A fully human antibody or antibody fragment targeting MICA and its chimeric antigen receptor and applications

Abstract

This invention discloses a fully human antibody or antibody fragment targeting MICA, its chimeric antigen receptor, and its applications. The method for preparing MICA-targeting CAR-NK cells includes the following steps: obtaining a single-chain antibody targeting the MICA antigen using phage display technology; constructing a chimeric antigen receptor containing the single-chain antibody targeting MICA; and expressing a CAR fusion protein in NK cells to obtain MICA-targeting CAR-NK cells. The CAR fusion protein includes a single-chain antibody targeting MICA, a CD8 hinge and transmembrane region, a 4-1BB co-stimulatory region, and a CD3ζ intracellular signaling activation domain. The technical solution of this invention has a positive effect on the treatment of solid tumors with high MICA expression and has significant application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fully human antibody or antibody fragment targeting MICA, its chimeric antigen receptor, and its applications. Background Technology

[0002] Natural killer (NK) cells are an important component of the body's innate immunity. These cells can recognize and kill target cells infected by bacteria or transformed into tumors, regardless of MHC antigen presentation, providing ready-to-use cell therapy drugs for adoptive immunotherapy of tumors. Therefore, by using genetic modification techniques to express chimeric antigen receptor (CAR) fusion proteins in NK cells, target-specific CAR-NK cells are prepared. In clinical treatment, these cells have shown less immunotoxicity caused by cytokine release, offering a safety advantage over CAR-T cell therapy. Simultaneously, the limited lifespan of CAR-NK cells significantly reduces the risk of on-target / off-tumor toxicity to normal cells. Furthermore, it reduces the risk of allogeneic reactions and graft-versus-host disease (GvHD). CAR-NK cells can be generated from multiple sources, including NK92 cells, peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), and induced pluripotent stem cell (iPSC) CAR-NK cells. This therapy is an innovative cancer immunotherapy targeting solid tumors and hematological malignancies.

[0003] NK cell killing requires the recognition of fewer inhibitory receptors and more killing activation signals on the surface of target cells, and the characteristic NK cell surface receptor NKG2D (NK group 2 member D) is one of the receptors involved in this process. NKG2D works by binding to stress proteins that provide killing activation signals, including MICA / B (MHC class I polypeptide-related sequence A / B) and ULBPs (unique long 16-binding proteins). The expression of these proteins is often associated with bacterial infection, cancer cell transformation, and genomic damage, and is commonly found in hematopoietic tumors and various solid tumors such as colon cancer, ovarian cancer, uterine cancer, breast cancer, pancreatic cancer, melanoma, gastric cancer, and liver cancer, making them a very promising therapeutic target. However, in cancer cells, MICA / B expression is often downregulated by post-translational modifications—the extracellular domain of MICA / B is cleaved by several metalloproteinases, allowing cancerous cells to escape immune surveillance. The detached fragments will exist in the intercellular space in a free form and bind to NKG2D, further hindering NK cell killing. Antibody drugs targeting MICA and inhibiting its detachment mechanism are under development abroad, but their efficacy still relies on the inherent NKG2D recognition of NK cells and is limited to interference from detached MICA.

[0004] Therefore, developing a CAR-NK preparation and treatment scheme that specifically recognizes MICA and can bypass the inherent NKG2D recognition and killing is an urgent problem to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a fully human antibody or antibody fragment targeting MICA, its chimeric antigen receptor, and its application. CAR-NK cells prepared based on the chimeric antigen receptor provided by this invention can recognize the NKG2D ligand MICA with high specificity, thus solving the technical problems of off-target toxicity in existing targeted therapies.

[0006] To achieve the above objectives, the present invention provides a fully human antibody or antibody fragment targeting MICA, the antibody or antibody fragment comprising: a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising three complementarity-determining regions: HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 comprises the sequence shown in SEQ ID NO: 3; the amino acid sequence of HCDR2 comprises the sequence shown in SEQ ID NO: 4; and the amino acid sequence of HCDR3 comprises the sequence shown in SEQ ID NO: 5.

[0007] The light chain variable region includes three complementary determinant regions: LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 includes the sequence shown in SEQ ID NO: 6; the amino acid sequence of LCDR2 includes the sequence shown in SEQ ID NO: 7; and the amino acid sequence of LCDR3 includes the sequence shown in SEQ ID NO: 8.

[0008] Preferably, the MICA binding domain of the antibody or antibody fragment is scFv, and the amino acid sequence of the scFv includes the sequence shown in SEQ ID NO: 1, or a functional sequence having 95%-99% identity with that sequence.

[0009] Preferably, the amino acid sequence of the scFv includes a sequence obtained by modifying sequence SEQ ID NO: 1, wherein the modified amino acid is at least one, two, or three, but not more than 30.

[0010] Preferably, the anti-MICA binding domain of the antibody or antibody fragment is scFv, and the nucleotide sequence of the scFv includes the sequence shown in SEQ ID NO: 2, or a functional sequence having 95%-99% identity with that sequence.

[0011] Preferably, the nucleotide sequence of the scFv includes a sequence obtained by modifying sequence SEQ ID NO: 2, wherein the modified nucleotides are at least one, two, or three but not more than 30.

[0012] The present invention also provides a chimeric antigen receptor containing the above-described fully human antibody or antibody fragment targeting MICA.

[0013] Preferably, the chimeric antigen receptor comprises, in sequence: a signal peptide sequence, a MICA binding domain, a detection tag, a hinge region, and a transmembrane domain, as well as a functional signal transduction domain.

[0014] The signal peptide sequence contains EF-1α, and the amino acid sequence of the signal peptide is the sequence shown in SEQ ID NO: 9;

[0015] The detection tag contains C-myc, and the amino acid sequence of the detection tag is the sequence shown in SEQ ID NO: 10;

[0016] The MICA binding domain includes scFv, and the scFv includes the fully human antibody or antibody fragment targeting MICA.

[0017] The hinge region and transmembrane domain comprise: a hinge region Hinge™ and a transmembrane structure CD8, wherein the amino acid sequence of the hinge region and transmembrane domain is the sequence shown in SEQ ID NO: 11;

[0018] The functional signal transduction domain comprises 4-1BB and CD3Zeta connected in sequence, wherein the amino acid sequence of 4-1BB is the sequence shown in SEQ ID NO: 12, and the amino acid sequence of CD3Zeta is the sequence shown in SEQ ID NO: 13.

[0019] The present invention also provides a host cell comprising the chimeric antigen receptor described above.

[0020] Preferably, the host cell is an NK cell.

[0021] The present invention also provides a lentiviral plasmid comprising a nucleotide sequence capable of encoding the above-described fully human antibody or antibody fragment targeting MICA.

[0022] The present invention also provides the application of the above-mentioned fully human antibodies or antibody fragments targeting MICA, or chimeric antigen receptors, or host cells, or lentiviral plasmids in the preparation of antitumor drugs, wherein the tumor is a MICA antigen overexpression-related tumor.

[0023] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0024] (1) The fully human antibody or antibody fragment provided by the present invention can bind to human MICA protein with high specificity and an Ec50 value of 0.67 nM.

[0025] (2) The chimeric antigen receptor provided by the present invention can target tumor cells that highly express MICA. By using chimeric antigen receptor technology, human antibody fragments that bind to MICA and are integrated into CARs are engineered to be expressed in NK cells. The resulting chimeric antigen receptor NK cells can be used to treat cancers associated with MICA antigen overexpression. Attached Figure Description

[0026] Figure 1 This is a bar graph showing the results of screening fully human scFv antibodies targeting MICA in this invention.

[0027] Figure 2 This invention aims to detect the binding activity of the fully human scFv antibody targeting MICA with the MICA antigen protein at the ELISA level.

[0028] Figure 3a Flow cytometry results of peripheral blood phenotypes used for NK cell expansion, from left to right: CD56+ CD3 + Cell population results.

[0029] Figure 3b The flow cytometry results show the phenotypes of NK cells after 14 days of in vitro expansion and proliferation. The x-axis represents CD3. + Cells, with CD56 on the ordinate. + cell.

[0030] Figure 4 This is a schematic diagram of the sequence structure of the IC1-CAR chimeric antigen receptor. Its basic design structure is EF1αpromoter-SP-IC1 scFv-myc-CD8-4-1BB-CD3ζ.

[0031] Figure 5 The distribution of flow cytometry results for NK expression of IC1-CAR chimeric antigen receptor is shown, with the horizontal axis representing the staining results of the myc tag.

[0032] Figure 6 This is a comparison of the killing activity of IC1-CARNK cells and negative control cells against K562-luc tumor cells. Detailed Implementation

[0033] Terminology Definition

[0034] An "antibody" is a glycoprotein, or its antigen-binding portion, that comprises at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Antibodies include single-chain antibodies.

[0035] The “heavy chain” consists of the heavy chain variable region (VH) and the heavy chain constant region.

[0036] A “light chain” consists of a light chain variable region (VL) and a light chain constant region.

[0037] “scFv”: Single-chain antibody fragment, which is composed of the variable regions of the antibody heavy chain and light chain linked by a short peptide (linker) of 15 to 20 amino acids.

[0038] The heavy chain variable regions and light chain variable regions can be further divided into hypervariable regions, called complementarity-determining regions (CDRs), which are scattered in more conserved regions called framework regions (FRs). Each VH and VL 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 variable regions of the heavy and light chains contain binding domains that can interact with the antigen. In this invention, the CDR1, CDR2, and CDR3 of the heavy chain variable regions are represented as HCDR1, HCDR2, and HCDR3, respectively; and the CDR1, CDR2, and CDR3 of the light chain variable regions are represented as LCDR1, LCDR2, and LCDR3, respectively.

[0039] "Constant regions" can mediate the binding of immunoglobulins to host tissues or factors.

[0040] An "antigen-binding domain" refers to one or more segments of an antibody that retain the ability to specifically bind to an antigen (such as MICA).

[0041] "Monoclonal antibody" refers to an antibody molecule composed of a single molecule. Monoclonal antibody compositions exhibit specific binding and affinity for a specific epitope (the antigenic moiety that is specifically recognized by the antigen receptor).

[0042] "CAR-NK" refers to chimeric antigen receptor NK cell immunotherapy. This involves recombining the scFv that recognizes tumor-associated antigens and the intracellular signaling domain "immunoreceptor tyrosine activation motif" in vitro to generate a recombinant plasmid. This plasmid is then transfected into PBMC-expanded NK cells in vitro, enabling the NK cells to express tumor antigen receptors. After purification and large-scale expansion, these transfected NK cells are called chimeric antigen receptor NK cells.

[0043] "95%-99% identity" refers to a homology of more than 95% (ideally more than 98%).

[0044] "Modification" is a variation of an amino acid or nucleotide, including the deletion, insertion, and / or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids or nucleotides, as well as the addition of one or more (usually up to 20, preferably up to 10, more preferably up to 5) amino acids or nucleotides at the C-terminus and / or N-terminus, without altering the function of the protein or nucleic acid.

[0045] The term "chimeric antigen receptor" or "CAR" refers to a polypeptide composed of an extracellular target protein binding sequence, a transmembrane sequence, and an intracellular signaling sequence tandemly. When expressed in immune cells, it becomes specific for target cells and generates intracellular signals, typically producing a killing effect on the target cells.

[0046] NK natural killer cells

[0047] The term "natural killer (NK) cells" refers to innate immune lymphocytes that do not require prior stimulation and are not MHC-restricted. They share many similarities with cytotoxic T cells, including common precursor cells and shared signal activation mechanisms. Similar to T cells, they can be modified with CARs to enhance their anti-tumor activity. Compared to CAR-T cell therapy, CARs exhibit less immunotoxicity and have access to a wider range of cell sources, including but not limited to: umbilical cord blood, bone marrow, human embryonic stem cells, induced pluripotent stem cells, and mature NK cell lines such as NK92, NKG, and YT.

[0048] MICA

[0049] The term "MICA" refers to MHC class I polypeptide-related sequence A, which is the ligand for the NKG2D receptor on the surface of NK cells. In this invention, it is a protein sequence derived from, for example, mammalian Homo sapiens (NCBI accession number NP_000238.1).

[0050] Vectors, plasmids, expression vectors

[0051] A "vector" is a nucleic acid sequence used to introduce a linked nucleic acid fragment into a cell. A "plasmid" is one type, referring to a linear or circular double-stranded DNA molecule into which additional nucleic acid fragments can be linked. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which can also introduce additional DNA fragments. Some vectors are capable of autonomous replication after being introduced into a host cell. Other vectors integrate into the host cell's genome after introduction, thus replicating along with the host genome.

[0052] This invention provides a fully human antibody or antibody fragment targeting MICA, wherein the MICA binding domain of the antibody or antibody fragment is scFv, and the amino acid sequence of the scFv is shown in Table 1.

[0053] Table 1. Amino acid sequence of scFv

[0054]

[0055] The nucleotide sequence encoding scFv provided by this invention includes the sequence shown in SEQ ID NO: 2. After translation into an amino acid sequence based on the codons corresponding to the bases in each sequence, the corresponding amino acid sequence of scFv is the sequence shown in SEQ ID NO: 1. The sequences shown in SEQ ID NO: 3 to SEQ ID NO: 8 are the variable region sequences of SEQ ID NO: 1.

[0056] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0057] The sources of some materials are explained below:

[0058] MICA protein: Human MICA (purchased from Acrobiosysterms, catalog number CD9-H8259).

[0059] Phage antibody library of human single-chain antibodies: constructed by Changzhou Felos Pharmaceutical Technology Co., Ltd.

[0060] Streptavidin magnetic beads: purchased from Invitrogen.

[0061] Enzyme-linked immunosorbent assay (ELISA) microplate: 96 half-well low-permeability flat-bottom microplate (purchased from Corning).

[0062] Anti-M13 HRP antibody: purchased from Thermo Fisher.

[0063] M13 helper phage: purchased from Invitrogen.

[0064] SOC culture medium: purchased from Shanghai Sangon Biotech.

[0065] pCGMT phage vector: purchased from Addgene.

[0066] XL1-blue bacteria: purchased from Agilent Technologies, product number 200228.

[0067] LB solid culture medium plates: Dissolve 5g yeast extract, 10g peptone, 10g sodium chloride, and 10g agar powder in 1L of double-distilled water, autoclave at 121℃, and then pour onto plates.

[0068] Developer ABTS solution: purchased from Thermo Fisher, product number 002024.

[0069] Gelred nucleic acid dye: purchased from Thermo Fisher.

[0070] pFUSE expression vector: purchased from Invitrogen.

[0071] Plasmid extraction kit: purchased from QIAGEN.

[0072] Restriction endonucleases: purchased from Takara.

[0073] Recombinase: purchased from Novoprotein.

[0074] lipo3000 reagent: purchased from Thermo Fisher Scientific.

[0075] Polybrene reagent: purchased from Thermo Fisher Scientific.

[0076] RMPI 1640 medium: purchased from Gibco.

[0077] DMEM medium: purchased from Gibco.

[0078] Myc-Tag(9B11)Mouse mAb(PE Conjugate): Purchased from Cell Signaling.

[0079] 293Fectin transfection reagent: purchased from Invitrogen, catalog number 12347500.

[0080] 293 Freestyle suspension cells: purchased from Thermo Fisher.

[0081] Immunoglobulin IgG1 constant region Fc segment: purchased from Nanjing Genscript Biotech Co., Ltd.

[0082] BCA protein quantification kit: purchased from Pierce, catalog number 23252.

[0083] CBS antigen fixation solution: Dissolve 1.59g Na2CO3 and 2.93g NaHCO3 in 1L of water and adjust the pH to 9.6.

[0084] Anti-Human Fc HRP secondary antibody: purchased from Thermo Fisher.

[0085] rhIL-2 Jiangsu Jinsiliyintekang

[0086] Thermofisher L30000008 lipo300000 transfection kit.

[0087] Promega E6120 Fluorescent Mycoplasma Detection Kit

[0088] Flow cytometry detection of antibody CD3-PE antibody Biolegend 300307.

[0089] CD56-AF-488 biolegend 318311.

[0090] CD3-FITC / CD16+56-PE Fluorescent Monoclonal Antibody Kit (Kuangbo Biotechnology Z6410012)

[0091] Microplate reader: Molecular Device, Spectramax iD5.

[0092] 96-hole bottom plate: purchased from Corning.

[0093] Biacore Instrument: Purchased from GE, T200.

[0094] Protein A chip: purchased from GE.

[0095] Example 1: Screening of single-chain antibodies targeting MICA

[0096] 1. Establish a phage antibody library of fully human single-chain antibodies.

[0097] Primers were designed to amplify the heavy and light chain variable regions of the fully human antibody. The heavy and light chain variable regions were ligated together using (GGGGS) 3-linker via overlap extension PCR to obtain a full-length PCR product. The PCR product and phage vector were digested with SfiI, and the ligation transformation product was electroporated into XL1-blue competent cells. 20 mL of selective medium containing 50 μg / mL ampicillin and 10 μg / mL tetracycline was added, and the cells were incubated at 37°C with shaking for 2 h. Then, a 10% concentration of... 13 Incubate 50 μL of VCSM13 helper phage at room temperature for 1 h, gently shaking every 10 min. Continue incubation at 37°C with shaking for 2 h, then add kanamycin to a final concentration of 70 μg / mL and incubate overnight at 30°C. Collect the supernatant by centrifugation, add 10% PEG-8000 / sodium chloride solution (PEG is polyethylene glycol), place on ice for 1 h, centrifuge at 8000 rpm at 4°C, discard the supernatant, and fully dissolve the precipitate in 2 mL of 1% BSA (bovine serum albumin) in PBS (phosphate buffer). Collect the supernatant by centrifugation; this is the phage antibody library of fully human single-chain antibodies, used for subsequent screening of fully human antibodies.

[0098] 2. Antibody screening

[0099] Take 5 μg of MICA antigen protein, dilute to 1 ng / μL with CBS, and coat 50 μL per well onto an ELISA plate (8 wells in total). Incubate overnight at 4°C. Discard the supernatant, wash the plate twice with PBS, and then add 50 μL (containing 1 × 10⁻⁶ phages) to the plate.13 A phage antibody library expressing fully human single-chain antibodies was incubated at room temperature for 2 hours, and the supernatant was discarded. The cells were washed three times each with PBS and PBST. Phages bound to the MICA antigen were captured, while unbound phages were removed by rinsing with 0.5% Tween-20 PBS solution (phosphate buffer). Stable phages bound to the antigen were eluted with glycine hydrochloride solution (pH 2.2) and set aside. 20 mL of XL1-Blue bacterial culture was inoculated. Once the OD600 (absorbance at 600 nm) reached 0.6, the eluted phage solution was added, and the mixture was incubated with XL1-Blue bacteria at 37°C for 30 min. The bacterial culture was then spread onto ampicillin-resistant plates. The next day, the cells on the ampicillin-resistant plates were collected and further cultured until the OD600 was approximately 0.6. The culture was then diluted with 1×10⁻⁶ ppm of PBS. 12 After infection with M13 helper phages at a concentration of pfu / mL (pfu, plaque-forming unit), the cells are amplified and then screened for the next round, for a total of four rounds. In each round, the number of wells coated with antigen is reduced by half to select phages with stronger binding activity.

[0100] The XL1-Blue bacterial suspension infected with bacteriophages was thoroughly diluted, and then spread on 15 cm diameter LB solid medium plates containing ampicillin. Monoclonal antibodies were picked, and the panned bacteriophage libraries and monoclonal antibodies were validated by phage enzyme-linked immunosorbent assay (ELISA).

[0101] 3. Phage enzyme-linked immunosorbent assay (ELISA)

[0102] XL1-Blue monoclonal antibodies were inoculated into 2 mL 96-well bacterial culture plates (purchased from Corning). 500 μL of SB medium containing tetracycline resistance was added, and the plates were incubated at 37°C with a shaking speed of 200 rpm for 4-6 h. Once the OD600 value approached 0.6, 1 μL of helper phage was added, and the plates were incubated overnight at 30°C with shaking. The next day, the plates were centrifuged at 3000 g, and the supernatant was collected for later use. ELISA microplates were coated with MICA antigen and incubated overnight at 4°C. On the third day, the plates were washed twice with PBST (phosphate-buffered saline containing Tween-20), blocked with 5% skim milk, and then the phage supernatant prepared in the previous step was added. The plates were incubated at room temperature for 2 h, washed five times with PBST, and then HRP-conjugated Anti-M13 antibody was added. The plates were incubated at 37°C for 30 min, washed five times with PBST, and then 50 μL of ABTS developing solution was added for color development.

[0103] 4. Experimental Results

[0104] like Figure 1As shown in the figure, this is a bar chart of the results of screening fully human scFv antibodies targeting MICA in Example 1. Table 2 shows the input and recovery amounts and recovery rates of the phage library in each round of screening for fully human scFv antibodies targeting MICA in Example 1.

[0105] Table 2. Results of phage input recovery amount and recovery rate in each round of Example 1.

[0106]

[0107] from Figure 1 As shown in Table 2, after four rounds of screening in the phage antibody library containing fully human single-chain antibodies, the recovery rate of MICA antigen in the fourth round increased by more than 100 times compared to the first round. This indicates that phages expressing antibodies specifically binding to MICA antigen were continuously enriched after four rounds of screening. Ninety-five monoclonal antibodies were selected from the phage library obtained after the fourth round of screening for enzyme-linked immunosorbent assay (ELISA) verification. Monoclonal antibodies with a signal intensity greater than 1 were identified as positive clones. These positive clones were then sequenced to obtain their nucleotide coding sequences. The obtained sequences were compared and analyzed; the more duplicate clones there were, the higher the enrichment level of the antibody sequence, thus determining the effectively enriched nucleotide coding sequences.

[0108] Example 2: Preparation (expression and purification) of anti-MICA single-chain antibody

[0109] The plasmid was extracted from the monoclonal strain screened in Example 1, digested with restriction endonuclease SfiⅠ, and the fragment was inserted into the pFuse expression vector by fragment recombination, thereby obtaining the pFuse expression vector of the antibody of the present invention.

[0110] The 293Fectin transfection reagent was mixed with the eukaryotic antibody expression vector obtained above at a volume-to-weight ratio of 30 μL:30 μg. 30 μL of 293Freestyle suspension cells were added, and the mixture was incubated at 37°C for 48-72 hours at 125 rpm. After centrifugation, the supernatant was collected and purified using a Protein A column. Specifically, the column was soaked in 1M NaOH for 1 hour, washed with endotoxin-free water until neutral, and then washed with 10 column volumes of 1% (v / v) Triton × 100. The column was equilibrated with 5 column volumes of PBS (PBS phosphate buffer, pH 7.2), and the filtered cell supernatant was loaded onto the column. Flow-through was collected if necessary. After loading, the column was washed with 5-10 column volumes of PBS. Elution was performed using 5 column volumes of 0.1 M pH 3.0 Glycine-HCl. The eluent was collected and neutralized with 0.5 column volumes of 1 M Tris-HCl (1.5 M NaCl) pH 8.5 to obtain the anti-MICA single-chain antibody (i.e., the fully human anti-MICA scFv antibody). Finally, the concentration of the purified antibody was determined according to the instructions of the BCA method protein quantification kit.

[0111] Example 3: Detection of the binding activity of anti-MICA single-chain antibody to MICA antigen protein (ELISA)

[0112] Commercially available MICA protein was diluted to a final concentration of 1.0 μg / mL with PBS, and then 100 μL was added to each well of a 96-well ELISA plate. The plate was sealed with plastic film and incubated overnight at 4°C. The next day, the plate was washed twice with 0.5% Tween-20 in PBS (phosphate buffer), and blocking buffer [PBS containing 0.01% (v / v) Tween-20 and 1% (v / v) BSA] was added, followed by blocking at room temperature for 2 hours. The blocking buffer was discarded, and 100 μL of the purified anti-MICA single-chain antibody obtained in Example 2 was added to each well. After incubation at 37°C for 2 hours, the plate was washed 5 times with 0.5% Tween-20 in PBS (phosphate buffer). HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Sigma) was added, and the plate was incubated at 37°C for 2 hours, followed by washing 5 times with 0.5% Tween-20 in PBS (phosphate buffer). Add 100 μL of TMB substrate to each well and incubate at room temperature for 30 minutes. Then add 100 μL of stop solution (1.0 N HCl) to each well. Read the A450 nm value using an ELISA reader (iD5, Molecular Device). The results are as follows: Figure 2 As shown, the purified antibody specifically binds to the MICA antigen protein at the ELISA level, with an EC50 value of 0.67 nM. PDI-1 is a positive control antibody.

[0113] Example 4: Natural killer cell expansion and preparation

[0114] PBMCs isolated from frozen healthy donor peripheral blood were resuscitated and counted in a clean bench. The PBMCs were then transferred to T25 culture flasks, and the cell concentration was adjusted to 2.5 × 10⁻⁶ cells using RPMI 1640 complete medium. 6 / mL. After culturing in a cell culture incubator at 37℃ and 5% carbon dioxide for 24 hours, the cells were observed and an appropriate amount was taken for immunophenotypic analysis, such as Figure 3a As shown, CD3 in unamplified PBMC cells + Cell percentage 42.6%, CD56 + Cells accounted for 32.5%.

[0115] The revived PBMCs underwent their first feeder cell stimulation and were transferred at a rate of 5 × 10⁶ cells / year. 6 One PBMC cell was transferred to T75 and 1 × 10 feeder cells were added. 7 Adjust the culture solution volume to 15 mL, and maintain a final IL-2 cytokine concentration of 100 IU / mL. Continue culturing in a cell culture incubator at 37°C and 5% CO2. For the next 5 days, supplement with fresh complete culture medium and IL-2 at a final concentration of 100 IU / mL every 2 days. On day 7, perform a second feeder cell stimulation, count the cells, add feeder cells at a 1:1 ratio according to the cell number, and supplement with IL-2 to 100 IU / mL. Continue culturing in a cell culture incubator at 37°C and 5% CO2 for 7 days, supplementing with fresh complete culture medium every 2-3 days, and maintaining a final IL-2 concentration of 100 IU / mL.

[0116] Cryopreservation of NK cells: NK cells were collected on day 14 after expansion and resuspended in complete culture medium with 10% DMSO until the density was 5 × 10⁻⁶. 7 / mL, retain an appropriate amount of sample for immunophenotypic analysis, such as Figure 3a As shown, CD3 in unamplified PBMCs + The cell proportion was 42.6%, CD56 + The cell proportion was 32.5%. NK cells (CD56) were expanded after 14 days. + The proportion is 32.5% Figure 3a ) rose to 96% Figure 3b ), the rest are frozen for later use.

[0117] Example 5: Construction of Lentiviral Expression Vector

[0118] A lentiviral plasmid expressing the MICAIC1 antibody was constructed. Its basic structure consists of the EF1α promoter, CSF2RA signal peptide (SP), MICA single-chain antibody IC1, a myc expression detection tag, human CD8 hinge and transmembrane region gene sequences (CD8hinge / TM), human 4-1BB intracellular region gene sequences, and CD3ζ intracellular region gene sequences. This is simplified below to IC1-CAR (EF1αpromoter-SP-IC1 scFv-myc-CD8-4-1BB-CD3ζ), and its tandem structure is shown in the diagram below. Figure 4 As shown. The same structure was used to construct 7C6 positive control and scFv deletion negative control plasmids (Mock-CAR).

[0119] The above-mentioned genes were ligated into a vector plasmid using a homologous recombinase (nearshore organisms). The vector was then transformed into the Stbl3 Escherichia coli strain and inoculated into a solid culture medium containing ampicillin for propagation and screening. Positive clones were obtained and verified by sequencing. The plasmid was then extracted for subsequent virus packaging and gene expression.

[0120] Example 6 Lentiviral Packaging and Infection of NK Cells

[0121] 293T cells were added to 2 mL of complete culture medium at a concentration of 4 × 10⁻⁶. 6 Cells / well were seeded in 6-well plates and cultured overnight. Transfection was performed using lipo3000 (ThermoFisher). The specific transfection reagent preparation is briefly described below: OptiMEM medium was used to prepare transfection and packaging plasmids; and lipo3000 transfection reagent was added to the plasmid solution prepared in Example 5. After vortexing and incubation at room temperature for 20 minutes, the mixture was added dropwise to the cell culture medium in the 6-well plates. After culturing at 37°C and 5% CO2 for 72 hours, the supernatant was collected, filtered through a 0.45 μm filter, and stored at 4°C for later use.

[0122] One day in advance, the frozen NK cells from Example 4 were thawed and activated with 100 IU / mL IL-2. The cells were centrifuged at 300g for 5 min to settle, and then resuspended in fresh culture medium to 5 × 10⁻⁶ cells / mL. 6 5 × 10⁻⁶ cells / mL. Add 5 × 10⁻⁶ cells / mL to a 12-well container. 5NK cells were prepared by centrifuging at 800g for 1 hour with 2 mL of viral supernatant and polybrene at a final concentration of 8 μg / mL. The cells were then cultured at 37°C in a 5% CO2 incubator for 6 hours. Another 2 mL of viral supernatant and IL-2 at a final concentration of 500 IU / mL were added, and the culture was aliquoted into two 12-well plates. After centrifugation at 800g for 1 hour, the cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The culture was then replaced with fresh complete culture medium containing IL-2 at a final concentration of 200 IU / mL and cultured for 48 hours to obtain NK cells expressing IC1-CAR, Mock-CAR (negative control), and 7C6-CAR (positive control). Flow cytometry was used for validation. Experimental results are as follows: Figure 5 As shown, the expression rates of IC1-CAR and 7C6-CAR in NK cells were 15.3% and 64%, respectively.

[0123] Example 7 Natural killer cell killing experiment

[0124] Construction of K562-luc cell line: A lentiviral plasmid expressing luciferase (luc) was constructed. The viral expression plasmid and the packaging plasmid were transfected into K562 cells using the Lipo3000 reagent. Stable monoclonal cell lines that could stably express luciferase were screened using puromycin and used as K562-luc, the target cells for NK lymphocytes.

[0125] Centrifuge at 300g for 5 min to settle K562-luc cells, then resuspend in culture medium to 1×10⁻⁶. 5 Cells were added at a concentration of 50 μL (5000 cells per well) to a white 96-well plate. IC1 CARNK, 7C6 CAR NK, and negative control (Mock) cells were counted, diluted to appropriate concentrations for each effector-to-target ratio, and added to the wells, resulting in a final volume of 100 μL per well. After culturing at 37°C and 5% CO2 for 4 hours, the luciferase content in the cells was detected using the E6120 (promega) assay kit. The specific method was as follows: 100 μL of the reaction reagent was mixed 1:1 with the cells and added to each well of the 96-well plate. The reaction was incubated at room temperature for 2 minutes, and the luminescence value of each well was read using a multi-mode microplate reader. Cytotoxicity was compared using the following formula: ADCC% = 1 - (E - NK) / T × 100% (where: experimental group luminescence value E, NK blank group luminescence value NK, target cell blank luminescence value T). Results are as follows. Figure 6 As shown, IC1 CAR NK cells exhibited superior killing activity compared to the positive control (7C6 CAR NK), indicating promising application prospects.

[0126] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A fully human antibody or antibody fragment targeting MICA, characterized in that, The antibody or antibody fragment comprises: a heavy chain variable region and a light chain variable region; The heavy chain variable region includes three complementarity-determining regions: HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 3; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 4; and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

5. The light chain variable region includes three complementary determinant regions: LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 6; the amino acid sequence of LCDR2 is shown in SEQ ID NO: 7; and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

8.

2. The fully human antibody or antibody fragment targeting MICA according to claim 1, characterized in that, The MICA binding domain of the antibody or antibody fragment is scFv, and the amino acid sequence of the scFv is the sequence shown in SEQ ID NO:

1.

3. The fully human antibody or antibody fragment targeting MICA according to claim 1, characterized in that, The MICA binding domain of the antibody or antibody fragment is scFv, and the nucleotide sequence of the scFv is the sequence shown in SEQ ID NO:

2.

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor contains a fully human antibody or antibody fragment targeting MICA as described in any one of claims 1-3, wherein the fully human antibody is a single-chain antibody.

5. The chimeric antigen receptor according to claim 4, characterized in that, The chimeric antigen receptor comprises, in sequence: a signal peptide sequence, a MICA binding domain, a detection tag, a hinge region and a transmembrane domain, and a functional signal transduction domain. The signal peptide sequence contains CSF2RA; The detection tag contains C-myc, and the amino acid sequence of the detection tag is the sequence shown in SEQ ID NO: 10; The MICA binding domain includes scFv, and the scFv includes the fully human antibody or antibody fragment targeting MICA. The hinge region and transmembrane domain comprise: a hinge region Hinge™ and a transmembrane structure CD8, wherein the amino acid sequence of the hinge region and transmembrane domain is the sequence shown in SEQ ID NO: 11; The functional signal transduction domain comprises 4-1BB and CD3 Zeta connected in sequence, wherein the amino acid sequence of 4-1BB is the sequence shown in SEQ ID NO: 12, and the amino acid sequence of CD3 Zeta is the sequence shown in SEQ ID NO:

13.

6. A host cell, characterized in that, The host cell comprises the chimeric antigen receptor as described in claim 4 or 5, and the host cell is an NK cell.

7. A lentiviral plasmid, characterized in that, The lentiviral plasmid contains a nucleotide sequence capable of encoding a fully human antibody or antibody fragment targeting MICA as described in any one of claims 1-3.

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