KIR2DL fully humanized antibody and application thereof

Abstract

The invention relates to a KIR2DL fully humanized antibody and an application thereof. The amino acid sequence of the KIR2DL fully humanized antibody is one of SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21. The invention further discloses a kit for detecting the KIR2DL fully humanized antibody. Compared with the prior art, the KIR2DL fully humanized antibody can better block the interaction between the KIR2DL and the ligand HLA-C of the KIR2DL, so that the immune response can be accurately adjusted, the non-specific immunosuppression can be reduced, and in addition, the KIR2DL fully humanized antibody can be used for improving the immune response and reducing the non-specific immunosuppression through the unique biological function of the KIR2DL fully humanized antibody and the synergistic effect of the KIR2DL fully humanized antibody and the existing therapy. And a new hope and strategy are provided for tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of antibody preparation technology, and in particular to a KIR2DL fully human antibody and its applications. Background Technology

[0002] NK cell surfaces contain many important molecules that play crucial roles in NK cell activation, proliferation, differentiation, and effector function. KIR2DL is a newly discovered cell surface protein with immunosuppressive functions.

[0003] KIRs are a large family of receptors present in subsets of lymphocytes, including NK cells. The terminology used for KIRSs is based on the number of extracellular domains (KIR2D or KIR3D) and whether the cytoplasmic tail is long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS). In humans, the presence or absence of a given KIR varies from NK cell to NK cell within a single individual's NK cell population. Relatively high levels of KIR molecule polymorphism also exist in the human population, with some KIR molecules present in some individuals but not all. Some KIR gene products stimulate lymphocyte activity when bound to appropriate ligands. The confirmed stimulatory KIRs all have short cytoplasmic tails and charged transmembrane residues associated with aptamer molecules possessing an immunostimulatory motif (ITAM). Other KIR gene products are inhibitory in nature. All confirmed repressive KIRs possess long cytoplasmic tails and appear to interact with distinct subsets of HLA antigens depending on the KIR subtype. Repressive KIRs present one or more repressive motifs that recruit phosphatases in their intracytoplasmic portions. Known repressive KIR receptors include members of the KIR2DL and KIR3DL subfamilies. KIR receptors with two I8 domains (KIR2D) identify HLA-C allotypes: KIR2DL2 (formerly known as p58.2) or the closely related gene product KIR2DL3 recognizes antigenic determinants shared by group 2 HLA-C allotypes (Cw1, 3, 7, and 8); while KIR2DL1 (p581) recognizes antigenic determinants shared by complementary group 1 HLA-C allotypes (Cw2, 4, 5, and 6). Recognition by KIR2DLI depends on the presence of a lysine (Lys) residue at position 80 of the HLA-C allele. KIR2DL2 and KIR2DL3 recognition depends on the presence of an asparagine (Asn) residue at position 80. Importantly, most ILA-C alleles have an asparagine or lysine residue at position 80. A KIR with three I8 domains, KIR3DL1 (p70), recognizes the antigenic determinant site shared by the HLA-Bw4 allele. Finally, the homodimer KIR3DL2 (p140) of the molecule with three Ig domains recognizes both HLA-A3 and HLA-A11.

[0004] Although inhibitory KIRs and other type I inhibitory receptors can be co-expressed by NK cells, there are cells expressing single-KIRs in the NK expression profile of any given individual. Therefore, the corresponding NK cells are blocked only by cells expressing the type I allele.

[0005] KIR-mismatched NK cell populations or clones—that is, NK cell populations expressing KIR (which is incompatible with the host's HLA molecules)—have been shown to be the most likely mediators of the graft-versus-leukemia effect seen in allogeneic (allogeneic) transplantation. One approach to reproducing this effect in a given individual is to use agents that block the KIR / IILA interaction.

[0006] Monoclonal antibodies specific to KIR2DL1 have been shown to block the interaction between KIR2DL1 and the Cw4 (or analogue) allele. Monoclonal antibodies targeting KIR2DL2 / 3 have also been described, blocking the interaction between KIR2DL2 / / 3 and the HLACw3 (or analogue) allele. However, using such agents in clinical settings would require developing two therapeutic monoclonal antibodies (mAbs) to treat all patients, regardless of whether any given patient expresses type 1 or type 2 HLA-C alleles. Furthermore, the specific HILA type expressed by each patient must be determined beforehand before deciding which therapeutic antibody to use, resulting in significantly higher treatment costs.

[0007] Watzl et al., Tissue Antigens, 56, p. 240 (2000) prepared cross-reactive antibodies against multiple isotypes of KIRs, but these antibodies did not enhance NK cell activity. Experiments were conducted using various monoclonal antibodies against different KIRs. One antibody, NKVSF1, recognized the common antigenic determinant of CD158a (KIR2DL1), CDI58b (KIR2DL2), and p50.3 (KIR2DS4). However, it did not suggest that NKVSF1 could enhance NK cell activity, nor did it suggest that it could be used as a therapeutic agent. Therefore, to date, no practical and effective method for regulating NK cell activity has been found in this field, and HLA allele-specific methods using specific reagents are still needed.

[0008] Monoclonal antibody drugs have potential immunogenicity, which may induce the production of anti-drug antibodies (ADAs) in the human body. ADAs can reduce the efficacy of drugs, and in more severe cases, cause hypersensitivity reactions. The closer the antibody gene source is to the human gene, the lower the immunogenicity, and the higher the efficacy and safety. From murine monoclonal antibodies to fully human monoclonal antibodies, the immunogenicity of biologics has been continuously reduced, providing patients with safer and more durable treatment options. Generally speaking, chimeric antibodies are 70% human in composition, humanized antibodies can reach a humanization level of 95%, while the amino acid sequence of fully human antibodies is 100% derived from humans. This difference in humanization level also determines the difference in antibody efficacy. Because fully human antibodies are entirely derived from the human body, they have the lowest rejection rate and the best safety profile. Summary of the Invention

[0009] The purpose of this invention is to provide a fully human KIR2DL antibody and its applications. Compared to Yangshen (1-7F9), the only currently clinically approved antibody, the fully human KIR2DL antibody described in this invention can better block the interaction between KIR2DL and its ligand HLA-C. This helps to precisely regulate the immune response and reduce non-specific immunosuppression. Furthermore, the fully human KIR2DL antibody, through its unique biological functions and synergistic effects with existing therapies, provides new hope and strategies for tumor immunotherapy.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] The first objective of this invention is to provide a KIR2DL fully human antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein,

[0012] The heavy chain variable region CDR1 includes the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.7 and SEQ ID NO.13, CDR2 includes the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.8 and SEQ ID NO.14, and CDR3 includes the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.9 and SEQ ID NO.15;

[0013] The CDR1 of the light chain variable region includes the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.10 and SEQ ID NO.16, the CDR2 includes the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.11 and SEQ ID NO.17, and the CDR3 includes the amino acid sequences shown in SEQ ID NO.6, SEQ ID NO.12 and SEQ ID NO.18.

[0014] Furthermore, the CDR1 sequence of the heavy chain variable region is SEQ ID NO.1, the CDR2 sequence is SEQ ID NO.2, and the CDR3 sequence is SEQ ID NO.3.

[0015] The CDR1 of the light chain variable region is SEQ ID NO.4, the CDR2 is SEQ ID NO.5, and the CDR3 is SEQ ID NO.6; or,

[0016] The CDR1 of the heavy chain variable region is SEQ ID NO.7, the CDR2 is SEQ ID NO.8, and the CDR3 is SEQ ID NO.9;

[0017] The CDR1 of the light chain variable region is SEQ ID NO.10, the CDR2 is SEQ ID NO.11, and the CDR3 is SEQ ID NO.12; or,

[0018] The heavy chain variable region has CDR1 as SEQ ID NO.13, CDR2 as SEQ ID NO.14, and CDR3 as SEQ ID NO.15;

[0019] The CDR1 of the light chain variable region is SEQ ID NO.16, the CDR2 is SEQ ID NO.17, and the CDR3 is SEQ ID NO.18.

[0020] As a preferred technical solution, the CDR1 sequence of the heavy chain variable region is SEQ ID NO.1, the CDR2 sequence is SEQ ID NO.2, and the CDR3 sequence is SEQ ID NO.3.

[0021] The CDR1 of the light chain variable region is SEQ ID NO.4, the CDR2 is SEQ ID NO.5, and the CDR3 is SEQ ID NO.6.

[0022] Furthermore, the KIR2DL fully human antibody or its antigen-binding fragment has an amino acid sequence selected from any of the following: SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21.

[0023] As a preferred technical solution, the amino acid sequence of the KIR2DL fully human antibody or its antigen-binding fragment is SEQ ID NO.19.

[0024] Further, the nucleotide sequence encoding the amino acid sequence of the KIR2DL fully human antibody or its antigen-binding fragment is one of the following sequences: SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24.

[0025] As a preferred technical solution, the nucleotide sequence encoding the amino acid sequence of the KIR2DL fully human antibody or its antigen-binding fragment is SEQ ID NO.22.

[0026] Furthermore, the amino acid sequence of the KIR2DL fully human antibody or its antigen-binding fragment is 100% derived from humans.

[0027] A second objective of the present invention is to provide a molecular expression vector comprising one of the nucleotide sequences of SEQ ID NO.22 to SEQ ID NO.24.

[0028] A third objective of the present invention is to provide a host cell containing the above-mentioned molecular expression vector, wherein the host cell is a mammalian cell.

[0029] Furthermore, the mammalian cells are selected from Expi 293F cells or CHO cells.

[0030] The fourth objective of this invention is to provide an application of a KIR2DL fully human antibody or its antigen-binding fragment in the preparation of a reagent for detecting T / NK cell surface proteins.

[0031] The fifth objective of this invention is to provide the use of a fully human KIR2DL antibody or its antigen-binding fragment in the preparation of a drug that blocks KIR2DL1, KIR2DL2 and KIR2DL3.

[0032] Furthermore, this invention also provides a method for preparing a fully human KIR2DL antibody, the specific steps of which are as follows:

[0033] S1. Based on the protein and gene sequence information of KIR2DL2, express and screen antigens, and link a Flag-tag to its C-terminus to obtain the modified nucleic acid sequence;

[0034] S2. The nucleic acid sequence obtained in step S1 is cloned into an expression vector, and the antigen is expressed using mammalian cells to obtain the KIR2DL / Flag antigen.

[0035] S3. Using normal donor PBMC cells as raw materials, RNA was extracted and reverse transcribed into cDNA. Antibody gene fragments were obtained by PCR and cloned into a phage vector for library construction.

[0036] S4. Use the KIR2DL2 / Flag antigen obtained in step S2 to screen for antibodies against the gene fragment library constructed in step S3.

[0037] S5. The antibody sequence obtained in step S4 is expressed through a mammalian cell system, and then a fully human antibody for KIR2DL2 is obtained through screening. The fully human antibody for KIR2DL2 has high sensitivity and specificity.

[0038] Further, in step S4, the sequence of the antibody gene fragment includes the nucleotide sequences shown in SEQ ID NO.22 to SEQ ID NO.24.

[0039] Furthermore, in step S5, further screening experiments include ELISA and FACS detection.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] 1. This invention provides antibodies, antibody fragments, and derivatives thereof, wherein the antibodies, fragments, or derivatives cross-react with at least two inhibitory KIR receptors on the surface of NK cells, neutralizing inhibitory signals of NK cells and enhancing NK cell activity. The antibody binds to common determinants of the human KIR2DL receptor. The antibody of this invention binds to at least two of the KIR2DL1, KIR2DL2, and KIR2DL3 receptors.

[0042] 2. This invention relates to a fully human KIR2DL antibody, its preparation method, and its applications. The amino acid sequence of the fully human KIR2DL antibody is one of SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21. Compared with *Gynostemma pentaphyllum*, the fully human KIR2DL antibody of this invention can better block the interaction between KIR2DL and its ligand HLA-C, which helps to precisely regulate the immune response and reduce non-specific immunosuppression. In addition, the fully human KIR2DL antibody, through its unique biological functions and synergistic effect with existing therapies, provides new hope and strategies for tumor immunotherapy.

[0043] 3. The preparation method of the present invention is simple, and the antibody is expressed efficiently in mammalian cell expression system. After translation, it can be processed and modified, and its activity is closer to that of natural antibodies.

[0044] 4. This invention utilizes fully human antibodies screened from a fully human library. The types of libraries and antibodies used in this invention differ from those in the comparative patent. The blocking effect of the fully human antibody sequence screened by this invention is better than that of Yangshen. The amino acid sequence of the fully human antibody is 100% derived from humans, thus resulting in the lowest rejection reaction, the best safety profile, and better drug-like properties. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the experimental results of binding detection between HUMAN-KIR2DL2 antigen (expressed KIR2DL2 / Flag antigen) and antibodies (KD6, F12 and C7);

[0046] Figure 2 This is a schematic diagram of the results of the binding assay between HUMAN-KIR2DL2 cell line (KIR2DL2 antigen on cell surface) and antibodies (KD6, F12 and C7);

[0047] Figure 3 This is a schematic diagram of the experimental results of binding detection between HUMAN-KIR2DL1 antigen (expressed KIR2DL1 / Flag antigen) and antibodies (KD6, F12 and C7);

[0048] Figure 4This is a schematic diagram of the experimental results of binding detection between HUMAN-KIR2DL3 antigen (expressed KIR2DL3 / Flag antigen) and antibodies (KD6, F12 and C7);

[0049] Figure 5 This is a schematic diagram of the results of the antibody blocking experiment at the protein level. Detailed Implementation

[0050] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0051] As used in this article, the terms "VH" and "VL" refer to the variable regions of the antibody heavy chain and light chain, respectively. Variable regions consist of discrete, well-defined subregions called complementarity-determining regions (CDRs, also known as HVRs) and framework regions (FRs). A CDR is an amino acid within the antibody variable region that confers antigen specificity and / or binding affinity, separated by FRs. Each antibody light chain variable region contains three CDRs (VLCDR1, VLCDR2, and VLCDR3), and each antibody heavy chain variable region contains three CDRs (VHCDR1, VHCR2, and VHCDR3). The complementarity-determining regions (CDRs) of the VH and VL regions alternate with more conserved regions of the framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0052] For the purposes of this invention, the term "KIR2DL2 / 3" refers to one or both of the KIR2DL2 and KIR2DL3 receptors. These two receptors have very high homology, may be alleles of the same gene, and are considered interchangeable. Therefore, for the purposes of this invention, KIR2DL2 / 3 is considered a single repressive KIR molecule, and thus antibodies that cross-react only with KIR2DL2 and KIR2DL3 and not with other repressive KIR receptors are not within the scope of this invention.

[0053] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule, such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0054] For any techniques or conditions not specified in this embodiment, the operation shall be carried out in accordance with conventional technical methods and instrument manuals in this field; for reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially.

[0055] In the following embodiments, the amino acid sequences are shown in Table 1:

[0056] Table 1. Amino acid sequence of the CDR region of the KIR2DL fully human antibody of this invention.

[0057]

[0058]

[0059]

[0060]

[0061] In the following embodiments, the sources of the materials are as follows:

[0062] The PBMC cells of the immunized mice were purchased from Miaoshun Biotechnology Co., Ltd.; the pComb3XSS was purchased from Addgene; the SS320 bacterial culture was purchased from Lucigen; the secondary antibody Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (imported) was purchased from Thermo; and the TMB chromogenic solution was purchased from Beyotime.

[0063] For any techniques or conditions not specified in this embodiment, the operation shall be carried out in accordance with conventional technical methods and instrument manuals in this field; for reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially.

[0064] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0065] Example 1

[0066] This embodiment provides a method for preparing a fully human KIR2DL antibody, the specific steps of which are as follows:

[0067] S1. Based on the protein and gene sequence information of KIR2DL2, its extracellular sequence was expressed, and a Flag-tag was linked to its C-terminus. The expression vector was cloned into pCDNA3.4, and antigen expression was performed using mammalian cells Expi 293F to obtain the KIR2DL / Flag antigen for subsequent screening and detection.

[0068] S2. Using PBMC cells from immunized mice as raw materials, RNA was extracted from the cells using the Trizol method and reversed into cDNA using oligo(dT). Gene fragments were obtained by PCR amplification and then cloned into the phage vector pComb3XSS for library construction.

[0069] S3. Use the KIR2DL2 / Flag antigen obtained in step S1 to screen for antibodies;

[0070] S3-1, First Round of Screening

[0071] S3-1-1. Prepare two labeled immunoassay tubes (one for positive screening and one for negative screening). Place them in a clean bench and wash twice with PBST and twice with PBS.

[0072] Antigen coating: Add 50 μg of the KIR2DL2 / Flag antigen expressed in step S1 to a cleaned immunosorbent tube, dilute and dissolve with 2 mL of PBS, and rotate overnight at 4°C to allow the antigen protein to adsorb (coat) onto the inner wall of the immunosorbent tube.

[0073] S3-1-2. On the second day, recover the antigen, wash 3 times with PBST, wash twice with PBS, and add 5×10 to the negative sieve tube. 12 (5×10 12 / 1.5×10 13 The packaged phage obtained in step S2 was sealed with 1% BSA to a final volume of 4 mL. Simultaneously, only 4 mL of 1% BSA was added to the positive sieve tube for sealing, and the tubes were incubated at room temperature for 1 hour by rotation.

[0074] S3-1-3. After sealing, take the supernatant phage from the negative sieve tube and add it to the positive sieve immunotherapy tube. Incubate at room temperature for 1 hour by rotation. Remove the supernatant, wash 8 times with PBST, and wash twice with PBS.

[0075] S3-1-4. Elute the bound phages, add 1 mL of trypsin to elute, and incubate at room temperature for 30 min by rotation.

[0076] S3-1-5. Infection: Add 500 μL of the phage elution mixture to 5 mL of shaken SS320 bacterial culture (OD600: 0.4-0.6), mix well, and incubate at 37°C for 30 min. Simultaneously, add 10 μL of the remaining 500 μL of phage elution to the shaken SS320 bacterial culture, perform a tenfold serial dilution (8-9 dilutions), and incubate at 37°C for 30 min.

[0077] S3-1-6. After infection, the bacterial suspension is centrifuged at 3000 rpm for 5 min to enrich the bacterial cells. The supernatant is discarded, and the bacterial cells are resuspended in approximately 300 μL of culture medium. The suspension is then plated (with A). + and T + The 2YT flat plate was placed upside down in a 37°C constant temperature incubator overnight, and the temperature was adjusted simultaneously.

[0078] S3-2, Second Round of Selection

[0079] The specific steps are the same as the first round of screening, with the differences shown in Table 2 below.

[0080] S3-3, Third Round of Selection

[0081] The specific steps are the same as the first round of screening, with the differences shown in Table 2 below, to obtain potential positive phage clones.

[0082] Table 2. Differences among the three rounds of solid phase screening

[0083] Round Input phage(pfu) Coating antigen amount (μg) Round 1 <![CDATA[~5×10 12 ]]> 50 2 rounds <![CDATA[~5×10 11 ]]> 30 3 rounds <![CDATA[~5×10 10 ]]> 15

[0084] S4. The positive clones prepared in step S3 are screened using the ELISA method to obtain antibody sequences (KD6, F12, and C7). The specific detection process is as follows:

[0085] S4-1. Coat the ELISA plate with the KIR2DL2 / Flag antigen expressed in step S1 at a concentration of 1 μg / mL and incubate overnight at 4°C.

[0086] S4-2, Wash three times with PBST on the second day, then block with 1% BSA at room temperature for 1 hour;

[0087] Wash three times with S4-3 and PBST, add 200 μL of overnight shaken monoclonal supernatant to each well, and incubate at 37°C for 1.5 h;

[0088] Wash three times with S4-4 and PBST, add Anti-HIS-HRP secondary antibody diluted 1:10000 to each well, and incubate at 37℃ for 1 h;

[0089] Wash three times with S4-5 PBST, add 100 μL of TMB substrate, incubate at 37 °C for 10 min, add 50 μL of 0.1 M H2SO4 to stop the reaction, and measure OD at 450 nm.

[0090] S5. Plasmid construction and extraction: Construction of positive antibody sequences and cloning into expression vectors.

[0091] The antibody sequences (SEQ ID NO.22~SEQ ID NO.24) obtained by screening were cloned into the pCDNA3.4 expression vector to obtain antibodies (KD6, F12 and C7).

[0092] S6. Cell transfection: Seed cells to an appropriate density the day before transfection, using a heavy chain to light chain molar ratio of 2:3. Culture at 37°C in a 5% CO2 cell incubator for 12 consecutive days.

[0093] S7. The antibody obtained in step S6 is expressed and then used to detect its binding with the KIR2DL2 protein (i.e., the KIR2DL / His antigen obtained in step S1). ELISA results show (e.g.) Figure 1 and Figure 2 As shown in the figure, the ELISA results of the selected KD6 and F12 antibody sequences were better than those of Yangshen (1-7F9).

[0094] To further verify the binding of positive antibodies, the antibodies obtained in step S6 were subjected to a cell binding assay with ECD cell lines overexpressing the full-length KIR2DL2 antigen. The selected antibodies were added to pre-coated cells at a starting concentration of 200 nM, serially diluted 3-fold, and incubated at 4°C for 1 hour. The cells were washed twice with a MACS buffer (PBS + 10% FBS + 2 mM EDTA), and then incubated with the secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (imported)) at 4°C for 30 minutes. The cells were washed twice with the MACS buffer and then analyzed by flow cytometry. The results showed... Figure 2 The results showed that the EC50 values ​​of the fully human antibodies (KD6, F12 and C7) obtained by CELL BINDING ASSAY screening were better than those of the control antibodies in terms of cell binding effect.

[0095] S8. Select antibody sequences (KD6, F12, and C7) with EC50 values ​​lower than those of *Cell Binding Assay* for subfamily crossover experiments. Start with 200 nM of the selected antibodies, and add them in 3-fold serial dilutions to pre-coated (Human-KIR2DL1-ECD; Human-KIR2DL3-ECD) solutions. Incubate at 4°C for 1 hour, wash twice with 1% PBST, add Anti-Human IgG Fc mAb-HRP secondary antibody, incubate at 4°C for 30 minutes, wash twice with 1% PBST, and analyze by flow cytometry. Results showed... Figure 3 and Figure 4 The results showed that the C7 sequence could not bind to KIR2DL1 but could bind to KIR2DL3, while the KD6, F12 antibody sequences and Yangshen could bind to KIR2DL1 / KIR2DL3.

[0096] S9. Select antibody sequences (KD6, F12) that bind to both the crossover experiment and subfamily binding for protein-level blocking experiments. Incubate the HLA-Cw7 ligand at a working concentration of 20 μg / mL with the KIR2DL / Flag antigen expressed in step S1 (plate-coated the previous day). Simultaneously, add the antibody to the antigen in a 3-fold serial dilution starting at 200 nM. Incubate at 37°C for 1 hour, wash three times with PBST, add Anti-HIS-HRP as the secondary antibody, incubate at 37°C for 60 min, wash three times with 1% PBST, add 50 μL of chromogenic buffer (TMB chromogenic buffer), incubate at 37°C for 5 min, add stop chromogenic buffer, and read the values ​​on the instrument. The results are as follows: Figure 5 The results showed that the KD6 sequence selected had a stronger blocking effect than Yangshen.

[0097] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A KIR2DL fully human antibody or its antigen-binding fragment, characterized in that, It contains a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region CDR1 includes the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.7 and SEQ ID NO.13, CDR2 includes the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.8 and SEQ ID NO.14, and CDR3 includes the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.9 and SEQ ID NO.15; The CDR1 of the light chain variable region includes the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.10 and SEQ ID NO.16, the CDR2 includes the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.11 and SEQ ID NO.17, and the CDR3 includes the amino acid sequences shown in SEQ ID NO.6, SEQ ID NO.12 and SEQ ID NO.

18.

2. The KIR2DL fully human antibody or its antigen-binding fragment according to claim 1, characterized in that, The CDR1 sequence of the heavy chain variable region is SEQ ID NO.1, the CDR2 sequence is SEQ ID NO.2, and the CDR3 sequence is SEQ ID NO.

3. The CDR1 of the light chain variable region is SEQ ID NO.4, the CDR2 is SEQ ID NO.5, and the CDR3 is SEQ ID NO.6; or, The CDR1 of the heavy chain variable region is SEQ ID NO.7, the CDR2 is SEQ ID NO.8, and the CDR3 is SEQ ID NO.9; The CDR1 of the light chain variable region is SEQ ID NO.10, the CDR2 is SEQ ID NO.11, and the CDR3 is SEQ ID NO.12; or, The CDR1 of the heavy chain variable region is SEQ ID NO.13, the CDR2 is SEQ ID NO.14, and the CDR3 is SEQ ID NO.15; The CDR1 of the light chain variable region is SEQ ID NO.16, the CDR2 is SEQ ID NO.17, and the CDR3 is SEQ ID NO.

18.

3. The KIR2DL fully human antibody or its antigen-binding fragment according to claim 2, characterized in that, The CDR1 sequence of the heavy chain variable region is SEQ ID NO.1, the CDR2 sequence is SEQ ID NO.2, and the CDR3 sequence is SEQ ID NO.

3. The CDR1 of the light chain variable region is SEQ ID NO.4, the CDR2 is SEQ ID NO.5, and the CDR3 is SEQ ID NO.

6.

4. The KIR2DL fully human antibody or its antigen-binding fragment according to claim 1, characterized in that, The KIR2DL fully human antibody or its antigen-binding fragment has an amino acid sequence selected from any of the following: SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.

21.

5. The KIR2DL fully human antibody or its antigen-binding fragment according to claim 4, characterized in that, The nucleotide sequence encoding the amino acid sequence of the KIR2DL fully human antibody or its antigen-binding fragment is one of the following sequences: SEQ ID NO.22, SEQ ID NO.23, or SEQ ID NO.

24.

6. A molecular expression vector, characterized in that, The vector contains one of the nucleotide sequences of SEQ ID NO.22 to SEQ ID NO.

24.

7. A host cell containing the molecular expression vector of claim 6, characterized in that, The host cell is a mammalian cell.

8. The host cell according to claim 7, characterized in that, The mammalian cells were selected from Expi293F cells or CHO cells.

9. The use of the KIR2DL fully human antibody or its antigen-binding fragment as described in any one of claims 1-5 in the preparation of a reagent for detecting T / NK cell surface proteins.

10. The use of a fully human KIR2DL antibody or antigen-binding fragment thereof as described in any one of claims 1-5 in the preparation of a medicament that blocks KIR2DL1, KIR2DL2 and KIR2DL3.

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