Preparation method of NK (natural killer) cells and application of NK cells in cancer treatment

Abstract

The invention relates to the field of biology, in particular to a preparation method of NK cells and application of the NK cells in cancer treatment. The method is an antibody-cell therapy, the persistence and killing efficiency of the NK cells are further improved through a targeted antibody, and wide clinical application prospects are shown.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a preparation method of NK cells and application thereof in treating cancer. BACKGROUND

[0002] Natural killer cells (NK cells) are the core effector cells of the innate immune system, and have unique tumor recognition and killing ability. As a subpopulation of lymphocytes, NK cells can directly recognize and kill tumor cells and virus-infected cells without prior sensitization through surface activating receptors (such as NKG2D, NCRs), and this rapid response characteristic makes it the first line of defense of the body's anti-tumor immunity. The killing mechanism of NK cells mainly includes inducing target cell apoptosis by releasing perforin and granzyme, triggering death receptor-mediated apoptosis through the Fas / FasL pathway, and secreting cytokines such as IFN-γ and TNF-α to regulate the immune microenvironment. Unlike T cells, the activation of NK cells is not limited by MHC, and it still maintains the killing potential for low immunogenic tumors, and this characteristic makes it have a unique advantage in tumor immunotherapy.

[0003] NKG2A (Natural Killer Group 2 Member A) is an inhibitory receptor of the C-type lectin family, mainly expressed on the surface of natural killer cells (NK cells) and part of cytotoxic T cells (CD8 + T cells). It forms a heterodimer (CD94 / NKG2A) with CD94, which recognizes non-classical MHC class I molecule HLA-E. HLA-E is highly expressed on the surface of various tumor cells, and by binding to NKG2A, it transmits an inhibitory signal, weakens the killing function of NK cells, and thus helps the tumor to escape immune surveillance. The intracellular segment of NKG2A contains an immunoreceptor tyrosine-based inhibitory motif (ITIM), which is phosphorylated and recruits SHP-1 / SHP-2 phosphatases when it binds to the highly expressed HLA-E on the surface of tumor cells, and then inhibits the activation of NK cells.

[0004] Key signaling pathways such as PI3K / AKT and MAPK lead to NK cell depletion, manifested as decreased degranulation ability, reduced secretion of cytokines such as IFN-γ, and weakened cytotoxicity. Studies have shown that the NKG2A / HLA-E pathway is abnormally activated in various malignant tumors, including lung cancer, colorectal cancer, and ovarian cancer, and its expression level is significantly correlated with poor patient prognosis, making it a novel target for tumor immunotherapy. Currently, monoclonal antibodies targeting NKG2A (such as monalizumab) have shown promising anti-tumor effects in clinical trials, especially when used in combination with PD-1 / PD-L1 inhibitors, which can synergistically enhance the anti-tumor activity of NK cells and T cells, providing a new approach to overcoming drug resistance in existing immunotherapies. Compared to other inhibitory receptors such as TIGIT, the NKG2A blocking strategy has advantages such as a unique mechanism of action and high safety. Combined with adoptive NK cell therapy, it can further enhance the persistence and killing efficacy of NK cells, showing broad clinical application prospects. Summary of the Invention

[0005] In view of the current state of the technology, the object of the present invention is to provide an antibody-cell therapy for cancer treatment, so as to achieve efficient, safe and durable therapeutic effects.

[0006] In a first aspect, the present invention provides an antibody specifically targeting NKG2A, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain and light chain variable regions each comprise three complementarity-determining regions (CDRs), namely HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0007] In some embodiments, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0008] In some embodiments, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0009] In some embodiments, the amino acid sequence of VH is shown in SEQ ID NO: 7, and the amino acid sequence of VL is shown in SEQ ID NO: 8.

[0010] In some embodiments, the antibody specifically targeting NKG2A comprises an Fc fragment.

[0011] In some embodiments, the antibody specifically targeting NKG2A is a full-length IgG1 antibody or an IgG4 antibody.

[0012] A second aspect of the invention provides a pharmaceutical composition comprising the aforementioned antibody specifically targeting NKG2A, and a second agent or therapy. In some embodiments, the second agent or therapy is a standard or commonly used agent or therapy for treating a disease or condition. In some embodiments, the second agent or therapy comprises immune cells. In some embodiments, the immune cells are NK cells.

[0013] In some embodiments, the above-described pharmaceutical composition also contains other therapeutic agents.

[0014] In some embodiments, the therapeutic agent is an immunotherapeutic agent, including, for example, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, TIM3 antibody, LAG-3 antibody, CD40 antibody, OX40 antibody, EGFR antibody, and VEGF antibody.

[0015] In some embodiments, the therapeutic agent is a chemotherapeutic agent, including, for example, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, zorubicin, dihydroxyanthraquinone, mitoxantrone, scintillans, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologs.

[0016] In some embodiments, the pharmaceutical composition is administered systemically (e.g., intravenously (iv)). In some embodiments, the pharmaceutical composition is administered locally (e.g., intratumorally).

[0017] A third aspect of the present invention provides the use of antibodies and NK cells specifically targeting NKG2A in the preparation of pharmaceutical compositions for treating cancer.

[0018] In some implementations, the cancer is a solid tumor or a hematologic tumor. Preferably, the cancer is selected from colon cancer, melanoma, prostate cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, glioblastoma, endometrial cancer, kidney cancer, rectal cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, chronic lymphocytic leukemia, or acute myeloid leukemia.

[0019] Beneficial effects: This invention extracts and prepares NK cells from blood. When these NK cells are used in combination with an antibody that specifically binds to NKG2A, they can inhibit the proliferation of tumor cells in vivo. Furthermore, the antibody that specifically binds to NKG2A provided by this invention can bind to human and / or monkey NKG2A with high affinity, while effectively inhibiting the binding of NKG2A to its ligand, showing great promise for clinical application. Attached Figure Description

[0020] Figure 1 The results shown are the changes in tumor volume in various mice after treatment with NK cells and / or NKG2A antibodies. Detailed Implementation

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Except as provided in the operational examples or otherwise indicated, all figures for the amount of expressed components or reaction conditions used herein should be understood to be modified by the term "about" in all cases. When used in conjunction with percentages, the term "about" may mean ±1%.

[0023] The term "antibody" is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and their antigen-binding fragments, as long as they exhibit the desired antigen-binding activity.

[0024] "Isolated" antibody is an antibody that has been identified, isolated, and / or recovered from components (e.g., natural or recombinant) of its production environment. Preferably, the isolated polypeptide does not associate with any other components from its production environment. Contaminating components of its production environment (such as contaminating components produced by recombinant transfected cells) are substances that generally interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In a preferred embodiment, the polypeptide will be purified to: (1) greater than 95% (by weight of antibody) as determined by, for example, the Lowry method, and in some embodiments, greater than 99% (by weight); (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) to homogenize by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining. Isolated antibodies include in situ antibodies from recombinant cells, since at least one component of the antibody's native environment will be absent. However, typically, isolated peptides, antibodies, or constructs are prepared through at least one purification step.

[0025] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the antibody's heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies in the same class) and contain antigen-binding sites.

[0026] The term "variable" refers to the fact that certain segments of the variable domain differ significantly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable domain. Instead, it is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, primarily employing a β-sheet configuration, linked by three CDRs that form loops that connect the β-sheet structure and, in some cases, form part of the β-sheet structure. The CDRs in each chain are tightly bound together by the FR regions and, together with the CDRs in the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, MD. (1991)). Constant domains do not directly participate in antibody-antigen binding, but exhibit a variety of effector functions, such as antibody participation in antibody-dependent cytotoxicity.

[0027] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, deamidation) that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized from hybridoma cultures, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibody used according to this application can be prepared by a variety of techniques, including, for example, hybridoma methods, recombinant DNA methods, phage display techniques, and techniques for producing human antibodies or human-like antibodies in animals having part or all of a human immunoglobulin locus or gene encoding a human immunoglobulin sequence.

[0028] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably and refer to antibodies that are essentially intact, as opposed to antibody fragments. Specifically, full-length 4-chain antibodies include those with heavy and light chains containing the Fc region. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or a variant of its amino acid sequence. In some cases, intact antibodies may have one or more effector functions.

[0029] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding and / or variable region of the complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments; biantibodies; linear antibodies, single-chain antibody molecules, single-domain antibodies (such as VHH), and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments (referred to as "Fab" fragments) and a residual "Fc" fragment (this name reflects its tendency to crystallize). The Fab fragment consists of the complete L chain and the variable region domain (VH) of the H chain, and a first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody produces a single large F(ab′)2 fragment, which roughly corresponds to two Fab fragments linked by disulfide bonds that have different antigen-binding activities and are still capable of crosslinking the antigen. The Fab′ fragment differs from the Fab fragment in that it has additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab′-SH is the designation of Fab′ in this paper, where one or more cysteine ​​residues in the constant domain are accompanied by a free thiol group. The F(ab′)2 antibody fragment was initially generated as a pair of Fab′ fragments with multiple hinge cysteine ​​residues between them. Other chemical conjugations of antibody fragments are also known.

[0030] The Fc fragment contains a carboxyl-terminal portion of two H chains linked together by disulfide bonds. The effector function of an antibody is determined by a sequence in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0031] When used herein, the terms “hypervariant region,” “HVR,” or “HV” refer to regions within the variable domain of an antibody where the sequence is hypervariable and / or forms structurally defined loops. Typically, single-domain antibodies contain three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) exhibits the greatest diversity among the three HVRs and is considered to play a unique role in conferring fine specificity to the antibody. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0032] The term “complementary determination region” or “CDR” is used to refer to the hypervariable region as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0033] As used herein, the terms “promote,” “increase,” “promoting,” and “increasing” are used interchangeably. These terms refer to an increase in a measured parameter in a treated cell, tissue, or individual compared to an untreated cell, tissue, or individual. The same cells, tissue, or individual may also be compared before and after treatment. In some embodiments, the increase in the treated cell, tissue, or individual compared to an untreated cell, tissue, or individual is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 2, 3, 4, or more times.

[0034] As used herein, the terms “treatment” or “improvement” are used interchangeably. These terms refer to the means of achieving a beneficial or desired outcome (including, but not limited to, therapeutic and / or preventative benefits).

[0035] In this application, unless otherwise specifically stated, the use of the singular includes the plural. In this application, unless otherwise stated, the use of “or” means “and / or”. Furthermore, the use of the term “including” and other forms such as “includes” and “included” is not restrictive. Additionally, unless otherwise specifically stated, terms such as “element” or “component” cover elements and components that include one unit as well as elements and components that include more than one subunit. Additionally, the use of the term “part” can include a portion of a part or an entire portion. Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply inclusion of the specified integer or group of integers, but not to exclude any other integer or group of integers.

[0036] In one or more specific embodiments of the present invention, the medicament of the present invention can be used to treat cancer. Examples of cancers that can be treated with the medicament of the present invention include solid tumors and hematologic malignancies. Solid tumors can be, for example, tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (such as the thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx, tongue and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testis, stomach, skin, ureter, vagina, and vulva. Cancers include hereditary cancers, such as squamous cell carcinoma, melanoma, retinoblastoma, and nephroblastoma. Furthermore, cancers include primary tumors in the said organs and corresponding secondary tumors (tumor metastases) in distant organs. Hematologic malignancies can include, for example, aggressive and painless forms of leukemia and lymphoma, namely non-Hodgkin's disease, chronic and acute myeloid leukemia (CML / AML), acute lymphoblastic leukemia (ALL), Hodgkin's disease, multiple myeloma, and T-cell lymphoma. They also include myelodysplastic syndromes, plasmacytoma, tumor-like syndromes, cancers of unknown primary origin, and AIDS-related cancers.

[0037] The compositions of the present invention can be administered daily or intermittently, with a frequency of once daily or two to three times daily. If each of the two active ingredients is a single formulation, their frequency of administration can be the same or different. Furthermore, the compositions of the present invention can be used alone or in combination with other anti-allergy medications. Considering all the foregoing factors, it is important to administer the lowest possible dose to achieve optimal efficacy without side effects, which can be readily determined by those skilled in the art. In some embodiments, the dosing regimen is repeated, for example, once, twice, three times, or more; for example, repeated over the remaining lifespan of the individual in need.

[0038] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0039] Example 1. Preparation and screening of anti-NKG2A antibodies

[0040] Mice were immunized multiple times via intraperitoneal injection using a mixture of NKG2A antigen (provided by Peptasys, catalog number: NKA-H5244) and adjuvant. After immunization, total RNA was extracted from mouse spleen tissue and cDNA templates were obtained via reverse transcription. Subsequently, using specifically designed light and heavy chain primers, antibody VH and VL fragments were amplified by PCR, and a scFv antibody phage display library was successfully constructed. The library was screened using ELISA to obtain positive clones targeting NKG2A. Further screening and identification yielded scFv antibodies with optimal binding activity. The CDR region of this scFv antibody was transplanted into a selected human lineage template to generate a humanized variable region. This variable region was then recombined with the human IgG1 heavy chain constant region and the human kappa light chain constant region to construct a complete full-length IgG1 antibody. After a series of functional verification experiments, the high-affinity anti-NKG2A antibody 21A8 was finally obtained. Its CDR region amino acid sequence is shown in SEQ ID NO: 1-6, and the VH and VL amino acid sequences are shown in SEQ ID NO: 7-8.

[0041] For comparative purposes, a specific NKG2A binding antibody (named Ref1) was used as a positive control antibody in subsequent experiments. The heavy chain sequence of this antibody is SEQ ID NO: 9, and the light chain sequence is SEQ ID NO: 10.

[0042] Example 2. Antibody Affinity Assay

[0043] The binding affinity of anti-NKG2A antibodies to NKG2A antigens from different species was determined using the Biacore™ 8K biosensor system based on surface plasmon resonance (SPR) technology. In the experiment, the antibody was first diluted to 2 μg / mL and injected onto the biosensor surface at a flow rate of 10 μL / min for 50 seconds. Subsequently, the binding of the antibody to human NKG2A antigen (hNKG2A, Biacore, catalog number: NKA-H5244), monkey NKG2A antigen (fasNKG2A, Biacore, catalog number: NKA-C5245), and mouse NKG2A antigen (mNKG2A, Biacore, catalog number: NKA-M52H5) at different concentrations was measured (binding time 180 seconds, dissociation time 600 seconds). By monitoring the binding and dissociation processes in real time, the binding rate (Kon) and dissociation rate (Koff) of the antibody were obtained, and the equilibrium dissociation constant (KD) was calculated accordingly.

[0044] The results are shown in the table below. Anti-NKG2A antibody 21A8 showed good affinity for human, monkey and mouse NKG2A antigens.

[0045] Table 1

[0046]

[0047] Example 3. Determination of blocking activity

[0048] The inhibitory effect of anti-NKG2A antibody 21A8 on the binding of NKG2A to HLA-E tetramer in NK cells was detected. The specific experimental method is as follows: NKL cells (Shanghai Binsui Biotechnology, catalog number BSCELL-0491) were resuspended in buffer containing 1% BSA to a concentration of 1.0 × 10⁻⁶. 6 At a concentration of [number] cells / mL, 100 μL was seeded into each well of a 96-well plate and blocked at 4°C for 30 minutes. Then, serially diluted anti-NKG2A antibody (maximum concentration 20 μg / mL, 4-fold dilution, 8 gradients) was added at 50 μL / well, along with 50 μL of SA-PE-labeled HLA-E tetramer (MBL, catalog number TS-ME01-1). Ref1 antibody was used as a positive control, and the blank control group contained only 100 μL of 1% BSA. After mixing, the cells were incubated at 4°C in the dark for 1 hour. After washing with PBS, the fluorescence signal was detected by flow cytometry, and the data were analyzed using FlowJo software to calculate the percentage of HLA-E tetramer-positive cells.

[0049] Plotting the percentage of positive cells on the Y-axis and the logarithm of antibody concentration on the X-axis, a fitted curve was obtained, and the binding IC50 value was calculated. The results are shown in the table below. Anti-NKG2A antibody 21A8 significantly inhibited the binding of NKG2A to HLA-E tetramer on the surface of NKL cells, and its blocking effect was superior to that of the positive control antibody Ref1.

[0050] Table 2

[0051] Antibody IC50 (pg / mL) 21A8 0.019 Ref 1 0.036

[0052] Example 4. Isolation, purification and culture of human peripheral blood NK cells

[0053] First, 20 mL of heparin-anticoagulated peripheral blood from a healthy volunteer was collected and diluted with an equal volume of RPMI 1640. This diluted solution was then slowly stacked along the wall of a centrifuge tube onto Ficoll lymphocyte separation medium. The mixture was centrifuged at 2000 rpm for 20 min at room temperature. Peripheral blood mononuclear cells (PBMCs) from the interface layer were collected, along with the precipitated red blood cells. Both were washed twice with PBS and the cells were counted. The red blood cells and mononuclear cells were then mixed at a 100:1 ratio, centrifuged at 1500 rpm for 15 min, and the supernatant was discarded. The cells were resuspended in 1 mL of PBS, followed by the addition of 20 μL of LosetteSep antibody complex (purchased from Stem Cell Technologies, 1×10⁻⁶). 6 After incubating the PBMC / 1μL antibody complex at room temperature for 20 min, add an equal volume of PBS containing 2% fetal bovine serum (FBS) and mix gently. Stack the mixture on an equal volume of Ficoll separation buffer and centrifuge at 2000 r / min for 20 min. Finally, aspirate the cells from the interface layer, wash twice with PBS containing 2% FBS and count them to obtain high-purity NK cells.

[0054] NK cells were cultured in complete medium containing 10% FBS and 500 U / mL IL-2 at 37°C in a 5% CO2 incubator. 0.2 mL of cell suspension was washed twice with PBS, and 20 μL each of FITC-CD3 mouse anti-human monoclonal antibody (purchased from BD) and PE-CD56 mouse anti-human monoclonal antibody (purchased from BD) were added. The cells were labeled at 4°C for 30 min. The cells were centrifuged at 1000 rpm for 8 min. The cells were resuspended in PBS, and the purity of NK cells (i.e., CD3+) was detected by flow cytometry. - CD56 + (Percentage of cells). The results showed that the purity of NK cells was as high as 96.52%.

[0055] Example 5. Anti-NKG2A antibody enhances the killing effect of NK cells on tumor cells.

[0056] NK cells (effective cells) prepared in Example 4 were seeded into 96-well plates and cultured in RPMI 1640 medium supplemented with human IL2 (10 ng / mL) and IL12 (20 ng / mL) for 4 days at 37°C in a 5% CO2 incubator. Target cells U-2OS-HLAE-EGFP cells (overexpressing HLA-E and EGFP) were seeded into 96-well plates at 5 × 10⁶ cells per well. 3 Cells were incubated for 6 hours. Subsequently, serially diluted anti-NKG2A antibody and pre-activated NK cells (1×10⁶ cells / year) were added. 3 Cells per well were added to 96-well plates containing target cells. Immunofluorescence intensity of the target cells was detected using IncuCyte (Sartorius AG), and cytotoxic activity was calculated. A control group was also included, containing human IgG1 antibody as an isotype control (ISO).

[0057] The results are shown in Table 3. 21A8 can enhance the killing effect of NK cells on human myeloma cells, and its killing effect is better than that of Ref1.

[0058] Table 3

[0059] Group IC50 (ng / mL) IgG1 / 21A8 234.8 Ref 1 327.5

[0060] Example 6. The effect of NK cells combined with anti-NKG2A monoclonal antibody on tumor inhibition in vivo.

[0061] 1×10 6 One MC38-HLAE cell (MC38 cells overexpressing HLA-E) was injected into

[0062] CD94 / NKG2A humanized mice (Biocytogen, catalog number: 121077) were subcutaneously inoculated until the tumor volume reached 150 mm. 3 Mice were randomly divided into 4 groups of 5 mice each. Group 1 (G1) was the control group, receiving only 200 μL of sterile PBS buffer (without NK cells) via tail vein injection. Group 2 (G2) was the NK cell group, receiving 1 × 10⁻⁶ NK cells via intraperitoneal injection on the day of grouping (D0). 7 NK cells. Groups 3 (G3) and 4 (G4) were antibody-NK cell combination groups, with Group 3 receiving 21A8 antibody and Group 4 receiving Ref1 antibody. 5 mg / kg of antibody was injected into mice via the tail vein twice a week for a total of three weeks (D0, D4, D7). Additionally, on the day of grouping, 1 × 10⁻⁶ NK cells were injected intraperitoneally. 7NK cells. Groups 5 (G5) and 6 (G6) were antibody treatment groups, with group 5 receiving 21A8 antibody and group 6 receiving Ref1 antibody. 5 mg / kg of antibody was injected into mice via tail vein twice a week for a total of 3 weeks (D0, D4, D7). Tumor volume was measured twice weekly. Tumor volume formula: Volume V = (Major axis a × Minor axis b) 2 ) / 2. In mice with tumor volumes exceeding 2000 mmHg. 3 The mice were euthanized.

[0063] Tumor volume changes in each group of mice are as follows: Figure 1 As shown, the tumor growth in mice in the anti-NKG2A antibody combined with NK cell therapy group (G3) was significantly better than that in the control group (G1), the NK cell therapy group (G2), and the antibody therapy group (G5 and G6). Furthermore, the tumor suppression effect shown by the 21A8 combined with NK cell therapy group was significantly better than that of the Ref1 combined with NK cell therapy group (G4).

Claims

1. An antibody specifically targeting NKG2A, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

2. The antibody specifically targeting NKG2A according to claim 1, comprising: VH, the amino acid sequence of which is shown in SEQ ID NO: 7; VL, the amino acid sequence of which is shown in SEQ ID NO:

8.

3. The antibody specifically targeting NKG2A according to any one of claims 1-2, wherein the antibody specifically targeting NKG2A comprises an Fc fragment.

4. The antibody specifically targeting NKG2A according to claim 3, wherein the antibody specifically targeting NKG2A is a full-length IgG1 antibody.

5. A pharmaceutical composition, characterized in that... It comprises an antibody specifically targeting NKG2A as described in any one of claims 1-4, and a second agent or therapy.

6. The pharmaceutical composition according to claim 5, wherein the second agent or therapy comprises immune cells.

7. The pharmaceutical composition according to claim 6, wherein the immune cell is an NK cell.

8. Use of a pharmaceutical composition of an antibody specifically targeting NKG2A and NK cells in the preparation of a medicament for treating cancer, wherein the antibody specifically targeting NKG2A comprises the antibody specifically targeting NKG2A according to any one of claims 1-4.

9. The use according to claim 8, wherein the cancer is a solid tumor or a hematologic tumor, preferably, the cancer is selected from colon cancer, melanoma, prostate cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, glioblastoma, endometrial cancer, kidney cancer, rectal cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, chronic lymphocytic leukemia, or acute myeloid leukemia.

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