Preparation method for and use of car-γδt cell for immunotherapy of t cell acute lymphoblastic leukemia

Abstract

A preparation method for and a use of a chimeric antigen receptor (CAR)-γδT cell for immunotherapy of T cell acute lymphoblastic leukemia, relating to the field of biomedicine. The provided anti-CD5 nanobody can specifically bind to a CD5 antigen, and has good affinity. By using the anti-CD5 nanobody as an antigen-binding domain to construct a CAR, the prepared CAR-γδT cell exhibits significant killing activity against CD5-positive tumor cell lines, such as T cell acute lymphoblastic leukemia cells.

Description

A method for preparing CAR-γδT cells for immunotherapy of T-cell acute lymphoblastic leukemia and its application Technical Field

[0001] This disclosure relates to the biomedical field, and in particular to a method for preparing CAR-γδT cells for immunotherapy of T-cell acute lymphoblastic leukemia and its application. Background Technology

[0002] CAR-T therapy has achieved significant success in clinical trials, and several CAR-T cell products have received regulatory approval. T-cell acute lymphoblastic leukemia (T-ALL), a highly aggressive malignant tumor arising from the transformation of T-cell progenitor cells, presents unique challenges for treatment, particularly due to the lack of specific target antigens that can effectively distinguish between normal and malignant T cells. T-ALL leukemia cells transformed from T cells exhibit surface markers similar to normal T cells, further complicating the selection of target antigens.

[0003] Studies have shown that CD5 is one of the ideal potential targets for CAR therapy because it is a common biomarker of malignant T cells in T-ALL. Furthermore, CD5 is not expressed on hematopoietic stem cells, thus reducing the risk of extratumor effects during targeted therapy. In addition, preclinical studies have shown that CD5-CAR-T cells can preferentially kill malignant T cells without harming the normal T cell population. Currently, there is a lack of highly specific and high-affinity anti-CD5 nanobodies on the market.

[0004] Despite its promising prospects, the naturally expressed CD5 on CAR-T cells can lead to cannibalism, thus reducing therapeutic efficacy. In this context, knocking out the CD5 gene in CAR-T / γδT cells using CRISPR-Cas9 technology avoids this cannibalism, thereby significantly enhancing the therapeutic effect of CD5-targeted CAR-T / γδT therapy.

[0005] T-ALL, a type of hematologic malignancy originating from T cells, faces significant challenges in obtaining healthy T cells when using autologous T cells to prepare CAR-T cell therapy. Therefore, CAR-γδT cell therapy has attracted widespread attention, offering hope for overcoming this obstacle. Unlike traditional αβT cells, γδT cells do not rely on classical human leukocyte antigen (HLA) molecules for recognition; their perception of infection or cancer depends more on variations prevalent across individuals. Therefore, γδT cells can be collected from healthy donors and used for xenograft transplantation, reducing the risk of graft-versus-host disease (GVHD). Preliminary studies have shown that CAR-modified γδT cells exhibit good cytotoxicity against various malignancies, including hematologic malignancies and solid tumors. However, optimizing γδT cell CAR design, enhancing in vivo expansion and persistence, and reducing off-target effects remain key areas of current research.

[0006] Therefore, this invention is proposed. Summary of the Invention

[0007] To address the aforementioned issues, the present disclosure aims to provide, for example, a method for preparing CAR-γδT cells for immunotherapy of T-cell acute lymphoblastic leukemia and its application.

[0008] To achieve the above objectives, this disclosure provides the following technical solution:

[0009] Embodiments of this disclosure provide an anti-CD5 nanobody, the nanobody comprising CDR1, CDR2 and CDR3 in the heavy chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:4 or 9.

[0010] Embodiments of this disclosure provide an antibody or an antigen-binding fragment thereof comprising the nanobody described in the foregoing embodiments.

[0011] Embodiments of this disclosure provide an isolated nucleic acid that encodes a nanobody or antibody or antigen-binding fragment thereof as described in the foregoing embodiments.

[0012] Embodiments of this disclosure provide a recombinant vector containing the isolated nucleic acid as described in the foregoing embodiments.

[0013] Embodiments of this disclosure provide a host cell containing the recombinant vector as described in the foregoing embodiments.

[0014] The embodiments of this disclosure provide a method for preparing an antibody or an antigen-binding fragment thereof, which includes: culturing host cells as described in the foregoing embodiments.

[0015] Embodiments of this disclosure provide a chimeric antigen receptor, wherein the antigen-binding domain of the chimeric antigen receptor includes a nanobody or an antibody or antigen-binding fragment thereof as described in the foregoing embodiments.

[0016] Embodiments of this disclosure provide a CAR-γδT cell that includes the chimeric antigen receptor as described in the foregoing embodiments;

[0017] The embodiments of this disclosure provide a cell injection solution whose active ingredients include: anti-CD5 nanobodies as described in the foregoing embodiments, or antibodies or their antigen-binding fragments as described in the foregoing embodiments, or isolated nucleic acids as described in the foregoing embodiments, or recombinant vectors as described in the foregoing embodiments, or host cells as described in the foregoing embodiments, or chimeric antigen receptors as described in the foregoing embodiments, or CAR-γδT cells as described in the foregoing embodiments.

[0018] The embodiments of this disclosure provide a kit comprising: an anti-CD5 nanobody as described in the foregoing embodiments, or an antibody or its antigen-binding fragment as described in the foregoing embodiments, or an isolated nucleic acid as described in the foregoing embodiments, or a recombinant vector as described in the foregoing embodiments, or a host cell as described in the foregoing embodiments, or a chimeric antigen receptor as described in the foregoing embodiments, or a CAR-γδT cell as described in the foregoing embodiments.

[0019] The embodiments of this disclosure also provide the use of anti-CD5 nanobodies as described in the foregoing embodiments, or antibodies or their antigen-binding fragments as described in the foregoing embodiments, or isolated nucleic acids as described in the foregoing embodiments, or recombinant vectors as described in the foregoing embodiments, or host cells as described in the foregoing embodiments, or chimeric antigen receptors as described in the foregoing embodiments, or CAR-γδT cells as described in the foregoing embodiments in the prevention or treatment of tumors or in the preparation of products for the prevention or treatment of tumors. Beneficial effects:

[0020] Embodiments of this disclosure provide an anti-CD5 nanobody that can specifically bind to the CD5 antigen and has good affinity.

[0021] The anti-CD5 nanobody was used as the antigen-binding domain to construct a chimeric antigen receptor, and the resulting CAR-γδT cells showed significant killing activity against CD5-positive tumor cell lines, such as T-cell acute lymphoblastic leukemia cells. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this disclosure and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 illustrates the use of ELISA to detect the reactivity of CD5-specific nanobodies with antigens in an embodiment of this disclosure.

[0024] Figure 2 shows the binding effect of anti-CD5 nanobody to T-cell acute lymphoblastic leukemia cell line (CCRF-CEM) analyzed by flow cytometry (FACS) in an embodiment of this disclosure.

[0025] Figure 3 shows the flow cytometry assessment of CD5-CAR-γδT. CD5- Cell transfection efficiency;

[0026] Figure 4 shows the detection of allogeneic CD5-CAR-γδT. CD5- Graph showing the killing efficiency of target cells;

[0027] Figure 5 shows the evaluation of CD5-CAR-γδT using the NCG mouse xenograft model. CD5- Cellular antitumor activity in vivo. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of this disclosure, but not all embodiments.

[0029] Therefore, the following detailed description of the embodiments of this disclosure provided in the accompanying drawings is not intended to limit the scope of the claimed disclosure, but merely to illustrate selected embodiments of the disclosure. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of this disclosure.

[0030] The embodiments of this disclosure are described in detail below with reference to the accompanying drawings; however, this disclosure may be implemented in a variety of different ways as defined and covered by the claims.

[0031] Embodiments of this disclosure provide an anti-CD5 nanobody, wherein the heavy chain variable region of the nanobody includes CDR1, CDR2 and CDR3, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID No:1-3 or SEQ ID No:6-8, respectively.

[0032] In some embodiments, the nanobody further includes a backbone region.

[0033] In this invention, the "backbone region" and the "FR region" refer to the region in the heavy chain variable region of the antibody, excluding the CDR region. The heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4), wherein the heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 backbone regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs with FRs (from the amino terminus to the carboxyl terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0034] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:4 or 9.

[0035] Embodiments of this disclosure provide an antibody or an antigen-binding fragment thereof, comprising the nanobody described in any of the foregoing embodiments.

[0036] In some embodiments, the antibody or its antigen-binding fragment further includes a constant region.

[0037] In some embodiments, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0038] In some embodiments, the species source of the constant region is cattle, horses, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0039] In some embodiments, the antibody is selected from any one of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, murine antibodies, chimeric antibodies, and full-length antibodies.

[0040] In this document, "antigen-binding fragment" refers to a portion of a complete antibody that specifically binds to the antigen to which the complete antibody is bound. Those skilled in the art will readily understand from the description of this invention that antigen-binding fragments can be prepared by methods known in the art, such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds, or by recombinant genetics techniques or by automated peptide synthesizers (such as Applied BioSystems' automated peptide synthesizers).

[0041] In some embodiments, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, and scFv.

[0042] Embodiments of this disclosure provide an isolated nucleic acid that encodes a nanobody or antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments.

[0043] In some embodiments, the separated verified sequence is as shown in SEQ ID NO:5 or 10.

[0044] Embodiments of this disclosure provide a recombinant vector containing isolated nucleic acids as described in any of the foregoing embodiments.

[0045] The recombinant vector is an expression vector or cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct. This construct can introduce the target DNA fragment directly or indirectly (e.g., packaged as a virus) into host cells through transformation, transfection, or transduction to express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can ligate the target DNA fragment into the plasmid circle. Another type of vector is a viral vector, which can ligate and package the target DNA fragment into the viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0046] Embodiments of this disclosure provide a host cell containing the recombinant vector as described in any of the foregoing embodiments.

[0047] Specifically, the host cell includes at least one of prokaryotic host cells, eukaryotic host cells, and bacteriophages. The prokaryotic host cell can be *Escherichia coli*, *Streptomyces*, or *Bacillus subtilis*, etc. The eukaryotic host cell can be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6 cells, *Saccharomyces cerevisiae*, *Pichia pastoris*, *Saccharomyces hansenii*, *Candida*, some insect cells, and plant cells. The 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins and are well known to those skilled in the art.

[0048] The embodiments of this disclosure provide a method for preparing an antibody or an antigen-binding fragment thereof, which includes: culturing host cells as described in any of the foregoing embodiments.

[0049] Specifically, the present invention does not specifically limit the culture conditions of the host cells. Based on conventional technical knowledge, culture conditions that enable the host cells to express and produce antibodies or their antigen-binding fragments can be obtained.

[0050] Embodiments of this disclosure provide a chimeric antigen receptor, wherein the antigen-binding domain of the chimeric antigen receptor includes a nanobody or an antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments.

[0051] In some embodiments, the chimeric antigen receptor further includes any one or more of a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain.

[0052] In some embodiments, the signal transduction structural domain includes CD3ζ.

[0053] In some embodiments, the signal transduction domain further includes a 4-1BB intracellular region.

[0054] Embodiments of this disclosure provide a CAR-γδT cell that includes a chimeric antigen receptor as described in any of the foregoing embodiments.

[0055] In some embodiments, the CAR-γδT cells include universal allogeneic CAR-γδT cells.

[0056] Autologous CAR-T cell therapy has advantages such as not causing immune rejection and being able to persist in the body for a relatively long time. However, autologous CAR-T cell therapy also has some limitations, such as high production costs, low T cell count in T-ALL patients who have undergone multiple lines of treatment or reduced T cell quality due to impaired T cell function after chemotherapy, inability to distinguish between normal T cells and tumor cells, and long manufacturing cycles that cause patients to miss the optimal treatment time. With the rapid development of universal allogeneic cell therapy technology, the development of this universal cell therapy can overcome most of the shortcomings of autologous CAR-T cell therapy, such as standardized processes for large-scale and industrialized manufacturing, reducing costs, and enabling the preparation of large numbers of CAR-T / γδT cells from a single donor.

[0057] The embodiments of this disclosure provide a cell injection solution whose active ingredients include: anti-CD5 nanobody as described in any of the foregoing embodiments, or antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments, or isolated nucleic acid as described in any of the foregoing embodiments, or recombinant vector as described in any of the foregoing embodiments, or host cell as described in any of the foregoing embodiments, or chimeric antigen receptor as described in any of the foregoing embodiments, or CAR-γδT cell as described in any of the foregoing embodiments.

[0058] The embodiments of this disclosure provide a kit comprising: an anti-CD5 nanobody as described in any of the foregoing embodiments, or an antibody or its antigen-binding fragment as described in any of the foregoing embodiments, or an isolated nucleic acid as described in any of the foregoing embodiments, or a recombinant vector as described in any of the foregoing embodiments, or a host cell as described in any of the foregoing embodiments, or a chimeric antigen receptor as described in any of the foregoing embodiments, or a CAR-γδT cell as described in any of the foregoing embodiments.

[0059] The embodiments of this disclosure provide the use of anti-CD5 nanobodies as described in any of the foregoing embodiments, or antibodies or their antigen-binding fragments as described in any of the foregoing embodiments, or isolated nucleic acids as described in any of the foregoing embodiments, or recombinant vectors as described in any of the foregoing embodiments, or host cells as described in any of the foregoing embodiments, or chimeric antigen receptors as described in any of the foregoing embodiments, or CAR-γδT cells as described in any of the foregoing embodiments in the prevention or treatment of tumors or in the preparation of products for the prevention or treatment of tumors.

[0060] In some embodiments, the product includes at least one of immune cells, reagents, kits, drugs, and drug compositions.

[0061] In some embodiments, the tumor includes T-cell acute lymphoblastic leukemia.

[0062] The term "treatment" in this invention includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0063] For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0064] To further illustrate this disclosure, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides an anti-CD5 nanobody, CAR-γδT cells, and related applications provided by this disclosure, but these should not be construed as limiting the scope of protection of this disclosure.

[0065] Example 1: Preparation of recombinant CD5 protein

[0066] The purchased human pGEM-CD5 plasmid was used as a template for PCR amplification. The full-length CD5 sequence was obtained by agarose gel electrophoresis. Then, using the pcDNA3.1 vector as a backbone, the extracellular CD5 sequence was cloned into the pcDNA3.1 expression vector carrying a His tag at the C-terminus. Subsequently, the expression was transiently transfected with 293F cells using FreeStyle... TM Cells were cultured in serum-free medium (Thermo Fisher) in shake flasks for 5-7 days, and the cell pellet was collected and purified using His tagging to obtain recombinant CD5 protein.

[0067] Example 2: Construction, panning, and preliminary ELISA screening of phage nanobody libraries

[0068] (1) Bactrian camel immunization

[0069] Two mg of purified CD5 extracellular domain recombinant protein was added to 2 mL of Freund's complete adjuvant and emulsified thoroughly using an emulsifier. Multiple subcutaneous injections were administered into the neck of Bactrian camels for immunization. Subsequent immunizations (2 mg protein) were administered every two weeks using Freund's incomplete adjuvant, for a total of four immunizations. Peripheral blood was collected after the final immunization to determine the titer. One week after the initial immunization, peripheral blood was collected from Bactrian camels to separate lymphocytes.

[0070] (2) Construction of nanobody libraries

[0071] Once the camels reached a certain immune titer, they underwent a final shock immunization. Seven days later, 200 mL of peripheral blood was collected using a blood collection bag for lymphocyte isolation. The isolated lymphocytes were then subjected to RNA extraction according to the Promega RNA extraction kit. Immediately after RNA extraction, cDNA was reverse transcribed using the TaKaRa reverse transcription kit, followed by nested PCR amplification of the VHH gene. The amplified VHH gene was inserted into the pMECS phage display vector and electroporated into TG1 competent cells. The electroporated culture was serially diluted (10-fold) using LB / Amp-GLU medium, and then 10... -4 10 -5 10 -6 10 -7 100 μL of the dilution buffer was spread onto LB / Amp-GLU plates and incubated upside down at 37°C for 8 hours. The colony counts at different dilutions were then used to calculate the antibody library capacity, which was 6.56 × 10⁻⁶. 9 Simultaneously, 50 colonies of similar morphology and size were randomly selected and cultured for 4-6 hours, followed by bacterial PCR to determine the library positivity rate, i.e., the insertion rate of the library reached 97%.

[0072] (3) Screening of CD5 nanobodies

[0073] First, the preparation, concentration, and phage library rescue of helper phages were performed. The panning steps for the nanobody phage library are as follows: ① Antigen coating: After diluting the CD5-His recombinant protein with PBS, 20 μg of each protein (the antigen coating amounts for the subsequent two rounds of panning were 10 μg / well and 5 μg / well, respectively) were coated into 96-well ELISA plates and incubated overnight at 4°C; ② Washing: After overnight coating, the liquid in the wells was discarded, and each well was washed 5 times with 200 μL PBST; ③ Blocking: 200 μL of 5% skim milk powder was added to each well and the plate was incubated at 37°C for 1 hour; ④ Washing: The liquid in the wells was discarded, and each well was washed 3 times with 200 μL PBST; ⑤ Incubation of recombinant phages: The recombinant phages were diluted with 5% skim milk powder to a concentration of 5 × 10⁻⁶. 11 Add 100 μL of pfu / mL to each well and incubate at room temperature for 2 h; ⑥ Wash: Discard the liquid in the well and wash each well 15 times with 200 μL PBST. Add 100 μL of freshly prepared 0.1 M triethylamine to each well, incubate at room temperature for 10 min, transfer the eluent to a 1.5 mL centrifuge tube, and quickly add an equal volume of 1 M Tris-HCl (pH = 7.4) for neutralization; ⑦ Recombinant phage titer determination: Collect the neutralized phage solution and determine the phage titer; Infect 2 mL of TG1 in the logarithmic growth phase with the remaining phage solution, incubate at 37℃ for 30 min; Add 8 mL of 2×YT / Amp GLU medium, and incubate at 37℃ and 220 rpm until the logarithmic growth phase; ⑧ Rescue: Add 8 mL of 2×YT ampicillin-resistant medium, add 4% glucose, and incubate at 37℃ and 220 rpm; ⑨ Phage concentration; ⑩ Repeat steps ①-⑨ above for the second and third rounds of screening.

[0074] (4) Detection of specific recombinant phage enrichment

[0075] Antigen Coating: Dilute the two antigens with PBS and coat each well with 400 ng of the solution into a 96-well ELISA plate. Incubate overnight at 4°C. Washing: After overnight coating, discard the liquid in the wells and wash each well three times with 200 μL PBST. Blocking: Add 200 μL of 5% skim milk powder to each well and incubate at 37°C for 1 h. Washing: Discard the liquid in the wells and wash each well three times with 200 μL PBST. Incubation of Recombinant Phage: Dilute the phage concentrate (1:10) and add 100 μL to each well. Incubate at 37°C for 1 h. Washing: Discard the liquid in the wells and wash each well three times with 200 μL PBST. Secondary Antibody: Dilute HRP-labeled mouse anti-M13 secondary antibody 1:2000, add 100 μL / well, and incubate at 37°C for 1 h. Washing: Discard the liquid in the wells and wash each well three times with 200 μL PBST. Color development: Add 100 μL of TMB colorimetric solution to each well and incubate at room temperature in the dark for 10-15 min. Termination and reading: After color development, add 50 μL of 2M H₂SO₄ to each well to terminate the reaction; read the absorbance at 450 nm. Analyze the data.

[0076] (5) Sequencing analysis of specific nanobodies

[0077] Clones with a negative value more than three times the negative value were identified as positive by ELISA test results and sent for bacterial culture sequencing and comparative analysis. Finally, two CD5 nanobody sequences were obtained. The nanobody sequences are shown in Table 1 below.

[0078] Table 1. Amino acid and nucleic acid sequences of anti-CD5 nanobodies

[0079] Example 3: Expression, purification, and reactivity with antigen of CD5-specific nanobodies

[0080] A Nanobody-hFc fusion protein expression platform was constructed based on the pcDNA3.1 eukaryotic expression vector, and CD5-specific nanobody sequences were cloned into the eukaryotic cell expression vector. The constructed expression vectors were expressed and purified using the HEK293T eukaryotic protein expression system. SDS-PAGE results showed that high-purity Nanobody-hFc fusion protein was obtained after affinity chromatography purification, with band sizes all around 55 kDa.

[0081] To assess the reactivity of the recombinant chimeric antibody with the antigen, 200 ng / well of recombinant CD5 protein was pre-coated onto an ELISA plate, incubated overnight at 4°C, and then the plate was blocked. Different amounts of recombinant antibody (dilution: 10) were then added. 2 ~10 -5 Add secondary antibody (μg / mL), wash, develop color, terminate the reaction, and measure the optical density (OD) at 450 nm using a microplate reader. 450The binding affinity was determined by measuring the binding properties using a four-parameter nonlinear regression curve. The results showed that both CD5 nanobodies exhibited high specificity binding to the recombinant CD5 protein (Figure 1).

[0082] Example 4: Analysis of antibody binding to endogenously expressed CD5 in cells

[0083] Flow cytometry analysis was performed on the CD5-positive human T-cell acute lymphoblastic leukemia cell line CCRF-CEM. The results showed that the CD5-positive CCRF-CEM cells were stained almost 100% positively by the obtained CD5 nanobodies. This indicates that the CD5 nanobodies can specifically recognize endogenously expressed CD5 molecules (Figure 2).

[0084] Example 5: CD5 Nanobody Affinity Assay

[0085] The affinity of nanobodies was determined by surface plasmon resonance, and the highest binding affinity was found to be Nb-62 (7.36 × 10⁻⁶). -11 ), followed by Nb-1 (1.96×10), -10 Furthermore, both antibodies exhibited relatively slow dissociation rates. In summary, Biacore data indicates that both nanobodies are high-affinity nanobodies; specific detection data are shown in Table 2.

[0086] Table 2 Summary of Affinity Data for Nanobodies

[0087] Example 6: CAR Structure Design and Lentiviral Packaging

[0088] According to Example 5, the monoclonal antibody sequence Nb-62 with higher affinity was selected to construct CD5-CAR, with the following structure: CD8αsignal peptide-CD5scFv-CD8αhinge-CD28αTm-4-1BB-CD3ζ-P2A-EGFP. HEK293T cells were used as cells for lentivirus packaging. Lentiviral packaging was performed using a three-plasmid packaging system (psPAX2, pMD2.G, CAR-γδT vector). After 48 hours, the supernatant viral solution was collected, concentrated by ultracentrifugation, pre-coated with RetroNectin protein, and added to infect γδT cells. After 48 hours of infection, the transfection efficiency of CAR-γδT cells was assessed by flow cytometry and found to be above 80.00% (Figure 3).

[0089] Using the Cas9 / RNP electroporation method, sgRNA targeting CD5 and Cas9 protein were electroporated into CAR-γδT cells to prepare CD5 knockout CAR-γδT cells (CD5-CAR-γδT cells). CD5-After 72 hours of amplification, the expression of CD5 in CAR-γδT cells was detected by flow cytometry. The flow cytometry results showed that 90% of CAR-γδT cells had the CD5 gene knocked out.

[0090] Example 7 Allogeneic CD5-CAR-γδT CD5- Evaluation of the in vitro killing effect of cells on target cells

[0091] To evaluate CAR-γδT cells (CD5-CAR-γδT cells constructed in Example 6) CD5- The function of cells to deliver CD5-CAR-γδT CD5- Cells were co-cultured with the T-ALL antigen-positive cell line CCRF-CEM-mCherry.ffLuc cells expressing luciferase to determine CD5-CAR-γδT CD5- The ability of cells to lyse T-ALL tumor cells using an antigen-specific mechanism was investigated. Untreated γδT cells (NT-γδT cells) were used as a control and seeded in 96-well plates at effector-to-target ratios of 1.25:1, 2.5:1, 5:1, and 10:1, respectively. No IL-2 was added to the culture system. After 24 hours of co-culture, luciferase levels were measured using a multifunctional enzyme-linked immunosorbent assay (ELISA) system to evaluate CD5-CAR-γδT cells. CD5- The killing effect of CAR-γδT cells on T-ALL antigen-positive cell lines was investigated. Results showed that CAR-γδT cells killed target cells expressing CD5 antigen-positive cells in a dose-dependent manner, indicating that CD5-CAR-γδT cells... CD5- It exhibits good killing activity against T-ALL antigen-positive cell lines (Figure 4).

[0092] Example 8: Anti-tumor experiment using a xenograft mouse model

[0093] Xenograft mouse model was used to evaluate CD5-targeting CAR-γδT cells (CD5-CAR-γδT cells constructed in Example 6). CD5- The in vivo antitumor activity of NCG cells was assessed by intravenous injection of 5 × 10⁻⁶ cells into the tail vein of NCG mice. 6 A mouse model of T-cell acute lymphoblastic leukemia was established using CCRF-CEM-mCherry.ffLuc cells to validate CD5-CAR-γδT cells. CD5- The in vivo effects were observed. On day 5 post-vaccination, NCG mice were randomly divided into 4 groups of 5 mice each, and each group was injected with 1×10⁻⁶ NCG via the tail vein. 7 Mock T cells, 1×10 7 NT-γδT cells, 1×10 7One CD5-CAR-γδT mouse was administered, and a control group was established that received PBS injections. The growth and survival of the mice were observed daily, and their lifespan was recorded. The results showed that CD5-CAR-γδT... CD5- The treatment group showed a significant delay in tumor progression and a marked increase in survival time in tumor-bearing mice (Figure 5).

[0094] Although the above embodiments have provided a detailed description of this disclosure, they are only some embodiments of this disclosure, not all embodiments. People can obtain other embodiments based on this disclosure without creative effort, and these embodiments all fall within the protection scope of this disclosure. Industrial applicability

[0095] In summary, this disclosure provides an anti-CD5 nanobody that specifically binds to the CD5 antigen with good affinity. Using this anti-CD5 nanobody as the antigen-binding domain, a chimeric antigen receptor was constructed to prepare the obtained CD5-CAR-γδT. CD5- The cells exhibit significant killing activity against CD5-positive tumor cell lines, such as T-cell acute lymphoblastic leukemia cells.

Claims

1. An anti-CD5 nanobody, characterized in that, The nanobody includes CDR1, CDR2 and CDR3 in the heavy chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:4 or 9.

2. The nanobody according to claim 1, characterized in that, The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID No:1-3 or SEQ ID No:6-8, respectively. Optionally, the nanobody further includes a backbone region; Optionally, the amino acid sequence of the heavy chain variable region of the nanobody is as shown in SEQ ID No:4 or 9.

3. An antibody or its antigen-binding fragment, characterized in that, It contains the nanobody as described in claim 1 or 2.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody or its antigen-binding fragment further includes a constant region; Optionally, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; Optionally, the species source of the constant region is cattle, horses, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; Optionally, the antibody is selected from any one of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, murine antibodies, chimeric antibodies, and full-length antibodies; Optionally, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, and scFv.

5. An isolated nucleic acid, characterized in that, It encodes the nanobody as described in claim 1 or 2, or the antibody or its antigen-binding fragment as described in claim 3 or 4.

6. A recombinant vector, characterized in that, It contains the isolated nucleic acid as described in claim 5.

7. A host cell, characterized in that, It contains the recombinant vector as described in claim 6.

8. A method for preparing an antibody or its antigen-binding fragment, characterized in that, It includes: Culture the host cells as described in claim 7.

9. A chimeric antigen receptor, characterized in that, The antigen-binding domain of the chimeric antigen receptor includes the nanobody as described in claim 1 or 2, or the antibody or its antigen-binding fragment as described in claim 3 or 4.

10. The chimeric antigen receptor according to claim 9, characterized in that, The chimeric antigen receptor further includes any one or more of the following: signal peptide, hinge region, transmembrane region, and signal transduction domain. Optionally, the signal transduction structure domain includes CD3ζ; Optionally, the signal transduction domain further includes a 4-1BB intracellular region.

11. A CAR-γδT cell, characterized in that, It includes the chimeric antigen receptor as described in claim 9 or 10.

12. A cell injection solution, characterized in that, Its active ingredients include: the anti-CD5 nanobody of claim 1 or 2, or the antibody or its antigen-binding fragment of claim 3 or 4, or the isolated nucleic acid of claim 5, or the recombinant vector of claim 6, or the host cell of claim 7, or the chimeric antigen receptor of claim 9 or 10, or the CAR-γδT cell of claim 11.

13. A reagent kit, characterized in that, It includes: the anti-CD5 nanobody of claim 1 or 2, or the antibody or its antigen-binding fragment of claim 3 or 4, or the isolated nucleic acid of claim 5, or the recombinant vector of claim 6, or the host cell of claim 7, or the chimeric antigen receptor of claim 9 or 10, or the CAR-γδT cell of claim 11.

14. The use of the anti-CD5 nanobody as described in claim 1 or 2, or the antibody or its antigen-binding fragment as described in claim 3 or 4, or the isolated nucleic acid as described in claim 5, or the recombinant vector as described in claim 6, or the host cell as described in claim 7, or the chimeric antigen receptor as described in claim 9 or 10, or the CAR-γδT cell as described in claim 11 in the prevention or treatment of tumors or in the preparation of products for the prevention or treatment of tumors.

15. The application according to claim 14, characterized in that, The products include at least one of the following: immune cells, reagents, kits, drugs, and drug compositions.

16. The application according to claim 13 or 14, characterized in that, The tumors include T-cell acute lymphoblastic leukemia.

Images