A fully human antibody or antibody fragment targeting CD7 and its chimeric antigen receptor and application

Abstract

The present invention discloses a fully human antibody or antibody fragment targeting CD7, a chimeric antigen receptor thereof, and applications thereof. The fully human antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, wherein 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 nucleic acid encoding the fully human antibody or antibody fragment can be used to prepare host cells, lentiviral plasmids, diagnose tumors, and target anti-tumor drugs. The fully human antibody or antibody fragment of the present invention can bind to the human CD7 protein with high specificity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a fully human antibody or antibody fragment targeting CD7, a chimeric antigen receptor thereof, and applications thereof. Background Art

[0002] T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematological malignancy characterized clinically by high relapse rates and low survival rates. T-ALL accounts for approximately 25% of adult acute lymphoblastic leukemia (ALL) cases and approximately 15% of childhood ALL cases. The disease presents with rapidly progressive clinical symptoms, including fever, infection, anemia, abnormal bleeding, and organ infiltration. In the absence of a unified first-line standard treatment regimen, the long-term prognosis of patients with this disease is not ideal, especially in relapsed or refractory cases, where the 5-year overall survival rate is less than 20%.

[0003] CD7 is a transmembrane glycoprotein widely expressed on the surface of T cells, natural killer (NK) cells, and their precursor cells. As a surface antigen of T cells and NK cells, CD7 is positively expressed in more than 95% of T-ALL and T-cell lymphoma (T-lymphoma) cases. This makes CD7 an ideal target for the treatment of T cell-related malignancies. Although normal T cells also express CD7, strategies targeting CD7 still have significant therapeutic potential in the context of cancer treatment.

[0004] Chimeric antigen receptor T cell (CAR-T cell) therapy is a rapidly developing cancer immunotherapy approach in recent years. Through genetic engineering, CAR-T cell therapy transforms a patient's autologous T cells into specialized cells capable of recognizing and killing tumor cells. The chimeric antigen receptors on CAR-T cells are composed of an antigen-binding region, a transmembrane region, and an intracellular signaling domain, enabling them to specifically recognize and kill tumor cells without relying on antigen presentation via the major histocompatibility complex (MHC).

[0005] CAR-T cell therapy has demonstrated significant clinical efficacy in B-cell-related leukemias and lymphomas. However, the application of CAR-T cell therapy for T-cell-related malignancies, particularly T-ALL, is still under exploration. Summary of the Invention

[0006] The purpose of the present invention is to overcome the lack of effective treatment for T-ALL in the prior art and to provide an application of CAR-T cell therapy for T cell-related malignancies, especially T-ALL. The fully human CD7 antibody or antibody fragment of the present invention has high targeting and can specifically bind to the human CD7 protein, thereby being used to treat hematological cancers related to CD7 expression.

[0007] In order to achieve the above objectives, the present invention provides a fully human antibody or antibody fragment against CD7, wherein the fully human antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region;

[0008] The heavy chain variable region includes three complementarity determining regions: HCDR1, HCDR2 and HCDR3;

[0009] The amino acid sequence of the HCDR1 includes the sequence shown in SEQ ID NO: 3;

[0010] The amino acid sequence of the HCDR2 includes the sequence shown in SEQ ID NO: 4;

[0011] The amino acid sequence of the HCDR3 includes the sequence shown in SEQ ID NO: 5;

[0012] The light chain variable region includes three complementarity determining regions: LCDR1, LCDR2, and LCDR3;

[0013] The amino acid sequence of the LCDR1 includes the sequence shown in SEQ ID NO: 6;

[0014] The amino acid sequence of the LCDR2 includes the sequence shown in SEQ ID NO: 7;

[0015] The amino acid sequence of LCDR3 includes the sequence shown in SEQ ID NO:8.

[0016] Preferably, the anti-CD7 binding domain of the fully human antibody or antibody fragment is scFv, and the amino acid sequence of the anti-CD7 binding domain is as shown in SEQ ID NO: 1.

[0017] Preferably, the anti-CD7 binding domain of the fully human antibody or antibody fragment is scFv, and the nucleic acid sequence of the scFv is as shown in SEQ ID NO: 2.

[0018] The present invention also provides a chimeric antigen receptor, which contains any one of the fully human antibodies or antibody fragments against CD7 described above.

[0019] Preferably, the chimeric antigen receptor comprises: a signal peptide sequence, a CD7 binding domain, a detection tag, a hinge region and a transmembrane domain, and a functional signal transduction domain connected in sequence:

[0020] The signal peptide sequence comprises CSF2RA, and the amino acid sequence of CSF2RA is as shown in SEQ ID NO: 9;

[0021] The detection tag comprises C-myc, and the amino acid sequence of C-myc is as shown in SEQ ID NO: 10;

[0022] The CD7 binding domain comprises scFv, and the scFv comprises the fully human antibody or antibody fragment against CD7;

[0023] The hinge region and transmembrane domain comprise: a hinge region Hinge TM and a transmembrane structure CD8, wherein the amino acid sequence of the hinge region Hinge TM and the transmembrane structure CD8 is as shown in SEQ ID NO: 11;

[0024] The functional signaling domain comprises: CD28, 4-1BB and CD3 Zeta connected in sequence, the amino acid sequence of the functional signaling structure CD28 is as shown in SEQ ID NO: 12, the amino acid sequence of the functional signaling structure 4-1BB is as shown in SEQ ID NO: 13, and the amino acid sequence of the functional signaling structure CD3 Zeta is as shown in SEQ ID NO: 14.

[0025] The present invention also provides a host cell, which comprises at least the chimeric antigen receptor as described above.

[0026] The present invention also provides a lentiviral plasmid comprising a nucleotide sequence capable of encoding any one of the above described fully human antibodies or antibody fragments against CD7.

[0027] The present invention also provides a use of a fully human antibody or antibody fragment targeting CD7, wherein the fully human antibody or antibody fragment targeting CD7 is used to prepare T cells for treating or preventing tumors.

[0028] Preferably, the T cell at least comprises the chimeric antigen receptor as described above.

[0029] Compared with the prior art, the beneficial effects of the present invention include at least:

[0030] (1) The fully human antibodies or antibody fragments of the present invention can bind to human CD7 protein with high specificity, with an EC50 value of 1.0 nM for the single-chain antibody and 0.7 nM for the full-length antibody.

[0031] (2) The chimeric antigen receptor of the present invention can specifically recognize the CD7 protein on the surface of tumor cells. The chimeric antigen receptor technology (CAR-T technology) is used to engineer the expression of human antibody fragments that bind to CD7 and are integrated into the CAR in T cells. The resulting chimeric antigen receptor T cells can be used to treat hematological cancers associated with CD7 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a bar graph showing the results of screening targeting CD7 protein using the phage display technology of the present invention.

[0033] Figure 2 This is a diagram showing the ELISA test results of three rounds of phage pool binding to CD7 protein in the present invention for screening targeting CD7 protein.

[0034] Figure 3 This is the ELISA test result of the anti-CD7 fully human scFv antibody of the present invention specifically recognizing the CD7 protein.

[0035] Figure 4 This is the flow cytometry result of the anti-CD7 fully human scFv antibody of the present invention specifically recognizing Jurkat cells overexpressing CD7.

[0036] Figure 5 This is the plasmid map of the chimeric antigen receptor of the present invention.

[0037] Figure 6 This is the flow cytometry result of the expression rate of CAR-VX-F7 on the surface of the host cell Jurkat of the present invention.

[0038] Figure 7 This is the activation result of the effector host cell Jurkat incubated with the target cell Raji of the present invention. DETAILED DESCRIPTION

[0039] Definition of terms

[0040] An "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Antibodies include single-chain antibodies.

[0041] A "heavy chain" consists of a heavy chain variable region (VH) and a heavy chain constant region.

[0042] A "light chain" consists of a light chain variable region (VL) and a light chain constant region.

[0043] "scFv": single-chain antibody fragment, composed of the variable region of the heavy chain and the variable region of the light chain of the antibody connected by a short peptide (linker) of 15 to 20 amino acids.

[0044] The "heavy chain variable region" and "light chain variable region" can be further divided into hypervariable regions called "complementarity determining regions" (CDRs), which are interspersed within more conserved regions called "framework regions" (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from amino-terminus to 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 antigens. In the present invention, the CDR1, CDR2, and CDR3 of the heavy chain variable region are represented as HCDR1, HCDR2, and HCDR3, respectively; the CDR1, CDR2, and CDR3 of the light chain variable region are represented as LCDR1, LCDR2, and LCDR3, respectively.

[0045] The "constant region" may mediate the binding of the immunoglobulin to host tissues or factors.

[0046] "Antigen binding domain" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, CD7).

[0047] "Monoclonal antibody" refers to a preparation of antibody molecules composed of a single molecule. A monoclonal antibody composition exhibits specific binding and affinity for a particular epitope (the portion of an antigen that is specifically recognized by an antigen receptor).

[0048] Chimeric antigen receptors (CARs) consist of an extracellular antigen-binding region (composed of light and heavy chains derived from monoclonal antibodies, connected by a flexible hinge region to form a single-chain antibody), a transmembrane region, and an intracellular signal transduction region.

[0049] "CAR-T technology" refers to chimeric antigen receptor T-cell immunotherapy. It involves in vitro genetic recombination of a scFv that recognizes a tumor-associated antigen with the intracellular signaling domain, an immunoreceptor tyrosine-based activation motif, to generate a recombinant plasmid. This plasmid is then transfected into the patient's T cells via in vitro transfection, causing them to express the tumor antigen receptor. The transfected T cells, purified, and expanded on a large scale, are then called chimeric antigen receptor T cells.

[0050] "95-99% identity" refers to greater than 95% (optimally greater than 98%) homology.

[0051] "Modification" is a variant form of amino acids or nucleotides, including deletion, insertion and / or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, and most preferably 1 to 10) amino acids or nucleotides, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids or nucleotides to the C-terminus and / or N-terminus, but does not change the function of the protein or nucleic acid.

[0052] As a hallmark antigen on the surface of T cells, CD7 has become an important target for CAR-T cell therapy. Studies have shown that CAR-T cells targeting CD7 have shown strong anti-tumor activity in preclinical models. The present invention innovatively screened out a fully human CD7 antibody sequence and applied lentiviral gene editing technology to provide a method for preparing CD7-CAR-T cells. The application of these CD7-CAR-T cells is expected to become an effective treatment for T-ALL and other T-cell malignancies. The present invention provides a fully human antibody or antibody fragment against CD7, the anti-CD7 binding domain of the fully human antibody or antibody fragment is scFv, and the amino acid sequence of the scFv is specifically shown in Table 1.

[0053] Table 1 Amino acid sequence of scFv

[0054]

[0055] The nucleic acid sequence encoding scFv provided by the present invention includes the sequence shown in SEQ ID NO: 2. According to the codons corresponding to the bases in each sequence, after translation into amino acid sequences, the corresponding scFv amino acid sequences are the sequences shown in SEQ ID NO: 1.

[0056] The sources of some materials are as follows:

[0057] CD7 protein: Human CD7-His tag (purchased from Kaixia, product number CD7-HM401)

[0058] Phage antibody library of human single-chain antibody: constructed by Changzhou Felos Pharmaceutical Technology Co., Ltd.

[0059] Streptavidin magnetic beads: purchased from Invitrogen.

[0060] ELISA microplate: 96 half-well low-transparency flat-bottom microplate (purchased from Corning).

[0061] Anti-M13 HRP antibody: purchased from Thermo Fisher.

[0062] M13 helper phage: purchased from Invitrogen.

[0063] SOC medium: purchased from Shanghai Bioengineering.

[0064] pCGMT phage vector: purchased from Addgene.

[0065] XL1-blue bacteria: purchased from Agilent, catalog number 200228.

[0066] LB solid medium plate: Dissolve 5 g yeast extract, 10 g peptone, 10 g sodium chloride, and 10 g agar powder in 1 L of double-distilled water, autoclave at 121°C, and then pour onto the plate.

[0067] ABTS developer solution: purchased from Thermo Fisher, product number 002024.

[0068] Gelred nucleic acid dye: purchased from Thermo Fisher.

[0069] pFUSE expression vector: purchased from Invitrogen.

[0070] Plasmid extraction kit: purchased from TIANGEN.

[0071] Restriction endonucleases: purchased from Takara.

[0072] Recombinant enzyme: purchased from Novoprotein.

[0073] Lipo3000 reagent: purchased from Thermo Fisher.

[0074] Polybrene reagent: purchased from Thermo Fisher.

[0075] RMPI 1640 culture medium: purchased from Gibco.

[0076] DMEM culture medium: purchased from Gibco.

[0077] LVP2MIX transfection reagent: purchased from Suzhou Aikangde Company.

[0078] Alexa 647 anti-c-Myc: purchased from Biolegend.

[0079] PE anti-human CD69: purchased from Biolegend.

[0080] Goat anti-Human,Alexa Fluor TM 488: purchased from Thermo Fisher.

[0081] 293Fectin transfection reagent: purchased from Invitrogen, catalog number 12347500.

[0082] 293 Freestyle suspension cells: purchased from Thermo Fisher.

[0083] Immunoglobulin IgG1 constant region Fc segment: purchased from Nanjing GenScript Company.

[0084] BCA protein quantification kit: purchased from Pierce, catalog number 23252.

[0085] CBS antigen fixation solution: Dissolve 1.59 g Na2CO3 and 2.93 g NaHCO3 in 1 L of water and adjust the pH to 9.6.

[0086] Anti-Human Fc HRP secondary antibody: purchased from Thermo Fisher.

[0087] 96-well transparent bottom plate: purchased from Corning.

[0088] Biacore instrument: purchased from GE, T200.

[0089] Protein A chip: purchased from GE.

[0090] Experimental Example 1 Screening of anti-CD7 single-chain antibodies

[0091] 1. Establishment of a phage antibody library of fully human single-chain antibodies

[0092] Primers were designed to amplify the heavy chain variable region and light chain variable region of the fully human antibody. The heavy chain variable region and light chain variable region were connected by overlap extension PCR using (GGGGS) 3-linker to obtain a full-length PCR product. The PCR product and phagemid vector were digested with SfiI, and the ligation transformation product was electroporated into XL1-blue competent cells. 20 mL of selective culture medium with a final concentration of 50 μg / mL ampicillin and 10 μg / mL tetracycline was added, and the cells were shaken and cultured at 37°C for 2 h. Then, a concentration of 10 13 / mL VCSM13 helper phage 50μL, incubate at room temperature for 1h, and gently shake every 10min in the middle. Continue shaking culture at 37℃ for 2h, then add kanamycin with a final concentration of 70μg / mL, and culture overnight at 30℃. Collect the supernatant by centrifugation, add 10% PEG-8000 / sodium chloride solution (PEG is polyethylene glycol), place in an ice bath for 1h, centrifuge at 8000rpm, 4℃, discard the supernatant, and fully dissolve the precipitate with 2mL of 1% BSA (bovine serum albumin) in PBS solution (phosphate buffer), and collect the supernatant by centrifugation. This is the phage antibody library of the fully human single-chain antibody, which is used for subsequent screening of fully human antibodies.

[0093] 2. Antibody Screening

[0094] Take 5 μg of CD7 antigen protein, dilute it to 1 ng / μL with CBS, and coat 50 μL per well on the enzyme-labeled plate, for a total of 8 wells, and incubate overnight at 4°C. After discarding the supernatant, wash the plate twice with PBS and add 50 μL (containing 1×10 phage 13 / mL) / well phage antibody library expressing fully human single-chain antibodies, incubate at room temperature for 2 hours, discard the supernatant, wash 3 times with PBS and PBST respectively, capture the phage bound to the CD7 antigen, and remove the unbound phage after rinsing with 0.5% Tween-20 PBS solution (phosphate buffer), and elute the phage stably bound to the antigen with hydrochloric acid glycine solution (pH2.2) for use. Inoculate 20mL of XL1-Blue bacterial solution, and when OD600 (absorbance at 600nm wavelength, optical density) reaches 0.6, add the eluted phage solution mentioned above, and incubate with XL1-Blue bacteria at 37°C for 30min. Spread the bacterial solution on an ampicillin-resistant plate, and collect the bacteria on the ampicillin-resistant plate by elution the next day. Further expand the culture until OD600 is about 0.6, and then use 1×10 12 The cells were then infected with M13 helper phage containing 10 pfu / mL (plaque forming unit) and amplified before the next round of screening. A total of three rounds of screening were performed. The number of antigen-coated plates was reduced by half with each round to screen for phages with stronger binding activity.

[0095] The XL1-Blue bacterial solution infecting the phage was fully diluted, and then the bacterial solution was spread on a 15 cm diameter LB solid culture medium plate resistant to ampicillin. Monoclonal antibodies were picked, and the phage libraries and monoclones after each round of panning were verified by phage enzyme-linked immunosorbent assay.

[0096] 3. Phage enzyme-linked immunosorbent assay

[0097] Inoculate XL1-Blue monoclonal clones into a 2 mL 96-well bacterial culture plate (purchased from Corning). Add 500 μL of SB medium containing tetracycline resistance and shake at 200 rpm at 37°C for 4-6 hours. Once the OD600 value approaches 0.6, add 1 μL of helper phage and shake overnight at 30°C. The next day, centrifuge at 3000 g and remove the supernatant for later use. Prepare an enzyme-linked immunosorbent assay (ELISA) microplate and coat it with CD7 antigen overnight at 4°C. On the third day, wash twice with PBST (phosphate-buffered saline containing Tween-20) and block with 5% skim milk. Then add the phage supernatant prepared in the previous step and incubate at room temperature for 2 hours. After washing the plate five times with PBST, add HRP-conjugated Anti-M13 antibody and incubate at 37°C for 30 minutes. Wash the plate five times with PBST and develop the color with 50 μL of ABTS.

[0098] 4. Experimental Results

[0099] like Figure 1 , which is a bar chart of the results of screening CD7 single-chain scFv antibody fragments in Experimental Example 1, and Table 2 is the output and recovery results of screening CD7 single-chain scFv antibody fragments in Experimental Example 1.

[0100] Table 2 Results of phage recovery and recovery rate in three rounds of Example 1

[0101]

[0102] from Figure 1 As can be seen from Table 2, after three rounds of screening of the CD7 antigen in the phage antibody library of the fully human single-chain antibody, the output of the third round increased by more than 100 times compared with the first round, indicating that after three rounds of screening, phages that can express antibodies that specifically bind to the CD7 antigen are continuously enriched. The phage pools from the three rounds of screening were diluted 5 times, 10 times, 50 times, and 100 times, respectively. The diluted phage pools were further tested for their binding ability to the CD7 protein by enzyme-linked immunosorbent assay, as shown in Figure 2. Figure 2 As shown, the binding ability of phage pools with different dilution ratios to CD7 protein was significantly enhanced after three rounds of screening.

[0103] 95 monoclones were selected from the phage library obtained after the third round of screening for enzyme-linked immunosorbent assay (ELISA) verification. Finally, monoclones with an ELISA signal intensity greater than 1 were identified as positive monoclones. The positive monoclones were sequenced to obtain nucleotide coding sequences, which were then compared and analyzed. The more repeated clones, the higher the enrichment of the antibody sequence, thereby determining the effectively enriched nucleotide coding sequences.

[0104] Experimental Example 2 Preparation (Expression and Purification) of Anti-CD7 Single-Chain Antibody

[0105] Plasmids were extracted from the monoclonal strain screened in Experimental Example 1, digested with the restriction endonuclease SfiI, and ligated into a pFuse expression vector via fragment recombination, thereby obtaining a pFuse expression vector for the antibody of the present invention. Primers were designed to amplify the heavy and light chain variable regions of the antibody, and the light and heavy chain sequences were ligated into pFUSE vectors carrying the IgG1 light and heavy chain constant regions, respectively, using homologous recombination to obtain a recombinant IgG1 expression plasmid pair.

[0106] The 293Fectin transfection reagent was mixed with the eukaryotic antibody expression vector obtained above at a volume-to-mass ratio of 30 μL: 30 μg, and 30 μL of 293Freestyle suspension cells was added. The cells were cultured in a shaker at 37°C at 125 rpm for 48-72 hours. After centrifugation, the supernatant was collected by filtration and the antibody protein was purified by protein A filler column. Specifically, the filler column was soaked in 1M NaOH for 1 hour, rinsed with endotoxin-free water until neutral, and the column material was washed with 10 column volumes of 1% (v / v) Triton×100. Five column volumes of PBS (PBS phosphate buffer, pH 7.2) were used for equilibrium, and the filtered cell supernatant obtained above was loaded onto the column. If necessary, the flow-through was collected. After the column was loaded, 5-10 column volumes of PBS were used for washing. Elution was performed with 5 column volumes of 0.1 M Glycine-HCl, pH 3.0, and the eluate 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-CD7 single-chain antibody (anti-CD7 fully human scFv antibody). Finally, the concentration of the purified antibody was detected according to the instructions of the BCA protein quantification kit.

[0107] Experimental Example 3 Anti-CD7 Antibody Binding Activity Detection to CD7 Antigen Protein (ELISA) The purchased commercial CD7 antigen protein was diluted with PBS to a final concentration of 1.0 μg / mL, and then added to a 96-well ELISA plate at 100 μl per well. Seal with plastic film and incubate overnight at 4°C. The next day, wash the plate twice with a 0.5% Tween-20 PBS solution (phosphate buffer) and add blocking solution [PBS containing 0.01% (v / v) Tween20 and 1% (v / v) BSA] to block at room temperature for 2 hours. Pour out the blocking solution and add 100 μl per well of the purified CD7 single-chain antibody obtained in Experimental Example 2. After incubation at 37°C for 2 hours, wash the plate 5 times with a 0.5% Tween-20 PBS solution (phosphate buffer). HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Sigma) was added and incubated at 37°C for 2 hours. The plate was then washed five times with 0.5% Tween-20 in PBS (phosphate buffered saline). 100 μl of TMB substrate was added to each well and incubated at room temperature for 30 minutes. 100 μl of stop solution (1.0 N HCl) was added to each well. The A450 nm value was read using an ELISA plate reader (iD5, Molecular Device). The results were as follows: Figure 3 As shown in FIG, the purified antibody specifically binds to the CD7 antigen protein at the ELISA level, wherein the human Fc protein is used as a negative control (NC). Figure 3As shown, the twice produced CD7 single-chain antibodies VX-F7-hFc (240621A01) and VX-F7-hFc (220707A02) and the full-range antibody VX-F7F7 IgG1 (240617A01) can all specifically bind to the CD7 protein.

[0108] Experimental Example 4 Flow cytometry (FACS) assay to detect the binding activity of antibodies to CD7-overexpressing Jurkat cells

[0109] Jurkat tumor cells were cultured in T25 cell culture flasks to a size of 3 × 10 6 cells / mL, centrifuged at 800 g for 10 minutes, the culture medium was aspirated, and the cells were washed twice with PBS buffer (purchased from Hyclone). After counting the cells, the cells were diluted to 2×10 6 cells / mL, add 1% goat serum blocking solution, incubate on ice for 30 minutes, and then wash twice with PBS buffer by centrifugation. The collected cells were suspended to 2×10 6 Cells / mL, 100 microliters per well were added to a 96-well FACS reaction plate, 100 microliters per well of the purified CD7 antibody test sample obtained in Experimental Example 2 was added to each well and incubated on ice for 2 hours. Washed twice by centrifugation with FACS buffer, 100 microliters of fluorescent (FITC) labeled secondary antibody (purchased from Biolegend) per well was added and incubated on ice for 1 hour. Washed 3 times by centrifugation with FACS buffer, the cells were suspended with 100 microliters of FACS buffer, and the fluorescence signal was detected by flow cytometry.

[0110] The experimental results are as follows Figure 4 As shown, both the single-chain and full-length antibodies VX-F7 targeting CD7 can specifically recognize and bind to Jurkat cells overexpressing the CD7 antigen.

[0111] Experimental Example 5: Verification of the ability of CD7-targeted CAR to specifically recognize CD7 antigens

[0112] 1. Anti-CD7 CAR lentiviral plasmid construction:

[0113] like Figure 5As shown, the Anti-CD7 CAR lentiviral plasmid includes a signal peptide sequence, a CD7 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 contains CSF2R; the detection tag contains C-myc; the CD7 binding domain contains scFv, which includes the anti-CD7 fully human antibody or antibody fragment; the hinge region and transmembrane domain contain: a hinge region HingeTM and a transmembrane structure CD8, and the functional signal transduction domain contains: CD28, 4-1BB and CD3Zeta connected in sequence.

[0114] Take 1 μg of the anti-CD7 monoclonal strain extract plasmid and lentiviral vector plasmid obtained in Experimental Example 1, add 1 μL SfiI for enzyme digestion, digest in a 50°C water bath for 2 hours, and then recover the enzyme digestion products on gel. Take the anti-human CD7 antibody single-chain sfiI enzyme digestion product and the lentiviral vector plasmid sfiI enzyme digestion product, connect at 16°C for 1 hour, and transfer the ligation product constructed in (2) into 100 μL Escherichia coli DH5α competent cells. Pick out the monoclonal clone and extract the plasmid using the Tiangen Plasmid Mini-Extraction Kit (DP106). The correctness of the sequence is verified by Sanger sequencing, which is the anti-CD7-CAR plasmid.

[0115] 2. Flow cytometry (FACS) detection of anti-CD7-CAR expression

[0116] 2.1 Lentiviral packaging

[0117] One day before transfection, 5 × 10 6 293T cells were seeded in a 6-well plate and mixed with DMEM+10% FBS medium to prepare the complex: (1) 1.5 μg anti-CD7-CAR plasmid, 3 μl LVP2MIX and 6 μl P3000 were diluted in 125 μl Opti-MEM culture medium and gently mixed. (2) 5 μl lipo3000 was diluted in 125 μl Opti-MEM culture medium, gently mixed and incubated at room temperature for 5 minutes. (3) After 5 minutes, they were gently mixed and incubated at room temperature for 20 minutes; the culture medium of the culture plate was aspirated and the cells were washed twice with PBS or serum-free medium (preferably); 3 ml DMEM+10% FBS was added and the complex (total volume 3.6 ml) was added to the culture wells, and the culture plate was shaken to distribute it evenly; the cells were placed in an incubator and incubated for 4 to 6 hours, and then the serum-containing culture medium was replaced to remove the complex (optional); after 24 to 72 hours, the expression of the transgenic gene can be observed. (4) After 72 hours, the virus was collected and filtered with a 0.45 μm filter and the virus titer was detected. The titer of the unconcentrated virus detected by the kit was >10 7 IFU / ml.

[0118] 2.2 Lentiviral infection

[0119] The collected lentivirus was used to infect the host Jurkat cells according to the MOI gradient.

[0120] Centrifuge the host cells at 300g for 5 minutes, resuspend the cells, add a certain volume of packaged virus solution, make up to 1ml with 1640+10% FBS, add infection enhancer polybrene to a final concentration of 8ug / ml, mix well, centrifuge at 800g for 1-2 hours, culture in an incubator, and observe the fluorescence signal preliminarily under a fluorescence microscope for 48-72 hours, and determine the infection efficiency by flow cytometry, such as Figure 6 As shown, anti-CD7-CAR was successfully expressed on the host Jurkat cells with an infection efficiency of 22.2%.

[0121] 2.3 Detection of the ability of CD7-targeted CAR to specifically recognize CD7 antigen using flow cytometry

[0122] (1) Prepare Jurkat cells: centrifuge the cells at 1000 rpm for 5 minutes, discard the supernatant, and resuspend to 1×10 6 / ml; CAR-jurkat cell count after sorting: 2.5×10 5 / ml, a total of 5ml; prepare a 12-well plate, add 1.5×10 5 CAR-jurkat cells and 3 × 10 5 Jurkat cells were added to 8 wells in total; 4.5×10 5 Jurkat cells; add 1640 + 10% FBS to a culture volume of 2 ml and culture in an incubator;

[0123] (2) Transfer the cells in the well plate to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes; discard the supernatant and resuspend in FACS buffer to 1×10 6 / ml density; add 20μl / 10 6 Incubate cells with anti-CD69-APC fluorescent antibodies at 4°C for 1 hour, protected from light. Wash cells twice with FACS buffer and centrifuge at 1000 rpm for 5 minutes. Resuspend in FACS buffer and analyze host cell activation by flow cytometry. Detection of CD69 expression on the Jurkat surface indicates activation of the effector Jurkat cells, indicating that they can recognize the CD7 antigen on the target cell surface. CD69 is the earliest surface antigen expressed by T lymphocytes after activation.

[0124] The experimental results are as follows Figure 7As shown, Jurkat host cells expressing the CAR gene were co-incubated with Jurkat cells expressing the CD7 antigen, and the empty Jurkat+Jurkat group was used as a negative control. Anti-CD7-CAR can activate Jurkat host cells with an activation efficiency of 48.6%, which has development potential.

[0125] In summary, the present invention provides a fully human antibody or antibody fragment against CD7, its chimeric antigen receptor and application, wherein the fully human antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions: HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises three complementarity determining regions: LCDR1, LCDR2 and LCDR3; the nucleic acid encoding the fully human antibody or antibody fragment can be used to prepare host cells, lentiviral plasmids, diagnose tumors and targeted anti-tumor drugs, etc. The fully human antibody or antibody fragment of the present invention can bind to the human CD7 protein with high specificity.

[0126] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. 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 against CD7, characterized in that: 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 amino acid sequence of the HCDR1 is shown in SEQ ID NO: 3; The amino acid sequence of the HCDR2 is shown in SEQ ID NO: 4; The amino acid sequence of the HCDR3 is shown in SEQ ID NO: 5; The light chain variable region includes three complementarity determining regions: LCDR1, LCDR2, and LCDR3; The amino acid sequence of the LCDR1 is shown in SEQ ID NO: 6; The amino acid sequence of the LCDR2 is shown in SEQ ID NO: 7; The amino acid sequence of the LCDR3 is shown in SEQ ID NO:

8.

2. The fully human antibody or antibody fragment against CD7 according to claim 1, wherein The anti-CD7 binding domain of the fully human antibody or antibody fragment is scFv, and the amino acid sequence of the anti-CD7 binding domain is shown in SEQ ID NO:

1.

3. The fully human antibody or antibody fragment against CD7 according to claim 1, wherein The anti-CD7 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:

2.

4. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the fully human antibody or antibody fragment against CD7 according to any one of claims 1 to 3.

5. The chimeric antigen receptor according to claim 4, wherein The chimeric antigen receptor comprises a signal peptide sequence, a CD7 binding domain, a detection tag, a hinge region and a transmembrane domain, and a functional signal transduction domain connected in sequence: The signal peptide sequence comprises CSF2RA, and the amino acid sequence of CSF2RA is shown in SEQ ID NO: 9; The detection tag comprises C-myc, and the amino acid sequence of C-myc is shown in SEQ ID NO: 10; The CD7 binding domain comprises scFv, and the scFv comprises the fully human antibody or antibody fragment against CD7; The hinge region and transmembrane domain comprise: a hinge region Hinge™ and a transmembrane structure CD8, the amino acid sequences of the hinge region Hinge™ and the transmembrane structure CD8 being shown in SEQ ID NO: 11; The functional signaling domain comprises: CD28, 4-1BB and CD3 Zeta connected in sequence, the amino acid sequence of CD28 is shown in SEQ ID NO: 12, the amino acid sequence of 4-1BB is shown in SEQ ID NO: 13, and the amino acid sequence of CD3 Zeta is shown in SEQ ID NO:

14.

6. A host cell, characterized in that The host cell at least comprises the chimeric antigen receptor according to claim 4 or 5, and the host cell is a T cell.

7. A lentiviral plasmid, characterized in that The lentiviral plasmid comprises a nucleotide sequence capable of encoding the fully human antibody or antibody fragment against CD7 according to any one of claims 1 to 3.

8. A use of a fully human antibody or antibody fragment against CD7, characterized in that: The fully human antibody or antibody fragment against CD7 is used to prepare T cells for treating or preventing T cell acute lymphoblastic leukemia, and the T cells at least contain the chimeric antigen receptor according to claim 4 or 5.

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