[0080] In some examples of the present application, the aforementioned nucleic acid molecule is DNA.

[0081] It should be noted that, for the nucleic acid molecules mentioned in the present application, a person skilled in the art should understand that, actually, either one of the complementary double strands, or both, are included. For the convenience, in the present specification and claims, although only one strand is given in most cases, actually, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in the present application include DNA forms or RNA forms, and the disclosure of one means the disclosure of the other.

[0082] Expression vector

[0083] In another aspect of the present application, an expression vector is provided, which carries the aforementioned nucleic acid molecule. Based on the aforementioned expression vector, the aforementioned antibody or antigen-binding fragment thereof can be prepared in large quantities in vitro.

[0084] It should be noted that, when the aforementioned nucleic acid molecule is connected to a vector, the aforementioned nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the aforementioned nucleic acid molecule. Of course, these control elements can be directly from the vector itself, or exogenous, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Of course, the aforementioned nucleic acid molecule can be operably connected to the control elements.

[0085] In some examples of the present application, the aforementioned vector refers to a nucleic acid carrier into which a polynucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector can be expressed in the host cell. The vector is well known to those skilled in the art, including but not limited to: plasmids, cosmids, fosmids, artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages, such as M13 phages or M13 phages; and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector can also contain a replication initiation site.

[0086] In the present application, the term "operably linked" refers to the linkage of an exogenous gene to a vector, so that the control elements within the vector, such as transcription control amino acid sequences, such as translation control amino acid sequences, and the like, can perform their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, bacteriophages, and the like. After the expression vector according to some embodiments of the present application is introduced into a suitable recipient cell, the aforementioned nucleic acid molecule can be effectively expressed under the mediation of the regulatory system, thereby achieving the aforementioned in vitro mass production of the protein encoded by the aforementioned nucleic acid molecule.

[0087] In some examples of the present application, the aforementioned vector is a eukaryotic vector or a prokaryotic vector.

[0088] In some examples of the present application, the aforementioned vector comprises at least one selected from the group consisting of a plasmid vector, an adenovirus vector, a lentivirus vector, and an adeno-associated virus vector.

[0089] Method for preparing an antibody or antigen-binding fragment thereof

[0090] In another aspect of the present application, a method for preparing the aforementioned antibody or antigen-binding fragment thereof is provided, comprising: introducing the aforementioned expression vector into a cell; and culturing the aforementioned cell under conditions suitable for protein expression and secretion, so as to obtain the aforementioned antibody or antigen-binding fragment thereof. The aforementioned antibody or antigen-binding fragment thereof can be prepared in large quantities in vitro by the method.

[0091] In some examples of the present application, the aforementioned cell is a eukaryotic cell.

[0092] In some specific examples of the present application, the aforementioned eukaryotic cell is a mammalian cell. When the cell is a mammalian cell, the expression efficiency of the aforementioned antibody or antigen-binding fragment thereof is higher.

[0093] It should be noted that the aforementioned eukaryotic cell does not include an animal reproductive cell, a fertilized egg, or an embryonic stem cell.

[0094] Recombinant cell

[0095] In another aspect of the present application, a recombinant cell carrying the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, or the aforementioned expression vector is provided. In some examples of the present application, the aforementioned recombinant cell is obtained by transfecting or transforming the aforementioned expression vector, and the aforementioned recombinant cell can efficiently express the aforementioned antibody or antigen-binding fragment under suitable conditions.

[0096] In some examples of the present application, the aforementioned recombinant cell is a prokaryotic cell, a eukaryotic cell, or a bacteriophage. It should be noted that the aforementioned eukaryotic cell does not include an animal reproductive cell, a fertilized egg, or an embryonic stem cell.

[0097] In some examples of the present application, the aforementioned prokaryotic cell is E. coli, B. subtilis, Streptomyces, or P. mirabilis.

[0098] In some examples of the present application, the aforementioned eukaryotic cell is a fungus, an insect cell, a plant cell, or a mammalian cell.

[0099] In some examples of the present application, the aforementioned fungus is P. pastoris, S. cerevisiae, S. pombe, or T. reesei.

[0100] In some examples of the present application, the aforementioned insect cell is a M. sexta cell; in some examples of the present application, the aforementioned plant cell is a tobacco plant cell; in some examples of the present application, the aforementioned mammalian cell is a BHK cell, a CHO cell, a COS cell, a myeloma cell, or a human embryonic kidney 293 cell; and the animal reproductive cells, zygotes, or embryonic stem cells are excluded.

[0101] In some examples of the present application, the aforementioned recombinant cell is a mammalian cell. When the cell is a mammalian cell, the expression efficiency of the aforementioned antibody or antigen-binding fragment thereof is higher.

[0102] In some examples of the present application, the aforementioned recombinant cell is a BHK cell, a CHO cell, a COS cell, or an NSO cell.

[0103] It should be noted that the aforementioned "suitable conditions" in the specification of the present application refer to conditions suitable for the expression of the bispecific antibody of the present application. Those skilled in the art can easily understand that the conditions suitable for the expression of the bispecific antibody include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the bispecific antibody according to the specific environment of the laboratory.

[0104] Immunoconjugate

[0105] In another aspect of the present application, an immunoconjugate is provided, comprising the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The aforementioned immunoconjugate can be used for treating diseases related to B7H7, such as tumors.

[0106] In some examples of the present application, the aforementioned immunoconjugate can further comprise a therapeutic agent. Among them, the aforementioned therapeutic agent comprises at least one of a cytokine, an immunomodulator, and a small molecule drug.

[0107] Composition

[0108] In another aspect of the present application, the present application provides a composition comprising: the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned immunoconjugate. The aforementioned composition can be used for treating a disease associated with B7H7, such as a tumor.

[0109] It should be noted that the aforementioned composition comprises components separated in time and / or space, as long as they can act together to achieve the purpose of the present application. For example, the components contained in the aforementioned composition can be administered to the subject as a whole, or administered to the subject separately. When the components contained in the aforementioned composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0110] Pharmaceutical use

[0111] In yet another aspect of the present application, the present application provides use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned immunoconjugate, or the aforementioned composition in the preparation of a medicament for treating or preventing a disease mediated by B7H7. The medicament prepared based on the aforementioned antibody or antigen-binding fragment thereof can block the binding of B7H7 to its receptor KIR3DL3, promote the killing of tumor cells by immune cells, and thus effectively treat and / or prevent a disease mediated by B7H7.

[0112] In some examples of the present application, the aforementioned disease mediated by B7H7 includes a tumor.

[0113] In some examples of the present application, the aforementioned tumor includes at least one of head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

[0114] Medicament

[0115] In yet another aspect of the present application, the present application provides a medicament comprising: the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned immunoconjugate, or the aforementioned composition, the aforementioned medicament for treating or preventing a disease mediated by B7H7. The aforementioned medicament can block the binding of B7H7 to its receptor KIR3DL3, promote the killing of tumor cells by immune cells, and thus effectively treat and / or prevent a disease mediated by B7H7.

[0116] In some examples of the present application, the aforementioned disease mediated by B7H7 includes a tumor.

[0117] In some examples of the present application, the aforementioned tumor comprises at least one of head and neck cancer, glioblastoma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, renal cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

[0118] In some examples of the present application, the aforementioned pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0119] In some examples of the present application, the aforementioned excipient comprises one or more pharmaceutically acceptable excipient, diluent, stabilizer, or carrier.

[0120] In some examples of the present application, the aforementioned pharmaceutical composition is an injection.

[0121] It is to be noted that the aforementioned pharmaceutical composition includes a combination separated in time and / or space as long as it can act together to achieve the purpose of the present application. For example, the components contained in the aforementioned pharmaceutical composition can be administered to a subject as a whole, or administered to a subject separately. When the components contained in the aforementioned pharmaceutical composition are administered to a subject separately, each component can be administered to a subject simultaneously or sequentially.

[0122] The pharmaceutical of the present application contains a safe and effective amount of the active ingredient (bispecific antibody) of the present application and a pharmaceutically acceptable excipient. Such excipients include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode, and the dosage form of the pharmaceutical of the present application is injection, oral preparation (tablet, capsule, oral liquid), transdermal preparation, sustained-release preparation. For example, it is prepared by conventional methods with normal saline or aqueous solution containing glucose and other auxiliary agents. The aforementioned pharmaceutical is preferably manufactured under sterile conditions.

[0123] The effective amount of the aforementioned active ingredient of the present application can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, by clinical trials). The aforementioned factors include, but are not limited to, the pharmacokinetic parameters of the aforementioned active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, several separate doses can be given daily, or the dose can be proportionally reduced, according to the exigencies of the therapeutic situation.

[0124] The aforementioned pharmaceutically acceptable excipient of the present application includes, but is not limited to, water, saline, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or combinations thereof. The selection of the carrier should be matched with the administration mode, which is well known to a person of ordinary skill in the art.

[0125] Use in preparing a kit

[0126] In yet another aspect of the present application, the present application provides use of the aforementioned antibody or antigen binding fragment thereof in preparing a kit, wherein the aforementioned kit is used for detecting B7H7.

[0127] As aforementioned, the antibody or antigen binding fragment thereof of the embodiments of the present application can specifically bind to B7H7, thus the aforementioned antibody or antigen binding fragment thereof can be used for detecting B7H7. Further, it can be used for preparing a B7H7 related kit and for scientific research, such as qualitatively or quantitatively detecting B7H7 protein molecules in a biological sample. More specifically, it can be used for a kit related to immunoblotting, immunoprecipitation, etc. which involves the use of the specific binding property of B7H7 and the antibody. These kits can comprise any one or more of the following: antagonists, bispecific antibodies of the present application or pharmaceutical reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents. The bispecific antibodies of the present application can be used in different types of diagnostic tests, for example, various diseases or the presence of drugs, toxins or other proteins, etc. can be detected in vitro or in vivo. For example, the serum or blood of a subject can be detected to test the B7H7 mediated related diseases.

[0128] Kit

[0129] In yet another aspect of the present application, the present application provides a kit comprising the aforementioned antibody or antigen binding fragment thereof of the first aspect. The aforementioned kit can be used for portable detection of B7H7.

[0130] Method for treating a disease

[0131] In yet another aspect of the present application, the present application provides a method for preventing and / or treating a B7H7 mediated disease. According to the embodiments of the present application, the aforementioned method comprises: administering a pharmaceutically acceptable amount of the antibody or antigen binding fragment thereof, expression vector, recombinant cell, immunoconjugate, composition or drug to a subject. Based on the aforementioned method, the related diseases mediated by B7H7 can be effectively treated.

[0132] It should be noted that the terms "subject", "individual" and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" include, but are not limited to, individuals having a tumor, individuals having an autoimmune disease, individuals having a pathogen infection, etc. The subject can be a human, but also includes other mammals, particularly mammals useful as laboratory models of human disease, e.g., mice, rats, etc.

[0133] The effective amount of the foregoing antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, immunoconjugate, composition or medicament of the present application can vary according to factors such as the mode of administration and the severity of the disease to be treated. The selection of an optimal effective amount can be made by one of ordinary skill in the art (e.g., by clinical trial). The foregoing factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient(s) such as the bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated; the age, body weight, and immune status of the patient, the route of administration, etc. For example, the dosage can be administered several times a day, or proportionally reduced, according to the exigencies of the therapeutic situation.

[0134] In some examples of the present application, the foregoing B7H7-mediated related disease includes a tumor.

[0135] In some examples of the present application, the foregoing tumor includes at least one of head and neck cancer, glioblastoma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

[0136] Therapeutic use for disease

[0137] In yet another aspect of the present application, the present application proposes the use of the foregoing antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, immunoconjugate, composition or medicament in the treatment or prevention of a B7H7-mediated disease. In some examples of the present application, the administration of an effective dose of the antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, immunoconjugate, composition or medicament to a subject can effectively treat a B7H7-mediated disease.

[0138] In some examples of the present application, the foregoing B7H7-mediated related disease includes a tumor.

[0139] In some examples of the present application, the foregoing tumor includes at least one of head and neck cancer, glioblastoma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

[0140] Table 1. B7H7 antibody sequences

[0141] The solutions of the present application will be explained below with reference to examples. Those skilled in the art will understand that the examples below are only for illustration of the present application and should not be considered as limiting the scope of the present application. If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained on the market.

[0142] Example 1 Antibody affinity maturation

[0143] In order to improve the drugability of the antibody and obtain a high affinity B7H7 antibody, the applicant carried out affinity maturation on the developed humanized B7H7 antibody h17H7B8 (application number: 202311708700.1). The antigen used for affinity maturation was human B7H7 protein with His tag (ACRO). The obtained affinity matured B7H7 antibody h17H7B8-V13 heavy chain variable region is shown as SEQ ID NO: 7, and the light chain variable region is shown as SEQ ID NO: 8.

[0144] Example 2 Antibody affinity detection

[0145] Surface plasmon resonance (SPR) technology was used to determine the affinity of the humanized B7H7 antibody.

[0146] The specific operation is as follows: open the Biacore 1K instrument (Cytiva), put the Protein A chip (Cytiva) into it, then balance in HBS-EP+ buffer, then make the tested antibody with a concentration of 0.5 μg / mL flow through the chip surface at a speed of 30 μL / min to capture the tested antibody; flow different concentrations of human B7H7 protein (ACRO) through the chip surface at a speed of 30 μL / min, set the binding time to 90 s, then dissociate for 600 s. Kinetic analysis was performed using a 1:1 binding model. The affinity values of the humanized B7H7 antibody binding to human B7H7 protein are shown in Table 2, indicating that the humanized B7H7 antibody has obvious binding activity to human B7H7 protein.

[0147] Table 2. Affinity values of humanized B7H7 antibody

[0148] Example 3 Antibody binding activity detection

[0149] Test Example 1 B7H7 antibody binding to human and cynomolgus monkey B7H7 protein

[0150] ELISA experiments were used to detect the binding of B7H7 antibody to human and cynomolgus monkey B7H7 protein.

[0151] Human and cynomolgus B7H7 protein (ACRO) was diluted with PBS buffer to 1 pg / mL, and added to a 96-well plate at a volume of 100 pL / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was discarded, and the plate was washed with PBST (0.1% Tween 20 in pH 7.2 PBS) buffer for 3 times, then 300 pL / well of 5% BSA was added, and incubated at room temperature for 2 h for blocking. The blocking solution was discarded, and 100 pL of the antibody to be tested diluted with 0.05% BSA was added, and incubated at room temperature for 1 h. After washing the plate with PBST for 3 times, 100 pL of HRP-labeled anti-human IgG antibody (Southern biotech) diluted with 0.5% BSA was added, and incubated at room temperature for 1 h. After washing the plate with PBST for 3 times, 80 pL / well of TMB was added, and incubated at room temperature for 10 min, and 80 pL / well of stop solution was added to stop the reaction. The absorbance value was read at 450 nm by an enzyme-labeled instrument. The specific experimental results are shown in FIG. 1, and the affinity matured antibody (h17H7B8-V13) of the present application can bind to human and cynomolgus B7H7 protein, and is stronger than the parent antibody h17H7B8.

[0152] Test Example 2 Binding of B7H7 antibody to HCC827 cell line

[0153] Flow cytometry experiments were used to detect the ability of B7H7 antibody to bind to HCC827 cell line.

[0154] The HCC827 cells were diluted with PBS to 1 x 10 6 cells / mL, and added to a 96-well plate at a volume of 90 pL / well, and 10 pL of goat serum (Shanghai Biotechnology) was added to each well, and blocked at 4°C for 30 min. A series of gradient-diluted antibodies to be tested were added, and incubated at 4°C for 30 min. After incubation, the cells were washed with PBS for 2 times. The cells were resuspended with 100 pL / well of PBS, and 0.1 pL / well of AF647-labeled goat anti-human IgG Fc fragment specific antibody (Jackson ImmunoResearch) was added, and incubated at 4°C in the dark for 30 min. The cells were washed with PBS for 2 times. The cells were resuspended with 200 pL / well of PBS, and detected by flow cytometry. FIG. 2 shows that the affinity matured antibody (h17H7B8-V13) of the present application can bind to the HCC827 tumor cell line with high expression of B7H7 well, and is stronger than the parent antibody h17H7B8.

[0155] Example 4 Detection of antibody blocking activity

[0156] Test Example 1 Blocking of B7H7 protein binding to KIR3DL3 protein by B7H7 antibody

[0157] ELISA experiment was used to detect the blocking of B7H7 antibody to the binding of B7H7 protein and KIR3DL3 protein, and the specific experimental operation was as follows:

[0158] Human B7H7 protein (ACRO) was diluted with PBS buffer to 5 μg / mL, and was added to 96-well plates at a volume of 100 μL / well, and was placed at 4°C overnight. The PBS buffer in the 96-well plate was discarded, and the plate was washed 3 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, and 300 μL / well of 5% BSA was added, and was incubated at room temperature for 2 h for blocking. The blocking solution was discarded, and the biotin-labeled KIR3DL3 protein (Kaiyuan Bio) was diluted with 0.05% BSA to 2 μg / mL, and 50 μL was added to each well, and 50 μL of 0.05% BSA-diluted antibody to be tested was added, and was incubated at room temperature for 1 h. After washing the plate 3 times with PBST, 100 μL of HRP-labeled Streptavidin secondary antibody (Southern biotech) diluted with 0.5% BSA was added, and was incubated at room temperature for 1 h. After washing the plate 3 times with PBST, 80 μL / well of TMB was added, and was incubated at room temperature for 10 min, and 80 μL / well of stop solution was added to stop the reaction. The absorbance value was read at 450 nm by an enzyme-labeled instrument. The specific experimental results are shown in Figure 3, and the affinity matured antibody (h17H7B8-V13) of the present application can effectively block the binding of B7H7 to its receptor KIR3DL3, and the blocking ability is comparable to that of the parent antibody h17H7B8.

[0159] Test Example 2: B7H7 antibody blocks the binding of B7H7 protein to CHO-K1-KIR3DL3 cells

[0160] Flow cytometry experiment was used to detect the blocking of B7H7 antibody to the binding of B7H7 protein and CHO-K1-KIR3DL3 cells, and the specific experimental operation was as follows:

[0161] CHO-K1-human KIR3DL3 cells were diluted with PBS to 2×10 6KIR3DL3, 2 pg / mL of B7H7 protein (ACRO) and 10 pL / well of rat serum were added to the 96-well plate, and then incubated at 4°C for 30 min. A series of gradient dilutions of the antibody to be tested and 2 pg / mL of biotin-labeled human B7H7 protein (ACRO) were added, and then incubated at 4°C for 30 min. After incubation, the cells were resuspended with 100 pL / well of PBS, and then 0.1 pL / well of APC-labeled streptavidin antibody (Biolegend) was added, and then incubated at 4°C for 30 min. After washing twice with PBS, the supernatant was discarded after centrifugation. The cells were resuspended with 200 pL / well of PBS, and then detected by flow cytometry. The specific experimental results are shown in FIG. 4. The affinity matured antibody (h17H7B8-V13) of the present application can well block the binding of B7H7 to its receptor KIR3DL3, and is stronger than the parent antibody h17H7B8.

[0162] Example 5: Detection of the activity of high-affinity humanized antibody in promoting PBMC killing function

[0163] Flow cytometry experiments were used to detect the activity of B7H7 antibody in promoting PBMC killing function.

[0164] K562-B7H7 cells were collected, resuspended with cell pellets, counted, and adjusted to a cell density of about 1.5 x 10 6 cells / mL. 1 mL of the cell suspension was taken into a 1.5 mL EP tube, 1 pL of Cell Trace Violet (Invitrogen) was added, mixed, and then labeled at 37°C for 20 min. The labeled cells were washed 3 times with 1640 culture medium, and the cell density was adjusted to 2 x 10 5 cells / mL. 300g centrifugation was used to collect PBMC (Miaoshun Biotechnology), resuspend the cell pellets with 1640 culture medium, count the cells, and adjust the PBMC concentration to 1 x 10 6 cells / mL. 100 pL / well of diluted K562-B7H7 cells and 100 pL / well of PBMC were added to a 96-well round-bottom plate, and a gradient dilution of the antibody to be tested was added. After centrifugation at 250g for 4 min at room temperature, the plate was incubated at 37°C in a 5% CO2 incubator for 4-6 h. After incubation, 1 pL of 7AAD (BD Bioscience) was added to each well, and then mixed. Flow cytometry was used for detection. The specific experimental results are shown in FIG. 5. The affinity matured antibody (h17H7B8-V13) of the present application can effectively promote the killing of K562 cell lines overexpressing B7H7 by PBMC in a dose-dependent manner, and is stronger than the parent antibody h17H7B8.

[0165] In conclusion, the monoclonal antibody prepared in the present application has high specificity and affinity, can effectively bind to human and cynomolgus monkey B7H7 protein, block the binding of B7H7 to its receptor KIR3DL3, and promote the killing ability of immune cells (such as PBMC) to tumor cells.

[0166] The above describes the preferred embodiments of the present application, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as the disclosed content of the present application, and all fall within the protection scope of the present application.

[0167] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0168] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. An antibody or antigen-binding fragment thereof, characterized in that, Comprises: a heavy chain complementarity determining region (HCDR) and a light chain complementarity determining region (LCDR); wherein, the HCDR comprises at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-3 or has at least 80% homology thereto; the LCDR comprises at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 4-6 or has at least 80% homology thereto.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, the HCDR comprises: a HCDR1, a HCDR2, and a HCDR3, wherein: the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1 or has at least 80% homology to SEQ ID NO: 1, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2 or has at least 80% homology to SEQ ID NO: 2, and the HCDR3 comprises an amino acid sequence selected from the group consisting of as set forth in SEQ ID NO: 3 or has at least 80% homology to SEQ ID NO: 3; preferably, the HCDR comprises: a HCDR1 of an amino acid sequence as set forth in SEQ ID NO: 1, respectively; preferably, the HCDR comprises: a HCDR2 of an amino acid sequence as set forth in SEQ ID NO: 2, respectively; preferably, the HCDR comprises: a HCDR3 of an amino acid sequence as set forth in SEQ ID NO: 3, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, the LCDR comprises: a LCDR1, a LCDR2, and a LCDR3, wherein: the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 4 or has at least 80% homology to SEQ ID NO: 4, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 5 or has at least 80% homology to SEQ ID NO: 5, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 6 or has at least 80% homology to SEQ ID NO: 6; preferably, the LCDR comprises: a LCDR1 of an amino acid sequence as set forth in SEQ ID NO: 4, respectively; preferably, the LCDR comprises: a LCDR2 of an amino acid sequence as set forth in SEQ ID NO: 5, respectively; preferably, the LCDR comprises: a LCDR3 of an amino acid sequence as set forth in SEQ ID NO: 6, respectively.

4. The antibody or antigen-binding fragment thereof of claim 2 or 3, wherein, the antibody or antigen-binding fragment thereof further comprises: a heavy chain framework region; optionally, the antibody or antigen-binding fragment thereof further comprises: a light chain framework region; optionally, at least one of the heavy chain framework region and the light chain framework region is independently from a rabbit-derived antibody, a murine-derived antibody, a sheep-derived antibody, a goat-derived antibody, a primate-derived antibody, or a mutant thereof; preferably, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of as set forth in SEQ ID NO: 7 or has at least 80% homology to SEQ ID NO: 7; preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 7; Preferably, the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 8 or having at least 80% homology to SEQ ID NO: 8; preferably, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO:

8.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein, The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region; Optionally, the antibody or antigen-binding fragment thereof further comprises a light chain constant region; Optionally, at least one of the heavy chain constant region and the light chain constant region is independently from at least one of a rabbit-derived antibody, a murine-derived antibody, a sheep-derived antibody, a goat-derived antibody, a primate-derived antibody, or a mutant thereof; preferably, at least one of the heavy chain constant region and the light chain constant region is from a human-derived antibody; Preferably, the heavy chain constant region is selected from a human IgG1 constant region; More preferably, the human IgG1 constant region has L234A / L235A mutations; Preferably, the light chain constant region is selected from a human Ig kappa constant region; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 or having at least 80% homology to SEQ ID NO: 9; preferably, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 9; Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 or having at least 80% homology to SEQ ID NO: 10; preferably, the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO:

10.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody comprises at least one selected from the group consisting of a polyclonal antibody and a monoclonal antibody; or The antigen-binding fragment comprises at least one selected from the group consisting of a F(ab’)2 fragment, a Fab’ fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, and a minimal recognition unit; Preferably, the antibody is a monoclonal antibody.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-6; Optionally, the nucleic acid molecule is DNA.

8. An expression vector, characterized by, Carrying the nucleic acid molecule according to claim 7.

9. A method of producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that, Comprising: Introducing the expression vector according to claim 8 into a cell; Culturing the cell under conditions suitable for protein expression and secretion so as to obtain the antibody or antigen-binding fragment thereof; Optionally, the cell is a eukaryotic cell.

10. A recombinant cell, characterized in that, The recombinant cell carries the nucleic acid molecule according to claim 7 or the expression vector according to claim 8.

11. An immunoconjugate, characterized in that, Comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, or the recombinant cell according to claim 10; Optionally, the immunoconjugate further comprises a therapeutic agent.

12. A composition characterized in that, Comprising: The antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 10, the immunoconjugate of claim 11, or the composition of claim 12 for use in the preparation of a medicament for the treatment or prevention of a B7H7-mediated related disease.

13. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 10, the immunoconjugate of claim 11, or the composition of claim 12 for the manufacture of a medicament for the treatment or prevention of a B7H7-mediated related disease.

14. Use according to claim 13, characterized in that, The B7H7-mediated related disease comprises a tumor; Optionally, the tumor comprises at least one of head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, renal cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

15. A medicament, characterized by comprising: Comprises: The antibody or antigen-binding fragment thereof of any one of claims 1-6, the nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 10, the immunoconjugate of claim 11, or the composition of claim 12 for use in the preparation of a medicament for the treatment or prevention of a B7H7-mediated related disease.

16. The medicament according to claim 15, characterized in that, The B7H7-mediated related disease comprises a tumor; Optionally, the tumor comprises at least one of head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, renal cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia. Optionally, further comprising a pharmaceutically acceptable excipient; Optionally, the excipient comprises: One or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers; Optionally, the medicament is an injection.

17. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6 for the preparation of a kit for detecting B7H7.

18. A kit comprising, The kit comprises: The antibody or antigen-binding fragment thereof of any one of claims 1-6.

19. The kit of claim 18, wherein The kit is for detecting B7H7.

20. A method of preventing and / or treating a B7H7-mediated disease, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-19, or a pharmaceutically acceptable salt thereof. Comprises: Administering to a subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment thereof of any one of claims 1-6, the expression vector of claim 8, the recombinant cell of claim 10, the immunoconjugate of claim 11, the composition of claim 12, or the medicament of claim 15.

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