HLA-G targeting antibody or antigen binding fragment thereof and application thereof

Abstract

The invention provides an HLA-G targeting antibody or an antigen binding fragment thereof and application thereof, and relates to the field of antibodies. Wherein the antibody or the antigen binding fragment thereof comprises a heavy chain complementarity determining region HCDR and a light chain complementarity determining region LCDR, and the HCDR comprises at least one selected from SEQ ID NO: 1-6 or an amino acid sequence having at least 80% homology with the HCDR; the LCDR comprises at least one of SEQ ID NO: 7-12 or an amino acid sequence having at least 80% homology with the at least one of SEQ ID NO: 7-12. The antibody or the antigen binding fragment has high specificity and high affinity, can bind to human and cynomolgus monkey HLA-G, specifically blocks the binding of the HLA-G and its receptors LILRB1 and LILRB2, promotes the killing of immune cells to tumor cells, and effectively treats and / or prevents HLA-G mediated related diseases, such as tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of antibodies, and in particular relates to antibodies or antigen-binding fragments thereof targeting HLA-G 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 treat various tumors. However, the overall response rate of patients is only 30%, and a large number of clinical patients still cannot benefit. Therefore, it is necessary to find new immune checkpoint molecules and new combination therapy strategies.

[0003] HLA-G is a non-classical MHC-I molecule, which is mainly expressed in placental tissue, and is not expressed or very lowly expressed in other normal tissues. However, in many tumor types, such as breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer and renal cell carcinoma, HLA-G is highly expressed and negatively correlated with the prognosis of patients.

[0004] It has been reported that HLA-G can bind to its receptors LILRB1 (ILT2) and LILRB2 (ILT4), thereby inhibiting the effector function of NK cells and T cells. Blocking the binding of HLA-G to LILRB1 or LILRB2 by blocking antibodies can significantly restore the anti-tumor function of NK cells and T cells. Therefore, targeting the HLA-G signaling pathway is very promising.

[0005] Currently, there are relatively few clinical studies on HLA-G, and it is urgent to develop an anti-HLA-G antibody with high affinity and good efficacy. SUMMARY

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

[0007] HLA-G is a non-classical MHC-I molecule, which is mainly expressed in placental tissue, and is not expressed or very lowly expressed in other normal tissues. However, in many tumor types, such as breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer and renal cell carcinoma, HLA-G is highly expressed and negatively correlated with the prognosis of patients.

[0008] It has been reported that HLA-G can bind to its receptors LILRB1 (ILT2), LILRB2 (ILT4), thereby inhibiting the effector function of NK cells and T cells. Blocking the binding of HLA-G to LILRB1 or LILRB2 by blocking antibodies can significantly restore the anti-tumor function of NK cells and T cells.

[0009] The inventors of the present application have successfully obtained blocking antibodies that specifically bind to HLA-G through screening. Specifically, the present study screened high-affinity monoclonal antibodies that can specifically bind to HLA-G and block the binding of HLA-G to its receptors LILRB1 or LILRB2 by hybridoma technology, and humanized the constant region of the murine anti-HLA-G monoclonal antibody and the framework region in the variable region of the murine antibody to obtain humanized anti-HLA-G antibodies.

[0010] It has been found through verification that the murine antibodies and humanized antibodies obtained by the present application can specifically target and bind to HLA-G, block the binding of HLA-G to its receptors LILRB1 or LILRB2, promote the killing of tumor cells by immune cells, and thus effectively treat and / or prevent HLA-G-mediated related diseases such as tumors.

[0011] Specifically, the present application provides the following technical solutions:

[0012] 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 selected from SEQ ID NO: 1-6 or an amino acid sequence having at least 80% homology thereto; and the LCDR comprises at least one selected from SEQ ID NO: 7-12 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 HLA-G, and specifically block the binding of HLA-G to its receptors LILRB1 and LILRB2, promote the killing of tumor cells by immune cells, and effectively treat and / or prevent HLA-G-mediated related diseases such as tumors.

[0013] 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.

[0014] In a 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.

[0015] 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 of the present application 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. In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof can be efficiently prepared by the aforementioned method.

[0016] 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 of the present application, the nucleic acid molecule of the second aspect of the present application, or the expression vector of the third aspect of the present application. 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.

[0017] 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 of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, or the recombinant cell of the fifth aspect of the present application. As previously described, the antibody or antigen-binding fragment can effectively specifically bind to human and cynomolgus monkey HLA-G protein. Therefore, the immunoconjugate comprising the antibody or antigen-binding fragment can also bind to human and cynomolgus monkey HLA-G protein, and the aforementioned immunoconjugate has good effects in preventing and / or treating HLA-G-mediated tumor diseases, such as breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, and renal cell carcinoma, etc.

[0018] In a seventh aspect, the present application provides a composition. 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 is capable of effectively binding to human and cynomolgus monkey HLA-G, and thus, the composition, such as a food composition, a pharmaceutical composition, etc., comprising the antibody or antigen binding fragment thereof is also capable of effectively treating or preventing a tumor disease, such as breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, and renal cell carcinoma, etc.

[0019] 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 disease associated with HLA-G. As previously described, the antibody or antigen binding fragment thereof is capable of effectively binding to human and cynomolgus monkey HLA-G, and thus, the medicament comprising the antibody or antigen binding fragment thereof and the like is also capable of significantly treating and / or preventing a tumor disease (e.g., breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, and renal cell carcinoma, etc.) mediated by HLA-G.

[0020] In a ninth aspect, the present application provides a medicament. According to embodiments 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 disease associated with HLA-G. As previously described, the antibody or antigen binding fragment thereof is capable of effectively binding to human and cynomolgus monkey HLA-G. Thus, the medicament comprising the antibody or antigen binding fragment thereof and the like is also capable of significantly treating and / or preventing a tumor disease (e.g., breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, and renal cell carcinoma, etc.) mediated by HLA-G.

[0021] In a tenth aspect of this application, the use of the antibody or antigen-binding fragment thereof described in the first aspect in the preparation of a kit for the detection of HLA-G is provided. As previously stated, the antibody or antigen-binding fragment thereof can effectively bind to HLA-G in humans and cynomolgus monkeys; therefore, a kit containing the antibody or antigen-binding fragment thereof can be used for the effective detection of HLA-G. In some examples of this application, the aforementioned kit can be used in scientific research, such as for the qualitative or quantitative detection of HLA-G protein in biological samples.

[0022] In the eleventh aspect of this application, a kit is provided. According to embodiments of this application, the kit comprises the antibody or its antigen-binding fragment described in the first aspect. As previously mentioned, the antibody or its antigen-binding fragment can effectively bind to HLA-G in humans and cynomolgus monkeys; therefore, the kit comprising the antibody or its antigen-binding fragment can be used for the effective detection of HLA-G. In some examples of this application, the kit can be used for scientific research, such as for the qualitative or quantitative detection of HLA-G protein in biological samples, and can also be used to assess an individual's condition, such as determining whether the individual's HLA-G level is higher or lower than normal after obtaining the individual's HLA-G level.

[0023] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of the ELISA results of mouse antibodies binding to human and cynomolgus monkey HLA-G proteins provided in the embodiments of this application;

[0026] Figure 2 This is a schematic diagram of the flow cytometry results of the JEG-3 tumor cell line with high HLA-G expression provided in the embodiments of this application;

[0027] Figure 3 This is a schematic diagram of the flow cytometry results of murine antibodies blocking the binding of LILRB1 or LILRB2 proteins to CHO-K1-HLA-G cells, as provided in the embodiments of this application.

[0028] Figure 4This is a schematic diagram of the ELISA results of murine antibody blocking the binding of HLA-G protein to LILRB1 protein or LILRB2 protein, provided in the embodiments of this application.

[0029] Figure 5 This is a schematic diagram of the flow cytometry results of the murine antibody promoting the killing of SK-OV-3 cells overexpressing HLA-G by PBMCs, as provided in the embodiments of this application.

[0030] Figure 6 This is a schematic diagram of the flow cytometry results of humanized antibodies binding to JEG-3 tumor cell lines that highly express HLA-G, as provided in the embodiments of this application.

[0031] Figure 7 This is a schematic diagram of the flow cytometry results of humanized antibodies blocking the binding of LILRB1 or LILRB2 proteins to CHO-K1-HLA-G cells, as provided in the embodiments of this application.

[0032] Figure 8 This is a schematic diagram of the ELISA results provided in the embodiments of this application, showing the effect of humanized antibodies blocking the binding of HLA-G protein to LILRB1 protein or LILRB2 protein;

[0033] Figure 9 This is a schematic diagram of the flow cytometry results of humanized antibodies promoting the killing of HLA-G-overexpressing K562 cells by PBMCs, as provided in the embodiments of this application. Detailed Implementation

[0034] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present application.

[0035] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0036] In this application, the term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Immunoglobulins can be classified into five classes: IgM, IgD, IgG, IgA, and IgE. The same class of immunoglobulins can also be divided into different subclasses based on their amino acid composition, such as IgG1, IgG2, IgG3, and IgG4. The immunoglobulin light chain is divided into κ chains or λ chains based on the difference in its constant region.

[0037] In this application, the variable region of the antibody heavy chain may further include a constant region of the heavy chain, wherein the constant region of the heavy chain includes human / mouse IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0038] In this application, the antibody light chain variable region may further include a light chain constant region, wherein the light chain constant region comprises human / mouse κ, λ chains or variants thereof.

[0039] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0040] In this application, the antibody may be derived from rabbit antibody, mouse antibody, sheep antibody, goat antibody, primate antibody (including human and monkey antibodies) or its mutants, as well as chimeric antibodies from different sources.

[0041] Taking murine antibodies as an example, this application describes the HLA-G monoclonal antibody. "Murine antibody" refers to a monoclonal antibody against human HLA-G prepared based on the knowledge and skills in the art. Preparation involves injecting the test subject with the antigen, followed by isolating a hybridoma expressing the antibody with the desired sequence or functional characteristics. In a preferred embodiment of this application, the murine HLA-G antibody or its antigen-binding fragment may further include a light chain constant region of murine κ, λ chains or variants thereof, or further include a heavy chain constant region of murine IgG1, IgG2, IgG3 or variants thereof.

[0042] In this application, the term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. The variable region gene is cloned from mouse hybridoma cells, and then the constant region gene of a human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic expression system. In a preferred embodiment of the invention, the antibody light chain of the HLA-G chimeric antibody further includes the Fc region of a human κ, λ chain, or a variant thereof. The antibody heavy chain of the HLA-G chimeric antibody further includes the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof.

[0043] In this 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 HLA-G; these fragments contain one or more CDR regions selected from SEQ ID NO:1 to SEQ ID NO:12 of the antibody described in this application. The Fv fragment contains variable regions of the antibody heavy chain and light chain, but no constant regions, and is the smallest antibody fragment with all 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 linked into a single polypeptide chain using different linkers, referred to as a single-chain antibody or single-chain Fv.

[0044] In this application, the amino acid sequences of the listed CDRs are all as shown in the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as the IMGT rule, the Chothia rule, etc. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described in any of the known schemes described above. Although the scope of protection claimed in this application is based on the sequences shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also be included in the scope of protection of this application.

[0045] In this application, the term "amino acid" is represented by a single-letter or three-letter code, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0046] 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 and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first 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 standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0 967 284, EP 1 085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0047] For peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0048] In this application, without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain sequences of the antibody or its functional fragments. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in this application can have at least 80% identity (or homology) with the reference sequences, meaning 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%, or 99.9% identity with each reference sequence. The sequence consistency described in this application can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0049] In this document, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (KD) binds to the antigen.

[0050] In this paper, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies bind at a dissociation constant of less than approximately 10-1. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (KD) binds to the antigen. Those skilled in the art can determine KD based on commonly used biological methods.

[0051] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0052] In this application, the term "composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining an active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely pulverized solid carrier, or both.

[0053] In this application, the term "pharmaceuticalally acceptable" means a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0054] In this application, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients that are incompatible with the compounds of this invention, such as any adverse biological effects they produce or interactions that occur harmfully with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0055] In this application, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, conjugates, or pharmaceutical compositions of this application can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the invention is not limited to these exemplified routes of administration. Preferably, the compositions of the invention are administered via intravenous or subcutaneous injection.

[0056] In this application, the term "treatment" is used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to disease but not yet diagnosed with it; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing symptoms associated with the disease. As used herein, "treatment" covers any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0057] In this application, the terms "effective amount" or "effective dose" refer to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0058] The amino acid sequences involved in this application are shown in Table 1.

[0059] This application discloses an antibody or antigen-binding fragment thereof targeting HLA-G, an expression vector, a preparation method, recombinant cells, an immunoconjugate, a composition, pharmaceutical use, a drug, a reagent kit, a treatment method for a disease, and a treatment for a disease, which will be described in detail below.

[0060] Antibody or its antigen-binding fragment

[0061] In one aspect of this application, an antibody or antigen-binding fragment thereof is provided, comprising: a heavy chain complementarity-determining region (HCDR) and a light chain complementarity-determining region (LCDR); wherein the aforementioned HCDR comprises an amino acid sequence selected from at least one of SEQ ID NO:1-6 or having at least 80% homology with it; and the aforementioned LCDR comprises an amino acid sequence selected from at least one of SEQ ID NO:7-12 or having at least 80% homology with it. In some examples of this application, antibodies or antigen-binding fragments thereof having the aforementioned HCDR and LCDR amino acid sequences are capable of binding to human and cynomolgus monkey HLA-G proteins with high affinity and specificity.

[0062] In some examples of this application, the aforementioned HCDRs include: HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in SEQ ID NO:1-2 or having at least 80% homology with SEQ ID NO:1-2, HCDR2 comprises an amino acid sequence as shown in SEQ ID NO:3-4 or having at least 80% homology with SEQ ID NO:3-4, and HCDR3 comprises an amino acid sequence selected from those shown in SEQ ID NO:5-6 or having at least 80% homology with SEQ ID NO:5-6. Antibodies or antigen-binding fragments based on the aforementioned HCDR sequences exhibit high binding affinity and specificity to HLA-G.

[0063] In some preferred embodiments of this application, the aforementioned HCDR includes: HCDR1 with amino acid sequences as shown in SEQ ID NO:1-2; HCDR2 with amino acid sequences as shown in SEQ ID NO:3-4; and HCDR3 with amino acid sequences as shown in SEQ ID NO:5-6. Experimental verification shows that antibodies or their antigen-binding fragments having amino acid sequences as shown in SEQ ID NO:1-6 exhibit high affinity and specificity for HLA-G binding.

[0064] In some examples of this application, the aforementioned LCDRs include: LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains an amino acid sequence as shown in SEQ ID NO:7-8 or having at least 80% homology with SEQ ID NO:7-8, LCDR2 contains an amino acid sequence as shown in SEQ ID NO:9-10 or having at least 80% homology with SEQ ID NO:9-10, and LCDR3 contains an amino acid sequence as shown in SEQ ID NO:11-12 or having at least 80% homology with SEQ ID NO:11-12. Antibodies or antigen-binding fragments based on the aforementioned LCDR sequences exhibit high binding affinity and specificity to HLA-G.

[0065] In some preferred embodiments of this application, the aforementioned LCDR includes: LCDR1 with amino acid sequences as shown in SEQ ID NO:7-8; LCDR2 with amino acid sequences as shown in SEQ ID NO:9-10; and LCDR3 with amino acid sequences as shown in SEQ ID NO:11-12. Experimental verification shows that antibodies or antigen-binding fragments having amino acid sequences as shown in SEQ ID NO:7-12 exhibit high affinity and specificity for HLA-G binding.

[0066] It should be noted that one or more amino acid residues in the aforementioned HLA-G-targeting antibodies HCDR and / or LCDR can be replaced by other amino acid residues from the same side chain family, and the retention function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conserved modifications does not exceed one or two.

[0067] In some examples of this application, the aforementioned antibody or its antigen-binding fragment further includes a heavy chain framework region.

[0068] In some examples of this application, the aforementioned antibody or its antigen-binding fragment further includes a light chain framework region.

[0069] In some examples of this 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 rabbit-derived antibodies, mouse-derived antibodies, sheep-derived antibodies, goat-derived antibodies, primate-derived antibodies, or mutants thereof.

[0070] In some preferred embodiments of this application, the heavy chain variable region of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from or having at least 80% homology with SEQ ID NO:13-14. In some more preferred embodiments of this application, the amino acid sequence of the heavy chain variable region of the aforementioned antibody or its antigen-binding fragment is as shown in SEQ ID NO:13-14.

[0071] In some preferred embodiments of this application, the light chain variable region of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:15-16 or having at least 80% homology with SEQ ID NO:15-16. In some more preferred embodiments of this application, the amino acid sequence of the light chain variable region of the aforementioned antibody or its antigen-binding fragment is as shown in SEQ ID NO:15-16.

[0072] Antibodies or antigen-binding fragments based on the above-mentioned heavy chain variable region and light chain variable region sequences have high affinity and specificity for HLA-G binding.

[0073] In some examples of this application, the aforementioned antibody or its antigen-binding fragment further includes a heavy chain constant region.

[0074] In some examples of this application, the aforementioned antibody or its antigen-binding fragment further includes a light chain constant region.

[0075] In some examples of this 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 rabbit-derived antibodies, mouse-derived antibodies, sheep-derived antibodies, goat-derived antibodies, primate-derived antibodies, or mutants thereof.

[0076] In some preferred embodiments of this application, at least a portion of the aforementioned heavy chain constant region and the aforementioned light chain constant region are derived from mouse or human antibodies.

[0077] In some preferred embodiments of this application, the aforementioned heavy chain constant region is selected from the human IgG1 constant region.

[0078] In some more preferred examples of this application, the aforementioned human IgG1 constant region has an L234A / L235A mutation.

[0079] It should be noted that L234A indicates that the leucine (Leu, L) at position 234 is mutated to alanine (Ala, A); similarly, L235A indicates that the leucine (Leu, L) at position 235 is mutated to alanine (Ala, A).

[0080] In some preferred examples of this application, the aforementioned light chain constant region is selected from the human Ig kappa constant region.

[0081] In some examples of this application, the heavy chain of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from or having at least 80% homology with SEQ ID NO:17-18. In some preferred examples of this application, the amino acid sequence of the heavy chain of the aforementioned antibody or its antigen-binding fragment is as shown in SEQ ID NO:17-18.

[0082] In some examples of this application, the light chain of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from or having at least 80% homology with SEQ ID NO:19-20. In some preferred examples of this application, the amino acid sequence of the light chain of the aforementioned antibody or its antigen-binding fragment is as shown in SEQ ID NO:19-20.

[0083] In some examples of this application, the aforementioned antibody includes at least one selected from polyclonal antibodies and monoclonal antibodies; or the aforementioned antigen-binding fragment includes at least one selected from F(ab')2 fragment, Fab' fragment, Fab fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, scFv-Fv fusion protein, and minimum recognition unit. In some preferred examples of this application, the aforementioned antibody is a monoclonal antibody.

[0084] Nucleic acid molecules

[0085] In another aspect of this application, a nucleic acid molecule is provided, which 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.

[0086] In some examples of this application, the aforementioned nucleic acid molecule is DNA.

[0087] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0088] expression carrier

[0089] In another aspect of this application, an expression vector is provided, which carries the nucleic acid molecule as described above. Based on the aforementioned expression vector, the aforementioned antibody or its antigen-binding fragment can be prepared in large quantities in vitro.

[0090] It should be noted that when linking the aforementioned nucleic acid molecules to the vector, the nucleic acid molecules and the control elements on the vector can be directly or indirectly connected, as long as these control elements can control the translation and expression of the aforementioned nucleic acid molecules. These control elements can originate directly from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the connection between the aforementioned nucleic acid molecules and the control elements must be operably established.

[0091] According to embodiments of the present invention, the aforementioned vector may refer to a cloning vector or an expression vector, which can be obtained by operatively ligating the aforementioned nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vectors used in this invention are not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0092] In this document, the term "operably ligated" refers to ligating a foreign gene to a vector such that the control elements within the vector, such as amino acid sequences controlling transcription and amino acid sequences controlling translation, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vectors of some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of proteins encoded by the aforementioned nucleic acid molecules.

[0093] In some examples of this application, the aforementioned vector is a eukaryotic vector or a prokaryotic vector.

[0094] In some examples of this application, the aforementioned vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0095] Preparation method of antibody or its antigen-binding fragment

[0096] In another aspect of this application, a method for preparing the aforementioned antibody or its antigen-binding fragment is provided, comprising: introducing the aforementioned expression vector into cells; culturing the aforementioned cells under conditions suitable for protein expression and secretion to obtain the aforementioned antibody or its antigen-binding fragment. This method enables the large-scale preparation of antibodies or their antigen-binding fragments in vitro.

[0097] In some examples of this application, the aforementioned cells are eukaryotic cells.

[0098] In some specific examples of this application, the aforementioned eukaryotic cells are mammalian cells. When the cell is a mammalian cell, the expression efficiency of the aforementioned antibody or its antigen-binding fragment is higher.

[0099] It should be noted that the aforementioned eukaryotic cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0100] Recombinant cells

[0101] In another aspect of this application, a recombinant cell is provided, carrying the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, or the aforementioned expression vector. In some examples of this application, the aforementioned recombinant cell is obtained by transfection or transformation of the aforementioned expression vector, and the aforementioned recombinant cell can efficiently express the aforementioned antibody or antigen-binding fragment under suitable conditions.

[0102] In some examples of this application, the aforementioned recombinant cells are prokaryotic cells, eukaryotic cells, or bacteriophages. It should be noted that the aforementioned eukaryotic cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0103] In some examples of this application, the aforementioned prokaryotic cells are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0104] In some examples of this application, the aforementioned eukaryotic cells are fungal, insect, plant, or mammalian cells.

[0105] In some examples of this application, the aforementioned fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomyces or Trichoderma.

[0106] In some examples of this application, the aforementioned insect cells are grass armyworm cells; in some examples of this application, the aforementioned plant cells are tobacco plant cells; in some examples of this application, the aforementioned mammalian cells are BHK cells, CHO cells, COS cells, myeloma cells, or human embryonic kidney 293 cells; and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0107] In some examples of this application, the aforementioned recombinant cells are mammalian cells. When the cell is a mammalian cell, the expression efficiency of the aforementioned antibody or its antigen-binding fragment is higher.

[0108] In some examples of this application, the aforementioned recombinant cells are BHK cells, CHO cells, COS cells, or NSO cells.

[0109] It should be noted that the "suitable conditions" mentioned above in this application refer to conditions suitable for the expression of the bispecific antibody of this application. Those skilled in the art will readily understand that suitable conditions for bispecific antibody expression 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 optimal conditions for bispecific antibody expression based on the specific environment of their laboratory.

[0110] Immunoconjugates

[0111] In another aspect of this application, an immunoconjugate is proposed, comprising: the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The aforementioned immunoconjugate can be used to treat HLA-G-related diseases, such as tumors.

[0112] In some examples of this application, the aforementioned immune conjugates may further include therapeutic agents. These therapeutic agents include cytokines, immunomodulators, and small molecule drugs, among others.

[0113] Composition

[0114] In another aspect of this application, a composition is provided comprising: the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned immunoconjugate. The aforementioned composition may be used to treat HLA-G-related diseases, such as tumors.

[0115] It should be noted that the aforementioned compositions include combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the aforementioned compositions may be administered to the subject as a whole or separately. When the components contained in the aforementioned compositions are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0116] Pharmaceutical uses

[0117] In another aspect of this application, the use of the aforementioned antibody or its antigen-binding fragment, 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 drug for the treatment or prevention of HLA-G-mediated diseases. Drugs prepared based on the aforementioned antibody or its antigen-binding fragment can block the binding of HLA-G to LILRB1 or LILRB2, significantly restoring the anti-tumor function of NK cells and T cells.

[0118] In some examples of this application, the aforementioned HLA-G-mediated diseases include tumors.

[0119] In some examples of this application, the aforementioned tumors include at least one of breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, leukemia, and renal cell carcinoma.

[0120] drug

[0121] In another aspect of this application, a drug is proposed, comprising: the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned immune conjugate, or the aforementioned composition, wherein the aforementioned drug is used for or to prevent HLA-G-mediated diseases. The aforementioned drug can block the binding of HLA-G to LILRB1 or LILRB2, significantly restoring the anti-tumor function of NK cells and T cells.

[0122] In some examples of this application, the aforementioned HLA-G-mediated diseases include tumors.

[0123] In some examples of this application, the aforementioned tumors include at least one of breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, leukemia, and renal cell carcinoma.

[0124] In some examples of this application, pharmaceutically acceptable excipients are further included.

[0125] In some examples of this application, the aforementioned excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

[0126] In some examples of this application, the aforementioned pharmaceutical composition is an injectable formulation.

[0127] It should be noted that the aforementioned pharmaceutical compositions include combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the aforementioned pharmaceutical compositions may be administered to the subject as a whole or separately. When the components contained in the aforementioned pharmaceutical compositions are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0128] The drug of this application contains a safe and effective amount of the active ingredient (bispecific antibody) of this application, as well as pharmaceutically acceptable excipients. These excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage form of the drug of this application is an injection, an oral formulation (tablet, capsule, oral liquid), a transdermal formulation, or a sustained-release formulation. For example, it may be prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The aforementioned drug is preferably manufactured under aseptic conditions.

[0129] The effective amount of the active ingredient described above may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, immune status, and route of administration. For example, due to the urgency of the treatment, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0130] The pharmaceutically acceptable excipients mentioned above in this application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the route of administration, as is well known to those skilled in the art.

[0131] Uses in the preparation kit

[0132] In another aspect of this application, the use of the aforementioned antibody or its antigen-binding fragment in the preparation of a kit for the detection of HLA-G is proposed.

[0133] As previously mentioned, the antibodies or antigen-binding fragments of the present application embodiments can specifically bind to HLA-G; therefore, the aforementioned antibodies or antigen-binding fragments can be used to detect HLA-G. Furthermore, they can be used to prepare HLA-G-related kits for scientific research, such as qualitative or quantitative detection of HLA-G protein molecules in biological samples. More specifically, they can be used in kits involving the specific binding properties of HLA-G and antibodies, such as immunoblotting and immunoprecipitation. These kits may contain any one or more of the following: antagonists, the bispecific antibodies of the present invention, or pharmaceutical reference materials; protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. The bispecific antibodies of this application can be used for different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, HLA-G-mediated diseases can be tested by examining the serum or blood of a subject.

[0134] Reagent test kit

[0135] In another aspect of this application, a kit is provided comprising the antibody or antigen-binding fragment thereof described in the first aspect. The aforementioned kit can be used for portable detection of HLA-G.

[0136] Disease treatment methods

[0137] In another aspect, this application provides a method for preventing and / or treating HLA-G mediated diseases. According to embodiments of this application, the aforementioned method includes administering to a subject a pharmaceutically acceptable amount of an antibody or its antigen-binding fragment, expression vector, recombinant cells, immunoconjugate, composition, or drug. Based on the aforementioned method, HLA-G mediated diseases can be effectively treated.

[0138] 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 implementation, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with tumors, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0139] The effective amount of the antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, immunoconjugate, composition, or drug described above in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0140] In some examples of this application, the aforementioned HLA-G-mediated diseases include tumors.

[0141] In some examples of this application, the aforementioned tumors include at least one of breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, leukemia, and renal cell carcinoma.

[0142] Uses in disease treatment

[0143] In another aspect of this application, the use of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, immunoconjugates, compositions, or drugs in the treatment or prevention of HLA-G mediated diseases is proposed. In some examples of this application, administration of an effective dose of the antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, immunoconjugate, composition, or drug to a subject can effectively treat HLA-G mediated diseases.

[0144] In some examples of this application, the aforementioned HLA-G-mediated diseases include tumors.

[0145] In some examples of this application, the aforementioned tumors include at least one of breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, leukemia, and renal cell carcinoma.

[0146] Table 1. HLA-G antibody sequences

[0147]

[0148]

[0149]

[0150]

[0151] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0152] Example 1: Preparation of Antibody

[0153] This embodiment was used to obtain a murine monoclonal antibody against human HLA-G. BALB / c mice were immunized three times with a His-tagged HLA-G protein (KaiKa Biotechnology) as the antigen and a complete Freund's adjuvant. Blood was collected from the immunized mice to detect the immune response. Serum was screened using ELISA and flow cytometry following standard procedures to obtain mice with anti-human HLA-G immunoglobulin. Spleen cells from the mice with the highest anti-HLA-G immunoglobulin levels were fused with murine myeloma cells SP2 / 0 (ATCC number CRL 1581). The fused hybridoma cells were screened for antibodies to obtain the murine monoclonal antibody. The positive hybridoma cells were sequenced to obtain the heavy chain variable region and light chain variable region sequences of the murine monoclonal antibody, whose amino acid sequences are shown in SEQ ID NO:13 and SEQ ID NO:15.

[0154] Example 2: Detection of binding activity of mouse HLA-G antibody

[0155] Test Example 1: Detection of the binding activity of mouse HLA-G antibody to human and cynomolgus monkey HLA-G proteins.

[0156] ELISA assays were used to detect the binding of HLA-G antibodies to HLA-G proteins in humans and cynomolgus monkeys. The specific experimental procedure is as follows:

[0157] Human and cynomolgus monkey HLA-G proteins (Kaikai Biotechnology) were diluted to 1 μg / mL with PBS buffer and added to 96-well plates at a volume of 100 μL / well. The plates were incubated overnight at 4°C. The PBS buffer was discarded, and the plates were washed three times with PBST (pH 7.2 PBS containing 0.1% Tween 20). 300 μL / well of 5% BSA was added for blocking, and the plates were incubated at room temperature for 2 h. The blocking buffer was discarded, and 100 μL of the antibody diluted with 0.05% BSA was added. The plates were incubated at room temperature for 1 h. After washing three times with PBST, 100 μL of HRP-labeled goat anti-mouse IgG Fab secondary antibody diluted with 0.5% BSA (Southern Biotech) was added. The plates were incubated at room temperature for 1 h. After washing three times with PBST, 80 μL / well of TMB was added. The plates were incubated at room temperature for 10 min, and the reaction was terminated by adding 80 μL / well of stop solution. The absorbance was read at 450 nm using a microplate reader. Specific experimental results are as follows: Figure 1 As shown, the murine antibody (49K8) of this application can bind well to human and cynomolgus monkey HLA-G protein.

[0158] Test Example 2: Detection of the activity of murine HLA-G antibody in JEG-3 tumor cell lines

[0159] The primary tumor cell line JEG-3 (ATCC number HTB-36), which highly expresses HLA-G, was diluted to 1×10⁻⁶ with PBS. 6 Cells were added at a rate of 90 μL / well to 96-well plates, with 10 μL of goat serum (Shanghai Sangon Biotech) added to each well. The plates were blocked at 4°C for 30 min. A series of serially diluted test antibodies were added, and the plates were incubated at 4°C for 30 min. After incubation, the cells were washed twice with PBS. Cells were resuspended in 100 μL / well of PBS, and 0.1 μL / well of APC-labeled goat anti-mouse IgG antibody (Biolegend) was added. The plates were incubated at 4°C in the dark for 30 min. Cells were washed twice with PBS. Cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry. Figure 2 The murine antibody (49K8) of this application shows that it can bind well to the JEG-3 tumor cell line that highly expresses HLA-G.

[0160] Example 3: Detection of blocking activity of murine HLA-G antibody

[0161] Test Example 1: Mouse-derived HLA-G antibody blocks the binding of LILRB1 or LILRB2 protein to CHO-K1-HLA-G cells.

[0162] HLA-G antibodies work by binding to the extracellular region of HLA-G, thereby blocking the signaling pathway of HLA-G binding to its receptors LILRB1 or LILRB2. Flow cytometry is used to detect the blocking effect of HLA-G antibodies on the binding of HLA-G to its receptors. The specific experimental procedure is as follows:

[0163] HEK293T cells were processed at a rate of 5 × 10 5 Seed cells / well in 6-well plates and cultured overnight in DMEM medium without antibiotics. Before transfection, discard the medium and add 1 mL of fresh DMEM medium without antibiotics. Add pLVXEF1ahuman HLA-GIRES-zsGreen (with the inserted nucleotide sequence encoding human HLA-G protein SEQ ID NO:21) to 200 μL of serum-free DMEM medium at a ratio of 2:1:1, along with pMD2G and psPAX2 vector (3 μg total). Then add 12 μg of polyetherimide (PEI, Polysciences Ltd.), mix well, and let stand for 16 min. Add all the liquid to the 6-well plates seeded with HEK293T cells. After culturing for 6 h, discard the medium and add fresh complete DMEM medium. 48 h after transfection, collect the cell culture supernatant and filter through a 0.45 μm filter (Millipore) to obtain lentiviral supernatant. Add all of the obtained lentiviral supernatant to a container containing 1×10⁻⁶ cells / well. 4 In a 6-well plate containing CHOK1 cells, polybrene (Sigma) was added to a final concentration of 4 μg / mL and cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells are CHOK1HLA-G cells.

[0164] CHO-K1-HLA-G cells were diluted to 2 × 10⁻⁶ with PBS. 6 Cells were added at a rate of 90 μL / well to a 96-well plate, followed by 10 μL / well of rat serum, and blocked at 4°C for 30 min. A series of serially diluted test antibodies and a final concentration of 2 μg / mL of biotin-tagged human LILRB1 or LILRB2 protein (ACRO) were added, and the plates were incubated at 4°C for 30 min. After incubation, the cells were washed twice with PBS, resuspended in 100 μL / well of PBS, and then 0.1 μL / well of APC-labeled Streptavidin antibody (Biolegend) was added. The plates were incubated at 4°C in the dark for 30 min. After washing twice with PBS, the cells were centrifuged and the supernatant was discarded. The cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry. Specific experimental results are shown below. Figure 3 As shown, the murine antibody (49K8) of this application can effectively block the binding of HLA-G to its receptors LILRB1 or LILRB2.

[0165] Test Example 2: Mouse-derived HLA-G antibody blocks the binding of HLA-G protein to LILRB1 or LILRB2 protein.

[0166] ELISA assays are used to detect the binding of HLA-G antibodies to HLA-G protein and LILRB1 or LILRB2 protein. The specific experimental procedure is as follows:

[0167] Human HLA-G protein (Kaikai Biotechnology) was diluted to 5 μg / mL with PBS buffer and added to 100 μL / well of a 96-well plate. The plate was incubated overnight at 4°C. The PBS buffer was discarded, and the plate was washed three times with PBST (pH 7.2 PBS containing 0.1% Tween 20). 300 μL / well of 5% BSA was added for blocking, and the plate was incubated at room temperature for 2 h. The blocking solution was discarded, and biotin-labeled LILRB1 or LILRB2 protein (ACRO) was diluted to 2 μg / mL with 0.05% BSA. 50 μL of the antibody diluted with 0.05% BSA was added to each well, and the plate was incubated at room temperature for 1 h. After washing three times with PBST, 100 μL of HRP-labeled Streptavidin secondary antibody diluted with 0.5% BSA (Southern Biotech) was added, and the plate was incubated at room temperature for 1 h. After washing the plate three times with PBST, add 80 μL / well of TMB and incubate at room temperature for 10 min. Then, add 80 μL / well of stop solution to terminate the reaction. Read the absorbance at 450 nm using a microplate reader. Specific experimental results are as follows: Figure 4 As shown, the murine antibody (49K8) of this application can effectively block the binding of HLA-G to its receptors LILRB1 or LILRB2.

[0168] Example 4: Activity assay of murine antibody promoting PBMC killing function

[0169] Flow cytometry was used to detect the activity of HLA-G antibodies in promoting PBMC killing.

[0170] HEK293T cells were processed at a rate of 5 × 10 5Seed cells / well in 6-well plates and cultured overnight in DMEM medium without antibiotics. Before transfection, discard the medium and add 1 mL of fresh DMEM medium without antibiotics. Add pLVXEF1ahuman HLA-GIRES-zsGreen (with the inserted nucleotide sequence encoding human HLA-G protein SEQ ID NO:21) to 200 μL of serum-free DMEM medium at a ratio of 2:1:1, along with pMD2G and psPAX2 vector (3 μg total). Then add 12 μg of polyetherimide (PEI, Polysciences Ltd.), mix well, and let stand for 16 min. Add all the liquid to the 6-well plates containing HEK293T cells. After culturing for 6 h, discard the medium and add fresh complete DMEM medium. 48 h after transfection, collect the cell culture supernatant and filter through a 0.45 μm filter (Millipore) to obtain lentiviral supernatant. Add all of the obtained lentiviral supernatant to a container containing 1×10⁻⁶ cells / well. 4 Add 4 μg / mL of polybrene (Sigma) to 6-well plates containing SK-OV-3 cells (ATCC ID: HTB-77) or K562 cells (ATCC ID: CCL-243) and incubate for 12 h. Then discard the supernatant and add fresh, complete IMEM medium. The resulting cells are SK-OV-3-HLA-G or K562-HLA-G.

[0171] SK-OV-3-HLA-G cells were collected, resuspended in 1640 medium, and counted. The cell density was adjusted to approximately 1.5 × 10⁻⁶ cells / year. 6 Cells / mL. Take 1 mL of cell suspension into a 1.5 mL EP tube, add 1 μL of Cell Trace Violet (Invitrogen), mix well, and label at 37℃ for 20 min. Wash the labeled cells three times with 1640 medium and adjust the cell density to 1×10⁻⁶ cells / mL. 5 PBMCs (Miaoshun Biotechnology) were collected by centrifugation at 300g, resuspended in 1640 medium, counted, and the PBMC concentration was adjusted to 5 × 10⁻⁶ cells / mL. 6 cells / mL. Add 100 μL / well of diluted SK-OV-3-HLA-G cells and 100 μL / well of PBMCs to each well of a 96-well round-bottom plate, along with serially diluted test antibodies. Centrifuge at 250g for 4 min at room temperature, then incubate at 37℃ in a 5% CO2 incubator for 4-6 h. After incubation, add 1 μL of 7AAD (BD Bioscience) to each well and mix well. Analyze the results using flow cytometry. Specific experimental results are shown below. Figure 5As shown, the murine antibody (49K8) of this application can effectively promote the killing of SK-OV-3 cell lines overexpressing HLA-G by PBMCs in a dose-dependent manner.

[0172] Example 5: Antibody Humanization

[0173] Referring to the HLA-G antibody heavy chain variable region sequence (as shown in SEQ ID NO: 13) and light chain variable region sequence (as shown in SEQ ID NO: 14) obtained in Example 1, a humanization template with the best match for its non-CDR region was designed and selected using MOE software. The CDR region of the murine antibody was transplanted onto the selected humanization template, replacing the CDR region of the human template, to obtain the humanized antibody. Then, based on the three-dimensional structure of the murine antibody, reversion mutations were performed on the embedded residues, residues that directly interact with the CDR region, and residues in the FR region that have an important influence on the conformation of VL and VH, to obtain the humanized antibody. In addition, proteins introduce some chemical modifications after translation in cells, called post-translational modifications (PTMs). For antibodies, some PTMs present in the CDR region are likely to have a significant impact on antigen binding, and may also change the physicochemical properties of the antibody, thereby increasing the difficulty and risk of antibody development. To avoid these potential PTM risks, during the humanization process, these PTM sites are mutated to remove the formation of specific PTMs. The final humanized HLA-G antibody has a heavy chain variable region sequence as shown in SEQ ID NO:14 and a light chain variable region sequence as shown in SEQ ID NO:16. The heavy chain constant region of the above-mentioned humanized antibody (h49K8) is the human IgG1 constant region, and a (L234A / L235A) mutation has been introduced. In addition, its light chain constant region is the human Ig kappa constant region.

[0174] Example 6: Detection of humanized antibody binding activity

[0175] Flow cytometry was used to detect the ability of humanized HLA-G antibodies to bind to the JEG-3 cell line.

[0176] The JEG-3 cells were diluted to 1 × 10⁻⁶ with PBS. 6Cells were added at a rate of 90 μL / well to 96-well plates, with 10 μL of goat serum (Shanghai Sangon Biotech) added to each well. The plates were blocked at 4°C for 30 min. A series of serially diluted test antibodies were added, and the plates were incubated at 4°C for 30 min. After incubation, the cells were washed twice with PBS. Cells were resuspended in 100 μL / well of PBS, and 0.1 μL / well of AF647-labeled goat anti-human IgG Fc fragment specific antibody (Jackson ImmunoResearch) was added. The plates were incubated at 4°C in the dark for 30 min. Cells were washed twice with PBS. Cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry. Figure 6 The humanized antibody (h49K8) of this application shows that it can bind well to JEG-3 tumor cell lines that highly express HLA-G.

[0177] Example 7: Detection of humanized antibody blocking activity

[0178] Test Example 1: Humanized HLA-G antibody blocks the binding of LILRB1 or LILRB2 protein to CHO-K1-HLA-G cells.

[0179] Flow cytometry was used to detect the blocking effect of humanized HLA-G antibodies on the binding of LILRB1 or LILRB2 proteins to CHO-K1-HLA-G cells. The specific experimental procedure is as follows:

[0180] CHO-K1-HLA-G cells were diluted to 2 × 10⁻⁶ with PBS. 6 Cells were added at a rate of 90 μL / well to a 96-well plate, followed by 10 μL / well of rat serum, and blocked at 4°C for 30 min. A series of serially diluted test antibodies and a final concentration of 2 μg / mL of biotin-tagged human LILRB1 or LILRB2 protein (ACRO) were added, and the plates were incubated at 4°C for 30 min. After incubation, the cells were washed twice with PBS, resuspended in 100 μL / well of PBS, and then 0.1 μL / well of APC-labeled Streptavidin antibody (Biolegend) was added. The plates were incubated at 4°C in the dark for 30 min. After washing twice with PBS, the cells were centrifuged and the supernatant was discarded. The cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry. Specific experimental results are shown below. Figure 7 As shown, the humanized antibody (h49K8) of this application can effectively block the binding of HLA-G to its receptors LILRB1 or LILRB2.

[0181] Test Example 2: Humanized HLA-G antibody blocks the binding of HLA-G protein to LILRB1 or LILRB2 protein.

[0182] ELISA assays are used to detect the binding of HLA-G antibodies to HLA-G protein and LILRB1 or LILRB2 protein. The specific experimental procedure is as follows:

[0183] Human HLA-G protein (Kaikai Biotechnology) was diluted to 5 μg / mL with PBS buffer and added to 100 μL / well of a 96-well plate. The plate was incubated overnight at 4°C. The PBS buffer was discarded, and the plate was washed three times with PBST (pH 7.2 PBS containing 0.1% Tween 20). 300 μL / well of 5% BSA was added for blocking, and the plate was incubated at room temperature for 2 h. The blocking solution was discarded, and biotin-labeled LILRB1 or LILRB2 protein (ACRO) was diluted to 2 μg / mL with 0.05% BSA. 50 μL of the antibody diluted with 0.05% BSA was added to each well, and the plate was incubated at room temperature for 1 h. After washing three times with PBST, 100 μL of HRP-labeled Streptavidin secondary antibody diluted with 0.5% BSA (Southern Biotech) was added, and the plate was incubated at room temperature for 1 h. After washing the plate three times with PBST, add 80 μL / well of TMB and incubate at room temperature for 10 min. Then, add 80 μL / well of stop solution to terminate the reaction. Read the absorbance at 450 nm using a microplate reader. Specific experimental results are as follows: Figure 8 As shown, the humanized antibody (h49K8) of this application can effectively block the binding of HLA-G to its receptors LILRB1 or LILRB2.

[0184] Example 8: Activity assay of humanized antibody promoting PBMC killing function

[0185] Flow cytometry was used to detect the activity of humanized HLA-G antibodies in promoting PBMC killing.

[0186] K562-HLA-G cells were collected, the cell pellet was resuspended in 1640 medium, and the cells were counted. The cell density was adjusted to approximately 1.5 × 10⁻⁶ cells / year. 6 Cells / mL. Take 1 mL of cell suspension into a 1.5 mL EP tube, add 1 μL of Cell Trace Violet (Invitrogen), mix well, and label at 37℃ for 20 min. Wash the labeled cells three times with 1640 medium and adjust the cell density to 1×10⁻⁶ cells / mL. 5 PBMCs were collected by centrifugation at 300g, resuspended in 1640 medium, counted, and the PBMC concentration was adjusted to 1×10⁻⁶ cells / mL. 6cells / mL. Add 100 μL / well of diluted K562-HLA-G cells and 100 μL / well of PBMCs to each well of a 96-well round-bottom plate, along with serially diluted test antibodies. Centrifuge at 250g for 4 min at room temperature, then incubate at 37℃ in a 5% CO2 incubator for 4-6 h. After incubation, add 1 μL of 7AAD (BD Bioscience) to each well and mix well. Analyze the results using flow cytometry. Specific experimental results are shown below. Figure 9 As shown, the humanized antibody (h49K8) of this application can effectively promote the killing of K562 cell lines overexpressing HLA-G by PBMCs in a dose-dependent manner.

[0187] Example 9: Humanized Antibody Affinity Detection

[0188] Surface plasmon resonance (SPR) technology was used to determine the affinity of humanized HLA-G antibodies.

[0189] The specific operation is as follows: Turn on the Biacore 1K instrument (Cytiva), place the Protein A chip (Cytiva) inside, and then equilibrate it in HBS-EP+ buffer. Next, flow the test antibody at a concentration of 1 μg / mL through the chip surface at a rate of 30 μL / min to capture the test antibody. Then, flow different concentrations of human HLA-G protein (Kaikai Biotechnology) or cynomolgus monkey HLA-G protein (Kaikai Biotechnology) through the chip surface at a rate of 30 μL / min, setting the binding time to 90 s, followed by dissociation for 600 s. Kinetic analysis was performed using a 1:1 binding model. The affinity values ​​of the humanized HLA-G antibody for human HLA-G protein and cynomolgus monkey HLA-G protein are shown in Table 2, indicating that the humanized HLA-G antibody has significant binding activity to both human and cynomolgus monkey HLA-G proteins.

[0190] Table 2. Affinity values ​​of humanized HLA-G antibodies

[0191] Antibody Antigen Ka (1 / Ms) Kd (1 / s) KD (M) h49K8 Human HLA-G 7.40E+05 4.05E-04 5.48E-10 h49K8 Monkey HLA-G 9.28E+04 5.96E-03 6.42E-08

[0192] In summary, the monoclonal antibody prepared in this application has high specificity and affinity, and can effectively bind to human and cynomolgus monkey HLA-G protein, block the binding of HLA-G to LILRB1 or LILRB2 protein, and promote the killing ability of immune cells (such as PBMCs) against tumor cells.

[0193] The preferred embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application.

[0194] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0195] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. An antibody or its antigen-binding fragment, characterized in that, include: Heavy chain complementarity-determining region (HCDR) and light chain complementarity-determining region (LCDR); in, The HCDR contains an amino acid sequence selected from at least one of SEQ ID NO:1-6 or having at least 80% homology with it; The LCDR contains an amino acid sequence selected from at least one of SEQ ID NO:7-12 or having at least 80% homology with it.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The HCDRs include: HCDR1, HCDR2, and HCDR3, wherein: The HCDR1 contains an amino acid sequence as shown in SEQ ID NO:1-2 or having at least 80% homology with SEQ ID NO:1-2. The HCDR2 comprises an amino acid sequence as shown in SEQ ID NO:3-4 or having at least 80% homology with SEQ ID NO:3-4, and The HCDR3 comprises an amino acid sequence selected from those shown in SEQ ID NO:5-6 or having at least 80% homology with SEQ ID NO:5-6; Preferably, the HCDR comprises: HCDR1 with amino acid sequences as shown in SEQ ID NO:1-2 respectively; Preferably, the HCDR comprises: HCDR2 with amino acid sequences as shown in SEQ ID NO:3-4 respectively; Preferably, the HCDR comprises HCDR3, which has an amino acid sequence as shown in SEQ ID NO:5-6.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The LCDR includes: LCDR1, LCDR2, and LCDR3, wherein: The LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:7-8 or having at least 80% homology with SEQ ID NO:7-8. The LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:9-10 or having at least 80% homology with SEQ ID NO:9-10, and The LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:11-12 or having at least 80% homology with SEQ ID NO:11-12; Preferably, the LCDR comprises: LCDR1 with amino acid sequences as shown in SEQ ID NO:7-8 respectively; Preferably, the LCDR comprises: LCDR2 with amino acid sequences as shown in SEQ ID NO:9-10 respectively; Preferably, the LCDR comprises: LCDR3 with amino acid sequences as shown in SEQ ID NO:11-12 respectively.

4. The antibody or its antigen-binding fragment according to claim 2 or 3, characterized in that, The antibody or its antigen-binding fragment further includes: a heavy chain framework region; Optionally, the antibody or its antigen-binding fragment further includes a light chain framework region; Optionally, at least a portion of the heavy chain framework region and the light chain framework region are independently derived from at least one of rabbit-derived antibodies, mouse-derived antibodies, sheep-derived antibodies, goat-derived antibodies, primate-derived antibodies, or mutants thereof; Preferably, the heavy chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence selected from or having at least 80% homology with SEQ ID NO:13-14; preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:13-14. Preferably, the light chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:15-16 or having at least 80% homology with SEQ ID NO:15-16; preferably, the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:15-16.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The antibody or its antigen-binding fragment further includes: a heavy chain constant region; Optionally, the antibody or its antigen-binding fragment further includes a light chain constant region; Optionally, at least a portion of the heavy chain constant region and the light chain constant region are independently derived from at least one of rabbit-derived antibodies, mouse-derived antibodies, sheep-derived antibodies, goat-derived antibodies, primate-derived antibodies, or mutants thereof; preferably, at least a portion of the heavy chain constant region and the light chain constant region are derived from mouse-derived or human-derived antibodies. Preferably, the heavy chain constant region is selected from the human IgG1 constant region; More preferably, the human IgG1 constant region has an L234A / L235A mutation; Preferably, the light chain constant region is selected from the human Ig kappa constant region; Preferably, the heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence selected from those shown in SEQ ID NO:17-18 or having at least 80% homology with SEQ ID NO:17-18; preferably, the amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:17-18. Preferably, the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence selected from those shown in SEQ ID NO:19-20 or having at least 80% homology with SEQ ID NO:19-20; preferably, the amino acid sequence of the light chain of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:19-20.

6. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody includes at least one selected from polyclonal antibodies and monoclonal antibodies; or The antigen-binding fragment includes at least one selected from the F(ab')2 fragment, Fab' fragment, Fab fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, scFv-Fv fusion protein, and the smallest 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 its antigen-binding fragment as described in any one of claims 1 to 6; Optionally, the nucleic acid molecule is DNA.

8. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 7.

9. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that, include: The expression vector according to claim 8 is introduced into cells; The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the antibody or its antigen-binding fragment; Optionally, the cells are eukaryotic cells.

10. A recombinant cell, characterized in that, The recombinant cells carry the nucleic acid molecule of claim 7 or the expression vector of claim 8.

11. An immunoconjugate, characterized in that, include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 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 immune conjugate further comprises a therapeutic agent.

12. A composition, characterized in that, include: The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6, the nucleic acid molecule carrying the nucleic acid molecule as described in claim 7, the expression vector as described in claim 8, the recombinant cell as described in claim 10, or the immunoconjugate as described in claim 11.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, carrying the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 10, the immunoconjugate according to claim 11, or the composition according to claim 12 in the preparation of a medicament for the treatment or prevention of HLA-G mediated diseases.

14. The use according to claim 13, characterized in that, The HLA-G-mediated diseases include tumors; Optionally, the tumor includes at least one of breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, leukemia, and renal cell carcinoma.

15. A drug, characterized in that, include: The drug is an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a nucleic acid molecule carrying the nucleic acid molecule according to claim 7, an expression vector according to claim 8, a recombinant cell according to claim 10, an immunoconjugate according to claim 11, or a composition according to claim 12, and is used for or to prevent HLA-G mediated diseases.

16. The medicament according to claim 15, characterized in that, The HLA-G-mediated diseases include tumors; Optionally, the tumor includes at least one of breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, leukemia, and renal cell carcinoma; Optionally, it may further include pharmaceutically acceptable excipients; Optionally, the excipients include: One or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers; Optionally, the drug is an injectable form.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the preparation of a kit for detecting HLA-G.

18. A reagent kit, characterized in that, The kit contains: The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6.

19. The reagent kit according to claim 18, characterized in that, The kit is used to detect HLA-G.

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