A fully human anti-CD20 antibody or antibody fragment and its chimeric antigen receptor and application

Abstract

This invention discloses a fully human anti-CD20 antibody or antibody fragment, its chimeric antigen receptor, and its applications. The fully human 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. The light chain variable region includes three complementarity-determining regions: LCDR1, LCDR2, and LCDR3. The fully human antibody or antibody fragment, or the nucleic acid encoding the antibody or antibody fragment, can be used to prepare host cells, lentiviral plasmids, and diagnostic tumors, as well as targeted anti-tumor drugs. The fully human antibody or antibody fragment of this invention can bind to the human CD20 protein with high specificity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fully human anti-CD20 antibody or antibody fragment, its chimeric antigen receptor, and its applications. Background Technology

[0002] Chimeric antigen receptor (CAR)-T cell therapy is an immunotherapy that uses genetic engineering to introduce specific receptors (CARs) into T cells, enabling them to recognize and attack cancer cells. Effective chimeric antigen receptor (CAR)-T cell therapy exhibits selective killing activity against cancer cells expressing CAR-target antigens. Cancer cells may evade CAR-T cell attack by reducing or ceasing the expression of these specific antigens—a natural escape pathway through which cancer cells can avoid recognition by CAR-T cells. In relapsed cases of leukemia and lymphoma, the relapse rate caused by CD19 antigen negativity ranges from 27% to 100%. The frequent occurrence of CD19-negative relapses indicates that CD19 antigens are easily lost; therefore, identifying alternative target antigens more resistant to gene expression downregulation is a potentially clinically viable treatment option. Clinical studies have shown a low incidence of CD20 antigen loss, suggesting that CD20 may be a suitable CAR target to reduce immune escape caused by antigen loss. Summary of the Invention

[0003] The purpose of this invention is to provide a fully human antibody or antibody fragment, along with its chimeric antigen receptor and application, that is highly targeted and specifically binds to the human CD20 protein, thereby enabling its use in the treatment of hematologic cancers associated with CD20 expression.

[0004] To achieve the above objectives, the present invention provides a fully human antibody or antibody fragment for anti-CD20, wherein the fully human antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region.

[0005] The heavy chain variable region includes three complementary determinant regions: HCDR1, HCDR2, and HCDR3;

[0006] The amino acid sequence of HCDR1 includes the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0007] The amino acid sequence of HCDR2 includes the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0008] The amino acid sequence of HCDR3 includes the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8;

[0009] The light chain variable region includes three complementary determining regions: LCDR1, LCDR2, and LCDR3;

[0010] The amino acid sequence of LCDR1 includes the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10;

[0011] The amino acid sequence of LCDR2 includes the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12;

[0012] The amino acid sequence of LCDR3 includes the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14.

[0013] Preferably, the anti-CD20 binding domain of the fully human antibody or antibody fragment is scFv, and the amino acid sequence of the anti-CD20 binding domain is the sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0014] Preferably, the anti-CD20 binding domain of the fully human antibody or antibody fragment is scFv, and the nucleic acid sequence of the scFv is the sequence shown in EQ ID NO: 15 and SEQ ID NO: 16.

[0015] The present invention also provides a chimeric antigen receptor comprising a fully human antibody or antibody fragment of anti-CD20 as described in any of the above claims.

[0016] Preferably, the chimeric antigen receptor comprises a signal peptide sequence, a CD20 binding domain, a detection tag, a hinge region, and a transmembrane domain connected in sequence, as well as a functional signal transduction domain; the signal peptide sequence comprises CSF2RA; the detection tag comprises C-myc; the CD20 binding domain comprises scFv, the scFv comprising the fully human antibody or antibody fragment of the anti-CD20; the hinge region and transmembrane domain comprise the hinge region Hinge™ and the transmembrane structure CD8; the functional signal transduction domain comprises CD28, 4-1BB, and CD3 Zeta connected in sequence.

[0017] Preferably, the amino acid sequences of the hinge region Hinge™ and the transmembrane structure CD8 are as shown in SEQ ID NO: 17, the amino acid sequence of CD28 is as shown in SEQ ID NO: 18, the amino acid sequence of 4-1BB is as shown in SEQ ID NO: 19, the amino acid sequence of CD3 Zeta is as shown in SEQ ID NO: 20, the amino acid sequence of CSF2RA is as shown in SEQ ID NO: 21, and the amino acid sequence of C-myc is as shown in SEQ ID NO: 22.

[0018] The present invention also provides a host cell that contains at least the chimeric antigen receptor described above.

[0019] The present invention also provides a lentiviral plasmid capable of encoding a nucleotide sequence of a fully human antibody or antibody fragment of anti-CD20 as described in any of the above claims.

[0020] The present invention also provides an application of a fully human anti-CD20 antibody or antibody fragment, wherein the fully human anti-CD20 antibody or antibody fragment is used to prepare T cells for the treatment or prevention of tumors.

[0021] Preferably, the T cell contains at least the chimeric antigen receptor as described above.

[0022] Compared to the prior art, the beneficial effects of the present invention include at least the following:

[0023] (1) The fully human antibody or antibody fragment of the present invention can bind to human CD20 protein with high specificity. The human antibody fragment that binds to CD20 and is integrated into the CAR is engineered and expressed in T cells using chimeric antigen receptor technology (CAR-T technology). The resulting chimeric antigen receptor T cells can be used to treat hematologic cancers related to CD20 expression.

[0024] (2) The chimeric antigen receptor of the present invention can target CD20, a cell surface marker molecule of malignant B-cell tumors. Attached Figure Description

[0025] Figure 1 This is a bar graph showing the results of screening CD20 single-chain scFv antibody fragments in Experimental Example 1 of the present invention.

[0026] Figure 2 This is a graph showing the results of ELISA detection of the binding activity between the anti-CD20 fully human scFv antibody and the CD20 antigen protein.

[0027] Figure 3This is a flow cytometry result of the anti-CD20 fully human scFv antibody of the present invention specifically recognizing CHO-K1-CD20 cells.

[0028] Figure 4 This is a flow cytometry result of the anti-CD20 fully human scFv antibody of the present invention specifically recognizing Raji cells overexpressing CD20.

[0029] Figure 5 This is a plasmid map of the Anti-CD20 CAR of the present invention.

[0030] Figure 6 The results are obtained by flow cytometry analysis of the expression rates of CAR-B5, CAR-F5, and CAR-Tab2 on the surface of the host cell jurakat, according to the present invention.

[0031] Figure 7 This diagram shows the activation results of the effector host cell jurakat, which was incubated with the target cell Raji according to the present invention. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0033] Terminology Definition

[0034] An "antibody" is a glycoprotein, or its antigen-binding portion, that contains 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 CD20).

[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] The "chimeric antigen receptor" consists of an extracellular antigen-binding region (composed of light and heavy chains derived from monoclonal antibodies, connected by a tough hinge region to form a single-chain antibody), a transmembrane region, and an intracellular signal transduction region.

[0043] "CAR-T technology" refers to chimeric antigen receptor T-cell immunotherapy. This involves recombining the scFv gene 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 the patient's T cells in vitro, causing the patient's T cells to express tumor antigen receptors. After purification and large-scale expansion, these transfected T cells are called chimeric antigen receptor T cells.

[0044] "95-99% identity" refers to a homology of more than 95% (ideally more than 98%).

[0045] "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.

[0046] CD20 and CD19 are both widely distributed marker antigen proteins on B cells. First-line therapy for B-cell malignancies typically uses anti-CD20 antibodies, such as rituximab. Treatment of relapsed and refractory (R / R) B-cell acute lymphoblastic leukemia (B-ALL) is a key focus of hematological research, with significant clinical application demand. Adaptive T cells expressing chimeric antigen receptors (CARs) are emerging as an effective clinical treatment technique. Single-target CARs targeting CD19, CD20, and CD22, dual-target CARs targeting CD19 and CD20 or CD22, and other combination therapies hold new therapeutic promise. CD20-targeted CAR-modified T-cell therapy has shown unprecedented efficacy in patients with no other treatment options.

[0047] This invention provides a fully human antibody or antibody fragment targeting CD20, wherein the anti-CD20 binding domain of the fully human antibody or antibody fragment is scFv, and the amino acid sequence of the scFv is shown in Table 1.

[0048] Table 1. Amino acid sequence of scFv

[0049]

[0050] The nucleic acid sequence encoding scFv provided by the present invention includes the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16. After translation into amino acid sequences based on the codons corresponding to the bases in each sequence, the corresponding amino acid sequences of scFv are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0051] The sources of some materials are explained below:

[0052] CD20 protein: Human CD20 (purchased from Acrobiosysterms, catalog number CD9-H8259).

[0053] Phage antibody library of human single-chain antibodies: constructed by Changzhou Felos Pharmaceutical Technology Co., Ltd.

[0054] Streptavidin magnetic beads: purchased from Invitrogen.

[0055] Enzyme-linked immunosorbent assay (ELISA) microplate: 96 half-well low-permeability flat-bottom microplate (purchased from Corning).

[0056] Anti-M13 HRP antibody: purchased from Thermo Fisher.

[0057] M13 helper phage: purchased from Invitrogen.

[0058] SOC culture medium: purchased from Shanghai Sangon Biotech.

[0059] pCGMT phage vector: purchased from Addgene.

[0060] XL1-blue bacteria: purchased from Agilent Technologies, product number 200228.

[0061] 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.

[0062] Developer ABTS solution: purchased from Thermo Fisher, product number 002024.

[0063] Gelred nucleic acid dye: purchased from Thermo Fisher.

[0064] pFUSE expression vector: purchased from Invitrogen.

[0065] Plasmid extraction kit: purchased from TIANGEN.

[0066] Restriction endonucleases: purchased from Takara.

[0067] Recombinase: purchased from Novoprotein.

[0068] lipo3000 reagent: purchased from Thermo Fisher Scientific.

[0069] Polybrene reagent: purchased from Thermo Fisher Scientific.

[0070] RMPI 1640 medium: purchased from Gibco.

[0071] DMEM medium: purchased from Gibco.

[0072] LVP2MIX transfection reagent: purchased from Suzhou Aikon Technology Co., Ltd.

[0073] Alexa 647anti-c-Myc: Purchased from Biolegend.

[0074] PE anti-human CD69: purchased from Biolegend.

[0075] Goat anti-Human, Alexa Fluor TM 488: Purchased from Thermo Fisher.

[0076] 293Fectin transfection reagent: purchased from Invitrogen, catalog number 12347500.

[0077] 293 Freestyle suspension cells: purchased from Thermo Fisher.

[0078] Immunoglobulin IgG1 constant region Fc segment: purchased from Nanjing Genscript Biotech Co., Ltd.

[0079] BCA protein quantification kit: purchased from Pierce, catalog number 23252.

[0080] CBS antigen fixation solution: Dissolve 1.59g Na2CO3 and 2.93g NaHCO3 in 1L of water and adjust the pH to 9.6.

[0081] Anti-Human Fc HRP secondary antibody: purchased from Thermo Fisher.

[0082] 96-hole bottom plate: purchased from Corning.

[0083] Biacore Instrument: Purchased from GE, T200.

[0084] Protein A chip: purchased from GE.

[0085] Example 1: Screening of anti-CD20 single-chain antibodies

[0086] 1. Establish a phage antibody library of fully human single-chain antibodies.

[0087] 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 using a (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, 50 μL of VCSM13 helper phage at a concentration of 10¹³ / mL was added, and the cells were incubated at room temperature for 1 h, gently shaking every 10 min. The cells were then incubated at 37°C with shaking for another 2 h, and finally, kanamycin was added to a final concentration of 70 μg / mL, and the cells were incubated overnight at 30°C. Centrifuge and collect the supernatant, add 10% PEG-8000 / sodium chloride solution (PEG is polyethylene glycol), place in an ice bath for 1 hour, centrifuge at 8000 rpm and 4℃, discard the supernatant, fully dissolve the precipitate in 2 mL of 1% BSA (bovine serum albumin) PBS solution (phosphate buffer), centrifuge and collect the supernatant, which is the phage antibody library of fully human single-chain antibodies, used for subsequent screening of fully human antibodies.

[0088] 2. Antibody screening

[0089] Take 5 μg of CD20 antigen protein, dilute to 1 ng / μL with CBS, and coat 50 μL per well onto an ELISA plate for a total of 8 wells. Incubate overnight at 4°C. After discarding the supernatant, wash the plate twice with PBS, then add 50 μL (containing 1 × 10¹³ phages / mL) / well of a phage antibody library expressing fully human single-chain antibodies. Incubate at room temperature for 2 hours, then discard the supernatant. Wash three times each with PBS and PBST. Phages that bind to the CD20 antigen are captured, while unbound phages are removed by rinsing with 0.5% Tween-20 PBS solution (phosphate buffer). Elute the phages that are stably bound to the antigen with glycine hydrochloride solution (pH 2.2) for later use. Inoculate 20 mL of XL1-Blue bacterial suspension. Once the OD600 (absorbance at 600 nm) reaches 0.6, add the eluted phage solution and incubate with the XL1-Blue bacteria at 37°C for 30 min. Spread the bacterial suspension onto ampicillin-resistant plates. The next day, elute and collect the bacteria from the ampicillin-resistant plates. Further expand the culture until the OD600 is approximately 0.6, then use 1×10⁻⁶... 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 three rounds. In each round, the number of wells coated with antigen is reduced by half to select phages with stronger binding activity.

[0090] The XL1-Blue bacterial suspension infected with bacteriophages was thoroughly diluted, and then spread on 15cm 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).

[0091] 3. Phage enzyme-linked immunosorbent assay (ELISA)

[0092] 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. Enzyme-linked immunosorbent assay (ELISA) microplates were coated with CD20 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.

[0093] 4. Experimental Results:

[0094] like Figure 1 As shown in the figure, this is a bar chart of the results of screening CD20 single-chain scFv antibody fragments in Experiment Example 1. Table 2 shows the input and recovery amounts and recovery rates of the phage library in each round of screening CD20 single-chain scFv antibodies in Experiment Example 1.

[0095] Table 2. Results of recovery amount and recovery rate of 3 rotaphages in Experiment Example 1.

[0096]

[0097] 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 number of phages recovered in the fourth round increased by more than 20 times compared to the first round. This indicates that after four rounds of screening, phages expressing antibodies specifically binding to the CD20 antigen were continuously enriched. 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.

[0098] Example 2: Preparation (expression and purification) of anti-CD20 single-chain antibody

[0099] 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.

[0100] 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 cultured in a shaker at 37°C for 48-72 hours at 125 rpm. After centrifugation, the supernatant was collected by filtration 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). The filtered cell supernatant was then loaded onto the column, and flowthrough 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-CD20 single-chain antibody (anti-CD20 fully human scFv antibody). Finally, the concentration of the purified antibody was determined according to the instructions of the BCA method protein quantification kit.

[0101] Experimental Example 3: Detection of the binding activity of anti-CD20 single-chain antibody against CD20 antigen protein (ELISA)

[0102] Immunodetection point proteins were 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 plates were sealed with plastic film and incubated overnight at 4°C. The next day, the plates were 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 purified CD20 single-chain antibody obtained in Experiment 2 was added to each well. After incubation at 37°C for 2 hours, the plates were 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 plates were 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, 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 CD20 antigen protein at the ELISA level, with Tab2 serving as the positive control antibody. The blank control, human Fc protein, serves as the negative control (NC).

[0103] Experiment Example 4: Flow Cytometry (FACS) assay to detect the binding activity of antibody to CD20-overexpressing cells.

[0104] A pCDH plasmid containing a nucleotide sequence encoding the full-length amino acid sequence of human CD20 was transfected into the CHOK1 cell line to obtain a stable CHOK1 cell line containing human CD20 (here referred to as the CHOK1-CD20 stable cell line). The CHOK1-CD20 stable cell line was cultured in T75 cell culture flasks until 90% confluence. The culture medium was aspirated, and the cells were washed twice with PBS buffer (Hyclone). Then, the cells were treated with enzyme-free cell dissociation solution (Versene solution, Life Technologies) and collected. After washing the cells twice with PBS buffer and performing cell counting, the cells were diluted to 2 × 10⁻⁶ cells with PBS buffer. 6 Cells / mL, add 1% goat serum blocking solution, incubate on ice for 30 minutes, then wash three times with PBS buffer by centrifugation. Resuspend the collected cells in FACS buffer (PBS + 2% FBS) to 2 × 10⁻⁶ cells / mL. 6Cells / mL were added at a rate of 100 μL per well to a 96-well FACS plate. 100 μL of purified CD20 antibody (obtained in Example 2) was added to each well, and the plate was incubated at 4°C for 2 hours. Cells were washed three times by centrifugation with FACS buffer. 100 μL of FITC-labeled secondary antibody (purchased from Biolegend) was added to each well, and the plate was incubated at 4°C for 1 hour. Cells were washed three times by centrifugation with FACS buffer, and then resuspended in 100 μL of FACS buffer. Flow cytometry was used to detect and analyze the results.

[0105] Experimental results: such as Figure 3 As shown, the results indicate that the test antibody F5 can specifically bind to the human CD20 antigen protein on the cell surface, while B5 hardly binds to the overexpressed CD20 antigen on the cell surface.

[0106] Experimental Example 5: Flow cytometry (FACS) assay to detect the binding activity of antibody to Raji tumor cells expressing CD20.

[0107] Raji tumor cells were cultured in T25 cell culture flasks to a scale of 3 × 10⁶ cells / year. 6 Centrifuge at 800g for 10 minutes, remove all culture medium, wash twice with PBS buffer (Hyclone), count cells, and then dilute to 2×10⁶ cells / mL with PBS buffer. 6 Cells / mL, add 1% goat serum blocking solution, incubate on ice for 30 minutes, then wash twice with PBS buffer by centrifugation. Resuspend the collected cells in FACS buffer (PBS + 2% FBS) to 2 × 10⁶ cells / mL. 6 Cells / mL were added at a rate of 100 μL per well to a 96-well FACS plate. 100 μL of purified CD20 antibody (obtained in Example 2) was added to each well, and the plate was incubated at 4°C for 2 hours. Cells were washed twice by centrifugation with FACS buffer, and 100 μL of FITC-labeled secondary antibody (purchased from Biolegend) was added to each well. The plate was incubated at 4°C for 1 hour. Cells were washed three times by centrifugation with FACS buffer, and then resuspended in 100 μL of FACS buffer. Flow cytometry was used to detect and analyze the results.

[0108] Experimental results: such as Figure 4 As shown, both the test antibodies F5 and B5 can specifically recognize Raji cells that overexpress CD20 antigen.

[0109] Experiment 6: Validation of the CD20-targeting CAR's ability to specifically recognize the CD20 antigen.

[0110] 1. Construction of Anti-CD20 CAR lentiviral plasmid:

[0111] For a map of lentiviral plasmids, please refer to [link / reference]. Figure 5The Anti-CD20 CAR plasmid includes a signal peptide sequence, a CD20 binding domain, a detection tag, a hinge region and a transmembrane domain, a functional signal transduction domain, a regulatory peptide, and a fluorescent reporter gene. The signal peptide sequence comprises CSF2R; the detection tag comprises C-myc; the CD20 binding domain comprises scFv, which comprises the fully human antibody or antibody fragment against CD20; the hinge region and transmembrane domain comprise a hinge region Hinge™ and a transmembrane structure CD8; and the functional signal transduction domain comprises CD28, 4-1BB, and CD3 Zeta connected in sequence.

[0112] (1) Take 1 μg of plasmid and lentiviral vector plasmid from the anti-CD20 monoclonal strain screened in Example 1, add 1 μL of LfiI for enzyme digestion, and digest in a 50℃ water bath for 2 h. Then, recover the digestion product by gel.

[0113] (2) Take the single-chain sfiI digestion product of anti-human CD20 antibody and the sfiI digestion product of lentiviral vector plasmid, and ligate them at 16℃ for 1h.

[0114] (3) Transform the ligation product constructed in (2) into 100 μL of Escherichia coli DH5α competent cells;

[0115] (4) Select single clones, extract plasmids using the Tiangen Plasmid Mini-Prep Kit (DP106), and verify the correctness of the sequence using Sanger sequencing to obtain the anti-CD20-CAR plasmid.

[0116] 2. Flow cytometry (FACS) detection of anti-CD20-CAR expression

[0117] 2.1 Lentiviral Packaging

[0118] One day before transfection, 5 × 10⁶ 293T cells were seeded into 6-well plates and mixed with DMEM + 10% FBS medium to prepare the complex: (1) 1.5 μg anti-CD20-CAR plasmid, 3 μl LVP2MIX and 6 μL P3000 were diluted in 125 μL Opti-MEM medium and gently mixed. (2) 5 μl lipo3000 was diluted in 125 μL Opti-MEM medium, gently mixed and incubated at room temperature for 5 minutes. (3) After 5 minutes, gently mix them and incubate at room temperature for 20 minutes; remove the culture medium from the culture plate and wash the cells twice with PBS or serum-free culture medium (preferably); add 3 ml DMEM + 10% FBS, add the complex (total volume 3.6 ml) to the culture wells, and shake the culture plate in a figure-eight motion to distribute it evenly; after incubating the cells in an incubator for 4-6 hours, replace the serum-containing culture medium to remove the complex (optional); observe the expression of the transformed gene after 24-72 hours. (4) Collect the virus after 72 hours, filter it with a 0.45 μm filter and detect the virus titer. The kit detects an unconcentrated virus titer >10. 7 IFU / ml.

[0119] 2.2 Lentiviral infection

[0120] The collected lentiviruses are used to infect host cells jurkat according to the MOI gradient.

[0121] Host cells were centrifuged at 300g for 5 minutes, resuspended, and a certain volume of packaged virus solution was added. The volume was then increased to 1ml with 1640 + 10% FBS. Polybrene, an infection enhancer, was added to a final concentration of 8ug / ml and mixed thoroughly. The mixture was centrifuged at 800g for 1-2 hours and incubated. After 48-72 hours, fluorescence signals were initially observed using a fluorescence microscope, and infection efficiency was determined by flow cytometry. Figure 6 As shown, anti-CD20-F5-CAR, anti-CD20-B5-CAR, and anti-CD20-Tab2-CAR were all expressed on Jurkat host cells, with infection efficiencies of 41.1%, 81.5%, and 75.9%, respectively.

[0122] 2.3 Detection of the CD20-specific recognition ability of CARs targeting CD20 using flow cytometry

[0123] (1) Preparation of Raji and K562 cells: Centrifuge cells at 1000 rpm for 5 minutes, discard the supernatant, and resuspend to 1×10⁶ / ml; the sorted CAR-jurkat cell count is 2.5×10⁶. 5 / ml, total 5ml; prepare a 12-well plate, add 1.5×10 ml to each well. 5 jurkat cells +3×105 Raji cells were used in 8 wells; 2.5 × 10⁻⁶ cells were added to the negative control wells. 4 Jurakat cells +5×10 5 K562 cells; add 1640 + 10% FBS to a culture volume of 2 ml, and incubate in an incubator;

[0124] (2) Transfer the cells from the well plate to centrifuge tubes and centrifuge at 1000 rpm for 5 minutes; discard the supernatant and resuspend in FACS buffer to 1×10⁻⁶. 6 Density / ml; add 20μl / 10 ml in the dark. 6 Cell anti-CD69-APC fluorescent antibody was incubated at 4°C for 1 hour, protected from light; cells were washed twice with FACS buffer and centrifuged at 1000 rpm for 5 minutes; cells were resuspended in FACS buffer and flow cytometry was used to detect host cell activation. The detection of CD69 molecule expression on the jurkat surface indicates that the effector cell jurkat is activated, meaning that the effector cell jurkat can recognize the CD20 antigen on the target cell surface, of which CD69 is the earliest surface antigen expressed after T lymphocyte activation.

[0125] The experimental results are as follows Figure 7 As shown, host cells expressing the CAR gene, juramat, were co-incubated with negative K562 cells and positive Raji cells, respectively, with the empty juramat+Raji group as a negative control. Anti-CD20-F5-CAR, anti-CD20-B5-CAR, and anti-CD20-Tab2-CAR could all activate juramat host cells. Among them, the positive control anti-CD20-Tab2-CAR showed a stronger self-activation phenomenon with an activation efficiency of 27%, while anti-CD20-F5-CAR had an activation efficiency of 50.3%, showing greater development potential.

[0126] In summary, this invention provides a fully human anti-CD20 antibody or antibody fragment comprising 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, and the light chain variable region includes three complementarity-determining regions: LCDR1, LCDR2, and LCDR3. The fully human antibody or antibody fragment of this invention can bind to human CD20 protein with high specificity. Using chimeric antigen receptor technology (CAR-T technology), the human antibody fragment integrating CD20 and being integrated into the CAR can be engineered and expressed in T cells. The resulting chimeric antigen receptor T cells can be used to treat hematological cancers associated with CD20 expression.

[0127] 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 of Anti-CD20, characterized in that, The fully human antibody or antibody fragment includes: a heavy chain variable region and a light chain variable region; The heavy chain variable region includes three complementary determinant regions: HCDR1, HCDR2, and HCDR3; 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: 5; The amino acid sequence of HCDR3 is shown in SEQ ID NO: 7; The light chain variable region includes three complementary determining regions: LCDR1, LCDR2, and LCDR3; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 9; The amino acid sequence of LCDR2 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR3 is shown in SEQ ID NO:

13.

2. The fully human antibody or antibody fragment against CD20 as described in claim 1, characterized in that, The anti-CD20 binding domain of the fully human antibody or antibody fragment is scFv, and the amino acid sequence of the anti-CD20 binding domain is shown in SEQ ID NO:

2.

3. The fully human antibody or antibody fragment against CD20 as described in claim 1, characterized in that, The anti-CD20 binding domain of the fully human antibody or antibody fragment is scFv, and the nucleic acid sequence of the scFv is shown in SEQ ID NO:

16.

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises a fully human antibody or antibody fragment of anti-CD20 as described in any one of claims 1 to 3, wherein the fully human antibody is a single-chain antibody.

5. The chimeric antigen receptor as described in claim 4, characterized in that, The chimeric antigen receptor comprises a signal peptide sequence, a CD20 binding domain, a detection tag, a hinge region, and a transmembrane domain, connected in sequence, as well as a functional signal transduction domain; the signal peptide sequence comprises CSF2RA; the detection tag comprises C-myc; the CD20 binding domain comprises scFv, wherein the scFv comprises the fully human antibody or antibody fragment of the anti-CD20; the hinge region and transmembrane domain comprise the hinge region Hinge™ and the transmembrane structure CD8; the functional signal transduction domain comprises CD28, 4-1BB, and CD3 Zeta connected in sequence.

6. The chimeric antigen receptor as described in claim 5, characterized in that, The amino acid sequences of the hinge region Hinge™ and the transmembrane structure CD8 are shown in SEQ ID NO: 17, the amino acid sequence of CD28 is shown in SEQ ID NO: 18, the amino acid sequence of 4-1BB is shown in SEQ ID NO: 19, the amino acid sequence of CD3 Zeta is shown in SEQ ID NO: 20, the amino acid sequence of CSF2RA is shown in SEQ ID NO: 21, and the amino acid sequence of C-myc is shown in SEQ ID NO:

22.

7. A host cell, characterized in that, The host cell contains at least the chimeric antigen receptor as described in any one of claims 4 to 6, and the host cell is a T cell.

8. A lentiviral plasmid, characterized in that, The lentiviral plasmid is capable of encoding the nucleotide sequence of a fully human antibody or antibody fragment against anti-CD20 as described in any one of claims 1 to 3.

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