Double-targeting chimeric antigen receptor specifically combined with CD70 and BCMA and application

Abstract

The invention relates to a CD70 and BCMA targeting dual-targeting chimeric antigen receptor and an application thereof. The CD70 and BCMA targeting dual-targeting chimeric antigen receptor has specific binding ability to CD70 and BCMA. The immune effector cell prepared on the basis of the double-targeting chimeric antigen receptor has the following advantages that the probability that tumor cells escape treatment by losing a single antigen is greatly reduced, and the targeting treatment effect is improved; a CD70 nano antibody sequence is used, so that the length of an insertion fragment is greatly shortened, and the development difficulty of a process end is reduced; and the polypeptide has excellent proliferation capacity, is beneficial to prolonging the in-vivo duration time, and plays a more remarkable anti-tumor role. The invention provides a treatment or improvement approach for diseases related to BCMA-CD70 expression.

Description

Technical Field

[0001] This invention relates to the field of biological immunotherapy technology, and more specifically, to a dual-targeting chimeric antigen receptor that specifically binds to CD70 and BCMA and its applications. Background Technology

[0002] BCMA, also known as TNFRSF17, is a B cell surface molecule belonging to the tumor necrosis factor receptor (TNFR) family and is a type I transmembrane receptor. BCMA is mainly expressed on the surface of mature B cells and plasma cells, and can bind to either BAFF or APRIL, two ligands that activate B cells, playing an important role in B cell maturation and autoimmune responses. BCMA is the most selectively expressed receptor on multiple myeloma cells, and its expression level gradually increases with B cell differentiation, also gradually increasing throughout the course of multiple myeloma.

[0003] CD70 is a type II transmembrane protein belonging to the tumor necrosis factor family. Under normal conditions, it is mainly expressed in activated T cells, B cells, and mature dendritic cells. Activation of its receptor CD27 can promote the activation, proliferation, and differentiation of T cells and B cells, regulating the immune response. Pathologically, CD70 can be expressed in both solid tumors and hematological malignancies, with 42%–63% of tumor samples from multiple myeloma patients being CD70 positive. Analysis of relapsed primary multiple myeloma samples confirmed that 19 out of 28 samples contained CD138+ plasma cells expressing CD70. In five patients who simultaneously measured BCMA and CD70, the antigen densities of the two were similar. CD70 is significantly upregulated in high-risk multiple myeloma patients, suggesting that it may be a potential immunotherapeutic target for treating MM, warranting further preclinical and clinical research.

[0004] High relapse rates are a major challenge and focus in the treatment of multiple myeloma. Although BCMA expression levels are similar across different stages of multiple myeloma (from untreated to relapse), making it an effective therapeutic target throughout the disease course, patients receiving BCMA-targeted CAR-T therapy, while achieving high complete remission and objective response rates, still face an increased risk of relapse with prolonged treatment. Due to tumor heterogeneity and the antigen escape problem caused by BCMA shedding, CAR-T products targeting multiple tumor antigens can effectively reduce relapse caused by BCMA loss. CD70 is highly expressed in high-risk multiple myeloma patients, with expression levels comparable to BCMA. As a target independent of BCMA, its potential for combined use is noteworthy. Furthermore, the BAFF / APRIL / BCMA functional axis and the CD70 / CD27 functional axis play important roles in lupus erythematosus, rheumatoid arthritis, and other autoimmune diseases through different mechanisms of action. Therefore, BCMA / CD70 CAR-T cell products can not only be used to treat relapsed multiple myeloma patients who have previously received BCMA-related treatments, but also reduce tumor relapse due to single antigen loss in high-risk multiple myeloma patients with BCMA-positive expression. Furthermore, BCMA / CD70 CAR-T also has the potential to treat autoimmune diseases, further enhancing its market value.

[0005] Chimeric antigen receptor T cell (CAR-T) therapy is a novel immunotherapy targeting specific antigens on the surface of tumor cells. VHH antibodies, also known as nanobodies, are naturally occurring antibodies lacking the light chain, found in alpaca peripheral blood. These antibodies contain only a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, making them the smallest known unit capable of binding to target antigens. Compared to traditional artificially modified scFvs, CAR-T therapy offers advantages such as smaller molecular weight, easier expression, higher specificity, higher affinity, weaker immunogenicity in humans, and a shorter development cycle. Combining the advantages of immunotherapy and VHH antibodies can lead to the development of highly effective CAR-T therapies.

[0006] There are currently no reports of dual-target chimeric antigen receptors that specifically bind to human BCMA and CD70 as described in this application. Summary of the Invention

[0007] The present invention provides a dual-targeting chimeric antigen receptor that targets CD70 and BCMA, wherein the dual-targeting chimeric antigen receptor comprises an anti-CD70 VHH antibody and an anti-BCMA antibody or an antigen-binding fragment thereof.

[0008] In one or more embodiments, the anti-CD70 VHH antibody comprises HCDR1-3 as shown in SEQ ID NO:11-13, and the anti-BCMA antibody or its antigen-binding fragment comprises HCDR1-3 as shown in SEQ ID NO:15-17 and LCDR1-3 as shown in SEQ ID NO:18-20; preferably, the anti-CD70 VHH antibody has an amino acid sequence as shown in SEQ ID NO:14, and the anti-BCMA antibody has an amino acid sequence as shown in SEQ ID NO:21.

[0009] In one or more embodiments, the dual-targeting chimeric antigen receptor comprises a first CAR and a second CAR. The first CAR comprises an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular co-stimulatory domain targeting CD70. The second CAR comprises an extracellular antigen-binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signal transduction domain targeting BCMA. In one example, a self-cleaving peptide is included between the first CAR and the second CAR. Preferably, the self-cleaving peptide is selected from P2A, T2A, E2A, and F2A.

[0010] In one or more embodiments, the hinge region is selected from the CD8α hinge region, CD8 hinge region, CD28 hinge region, IgD hinge region, IgG1 Fc CH2CH3 hinge region, and IgG4 Fc CH2CH3 hinge region; the transmembrane domain is selected from the transmembrane domains of the following proteins: CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, and the α, β, or ζ chain of the T cell receptor; the intracellular co-stimulatory domain is derived from 4-1BB, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, C The intracellular signal transduction domains are derived from at least one of the following intracellular domains: D134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, and 41BBL; wherein the intracellular signal transduction domains are derived from at least one of the following intracellular domains: CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0011] In one or more embodiments, the dual-target chimeric antigen receptor has an amino acid sequence as described in any of SEQ ID NO:1-5.

[0012] The present invention also provides a nucleic acid molecule having a sequence selected from any of the following:

[0013] (1) The coding sequence of the dual-targeting chimeric antigen receptor described in any of the embodiments herein;

[0014] (2)(1) complementary sequences.

[0015] The present invention also provides a nucleic acid construct comprising the nucleic acid molecule described in any of the embodiments herein.

[0016] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.

[0017] The present invention also provides a host cell selected from:

[0018] (1) Expressing and / or secreting the dual-targeting chimeric antigen receptor as described in any of the embodiments herein;

[0019] (2) Contains the nucleic acid molecule described in any of the embodiments herein; and / or

[0020] (3) Contains the nucleic acid constructs described in any of the embodiments herein.

[0021] In one or more embodiments, the host cell is an immune effector cell. Preferably, the immune effector cell is a T cell.

[0022] The present invention also provides a pharmaceutical composition comprising a chimeric antigen receptor as described in any embodiment herein, a nucleic acid molecule as described in any embodiment herein, a nucleic acid construct as described in any embodiment herein, or a host cell as described in any embodiment herein, and pharmaceutically acceptable excipients.

[0023] The present invention also provides the use of the chimeric antigen receptor described in any embodiment of the present invention, the nucleic acid molecule described in any embodiment of the present invention, the nucleic acid construct described in any embodiment of the present invention, or the host cell described in any embodiment of the present invention in the preparation of activated immune cells, or in the preparation of medicaments for the prevention or treatment of diseases or conditions related to CD70 or BCMA expression.

[0024] In one or more embodiments, the disease or condition is selected from one or more of the following: multiple myeloma, acute myeloid leukemia, non-Hodgkin's lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, renal cell carcinoma, pancreatic cancer, breast cancer, and glioblastoma.

[0025] The advantages of this invention are as follows: Based on scFv antibodies that specifically recognize human BCMA and VHH antibodies that recognize human CD70, this invention constructs dual-target BCMA / CD70 CAR-T cells using Daulstim CAR, Tandem CAR, and Loop CAR structures. This dual-target design allows for the simultaneous recognition of two different tumor antigens, significantly reducing the probability of tumor cells escaping treatment by losing a single antigen and improving the therapeutic effect of targeting BCMA-CD70. The increased length of the insert fragment in the dual-target CAR-T structure vector can pose challenges to its preparation; however, this invention uses a CD70 nanobody sequence to significantly shorten the insert fragment length, reducing the development difficulty in the manufacturing process. This invention uses human scFv instead of mouse scFv, reducing the immunogenicity of the CAR-T product. CAR-T cells constructed based on the dual-target CAR structure of this invention exhibit excellent proliferative capacity, which helps prolong the in vivo duration of CAR-T cell immunotherapy and exert a more significant anti-tumor effect. In summary, this invention uses these two antibodies to prepare immune effector cells targeting BCMA-CD70, providing a therapeutic or ameliorative approach for diseases related to BCMA-CD70 expression. Attached Figure Description

[0026] Figure 1 Schematic diagrams of different BCMA / CD70 CAR structures.

[0027] Figure 2 The positive rate of CAR expression in BCMA / CD70 CAR-T cells with different structures.

[0028] Figure 3 CD107a expression in BCMA / CD70 CAR-T cells with different structures.

[0029] Figure 4 Results of CAR+ cell targeting proliferation of BCMA / CD70 CAR-T cells with different structures. ****, P<0.001.

[0030] Figure 5 Results of CD3+ cell targeting proliferation of BCMA / CD70 CAR-T cells with different structures. **, P<0.05; ***, P<0.01; ****, P<0.001.

[0031] Figure 6 The results of experiments showing the killing effect of BCMA / CD70 CAR-T cells with different structures on target cells. Detailed Implementation

[0032] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

[0033] Example 1:

[0034] 1. Preparation of retroviral stock solution containing anti-human CD70 / BCMA chimeric antigen receptor element

[0035] (1) Preparation of CAR targeting human CD70 / BCMA antigen

[0036] BCMA scFv antibodies and human CD70 VHH antibodies were synthesized or cloned. These were then used to construct dual-target BCMA / CD70 CARs using Daulstim CAR, Tandem CAR, and Loop CAR structures. Various structures were designed based on different sequences of BCMA scFv and CD70 VHH, including the five chimeric antigen receptors described in this paper, named DaulstimCAR, Tandem CAR1, Tandem CAR2, Loop CAR1, and Loop CAR4. Their amino acid sequences are shown in SEQ ID NO:1 to SEQ ID NO:5, and their nucleotide sequences are shown in SEQ ID NO:6 to SEQ ID NO:10, respectively. Retroviral plasmids expressing the chimeric antigen receptors of Daulstim CAR, Tandem CAR1, Tandem CAR2, Loop CAR1, and Loop CAR4 clones were constructed using the reverse transcription vector MSGV as the backbone. Select the clones that were correctly sequenced, inoculate the bacterial culture into 200 ml of 2YT medium, shake the culture overnight, and complete the large plasmid extraction according to the NucleoBond Xtra Maxi EF kit instructions.

[0037] (2) Retroviral packaging

[0038] The retrovirus was packaged using the cationic polymer PEI as follows: 36 μl of PEI and the retrovirus packaging plasmid (6 μg of main viral plasmid, 3.8 μg of Gag-pol, and 1.5 μg of vsvg) were diluted separately with 600 μl of serum-free DMEM; then, PEI / DMEM was added to the plasmid / DMEM mixture, vortexed to mix, and incubated at room temperature for 15 minutes; the plasmid-PEI complex was added to pre-coated 293T cells. The medium was changed 16 h after transfection, and the first viral supernatant was collected after 48 h, and the second viral supernatant was collected after 72 h. The supernatant was filtered through a 0.45 μm filter, aliquoted into 1.5 mL centrifuge tubes (1 mL / tube), and stored at -80 °C for later use.

[0039] 2. Preparation of BCMA / CD70 CAR-T cells and determination of CAR positivity rate

[0040] (1) PBMC isolation and activation

[0041] After receiving a PBMC and verifying the patient's individual identification code, resuscitation was initiated. The cell density was adjusted to 1×10⁶ cells / mL using X-VIVO complete culture medium. 6 / mL. After 2 hours of resuscitation, PBMCs were gently pipetted, filtered through a 70μm cell sieve, and transferred to a 50ml centrifuge tube. The cells were centrifuged at 500g for 5 minutes at room temperature, and the supernatant was discarded. The cells were resuspended in an appropriate amount of DPBS and mixed well. 100μl of the resuspended cells were then used for NC-200 cell counting to calculate viability and CD3+ cell count. The desired volume of cells was then centrifuged at 500g for 5 minutes at room temperature, and the supernatant was discarded for sorting. The amount of magnetic beads used was calculated based on a 1:1 ratio of CD3 / CD28 beads to CD3+ cells: Bead quantity = [CD3+ cell count / 4 × 10⁻⁶]. 5 **μl.** Cleaning the magnetic beads: Take a sterile flow cytometry tube, add 2ml of DPBS and magnetic beads, and let it stand on a magnetic rack for 1 min. Discard the supernatant. Remove the flow cytometry tube from the magnetic rack, resuspend the cells in an equal volume of DPBS or X-VIVO15, add the magnetic beads and cells to the cell suspension, mix, and incubate on a rotary mixer. Incubate at room temperature for 30 min. After incubation, gently transfer the cells to a sterile flow cytometry tube, and rinse the 15ml centrifuge tube with 1ml of DPBS, adding the rinse solution to the same flow cytometry tube. Transfer the sterile flow cytometry tube to a magnetic rack, let it stand for 1 min, and discard any unabsorbed liquid. Remove the sterile flow cytometry tube from the magnetic rack, resuspend the cells in 1ml of CAR-T medium, and rinse the tube wall twice with CAR-T medium. Collect all the CAR-T medium and transfer it to the same centrifuge tube. Adjust the cell density to 1×10⁻⁶ cells using CAR-T medium. 6 Add IL-2 to a final concentration of 300 IU / ml and incubate at 37°C in a 5% CO2 incubator for one day.

[0042] (2) Virus stock solution infection and culture

[0043] The activated T cells were adjusted to 5 × 10 5 Add 1 ml of T cells and 1 ml of viral stock solution to each well of a 24-well plate, centrifuge at 2500 rpm for 1.5 h at 32°C. Discard the supernatant and add 1 ml of CAR-T medium (containing 300 IU / ml IL-2) to each well. Incubate the plate at 37°C in a 5% CO2 incubator. 24 h post-infection, transfer to 6-well plates. Observe cell density daily and supplement with T cell culture medium containing 300 IU / ml IL-2 as needed to maintain T cell density at 5 × 10⁶ cells / ml.5 Approximately 1 / ml, to promote cell proliferation.

[0044] (3) CAR positivity rate detection

[0045] The CAR positivity rate of retrovirally infected T lymphocytes was measured 72 hours after viral infection. NT values ​​of 1×10⁻⁶ cells were collected from the chimeric antigen receptor group targeting DaulstimCAR, TandemCAR1, TandemCAR2, LoopCAR1, and LoopCAR4 structures, and from the negative, uninfected control group. 6 Cells were centrifuged to remove the culture medium, washed once with 500 μl PBS, and resuspended in 100 μl of PBS in a flow cytometry tube (BD). FITC-Labeled Human BCMA / TNFRSF17 Protein (1:100) and Dylight 405 Goat anti-Alpaca IgG VHH (1:100) were added, and the cells were incubated at °C for 30 minutes. After washing with 500 μl PBS, the cells were resuspended in 200 μl PBS and analyzed by flow cytometry. The CAR-T positivity rate results are shown below. Figure 2 As shown.

[0046] 3. Functional analysis of anti-human BCMA / CD70 CAR-T cells

[0047] (1) Analysis of CD107a expression in anti-human BCMA / CD70 CAR-T cells

[0048] CD107a assay is a method for assessing the cytotoxic function of CAR-T cells. When CAR-T cells interact with target cells, degranulation of CAR-T cells leads to increased expression of CD107a on the cell membrane surface. Detecting the expression level of CD107a can effectively reflect the cytotoxic activity of CAR-T cells. CAR-T cells containing different CAR structures were compared with target cells (BCMA / CD70-positive multiple myeloma cell line U266) at a 1:1 effector-to-target ratio (both effector and target cells were 1×10⁻⁶). 5 (Number of samples) were added to Anti-Human CD107a (LAMP-1) PE flow cytometry antibody (1:200) and incubated at 37°C in a 5% CO2 incubator for 4 hours. Flow cytometry analysis was then performed to determine the proportion of CD107a-expressing cells among CD3+ cells in each group. The degranulation response of CAR-T cells after stimulation by target cells was evaluated. The flow cytometry analysis results of CD107a expression are shown below. Figure 3 As shown.

[0049] (2) Anti-human BCMA / CD70 CAR-T cell targeted proliferation ability

[0050] Targeted proliferation assays are an important method for evaluating CAR-T cell activity. This assay observes the proliferation of CAR-T cells with different CAR structures when co-cultured with target cells, revealing their effects on specific tumor cells. Counting and flow cytometry analysis provide data support for optimizing CAR-T cell therapy. In this experiment, CAR-T cells containing different CAR structures were mixed with target cells (BCMA / CD70-positive multiple myeloma cell line U266) at a 1:5 effector-target ratio in 24-well plates and cultured at 37°C in a 5% CO2 incubator. Samples were taken every 1-2 days for counting, and a portion of the cells was analyzed by flow cytometry using antibodies. The remaining cells were replenished with target cells for continued culture. The fold increase of CD3+ T cells and CAR-T cells was calculated based on the flow cytometry and cell counting results. The results are shown below. Figure 4 , Figure 5 As shown.

[0051] (3) Anti-human BCMA / CD70 CAR-T cell cytotoxicity experiment

[0052] The CAR-T cell cytotoxicity assay aimed to evaluate the in vitro function of CAR-T cells by detecting their cytotoxic effect on target cells. Different effector-to-target ratios (based on 1×10⁴ CAR-T cells, effector-to-target ratios of 1:1, 1:5, and 1:10) were used. CAR-T cells containing different CAR structures were co-cultured with target cells (U266-LUC-GFP BCMA / CD70-positive target cells stably expressing firefly luciferase). A positive control containing only target cells was also included. After overnight incubation at 37°C, 100 μl of luciferase reaction substrate was added to the culture system, fluorescence values ​​were detected, and the cytotoxicity was calculated using the following formula: Cytotoxicity = [(fluorescence value of positive control well - fluorescence value of experimental well) / fluorescence value of positive control well] × 100%. The experimental results are as follows: Figure 6 As shown.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0054] The sequence of this article is as follows:

[0055] 1. BCMA-CD70-LoopCAR1 amino acid sequence:

[0056] MALPVTALLLPLALLLHAARPEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPPGYTFGQGTKLEIKGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAVGWFRQAPGKEREGVSCISSSSDGSTYYVDSVLGRFTISRNNAENTVYLQMNSLKPEDTAVYYCSTDVLTSCRSDRYLEVWGQGTLVTVSGGGGGSQVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLMVYAVNWFDPWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0057] 2. Amino acid sequence of CD70 - BCMA - LoopCAR4:

[0058] MALPVTALLLPLALLLHAARPEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPPGYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAVGWFRQAPGKEREGVSCISSSSDGSTYYVDSVLGRFTISRNNAENTVYLQMNSLKPEDTAVYYCSTDVLTSCRSDRYLEVWGQGTLVTVSGGGGGSQVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLMVYAVNWFDPWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0059] 3. Amino acid sequence of CD70-BCMA-TanCAR1:

[0060] MALPVTALLLPLALLLHAARPEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPPGYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLMVYAVNWFDPWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAVGWFRQAPGKEREGVSCISSSSDGSTYYVDSVLGRFTISRNNAENTVYLQMNSLKPEDTAVYYCSTDVLTSCRSDRYLEVWGQGTLVTVSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0061] 4. Amino acid sequence of CD70 - BCMA - TanCAR2:

[0062] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAVGWFRQAPGKEREGVSCISSSSDGSTYYVDSVLGRFTISRNNAENTVYLQMNSLKPEDTAVYYCSTDVLTSCRSDRYLEVWGQGTLVTVSGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPPGYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLMVYAVNWFDPWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0063] 5. Amino acid sequence of CD70 - BCMA - DualstimCAR:

[0064] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAVGWFRQAPGKEREGVSCISSSSDGSTYYVDSVLGRFTISRNNAENTVYLQMNSLKPEDTAVYYCSTDVLTSCRSDRYLEVWGQGTLVTVSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPPGYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLMVYAVNWFDPWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0065] 6. Amino acid sequence of BCMA - CD70 - LoopCAR1:

[0066]

[0067] 7. CD70-BCMA-LoopCAR4 amino acid sequence:

[0068]

[0069] 8. CD70-BCMA-TanCAR1 amino acid sequence:

[0070]

[0071] 9. CD70-BCMA-TanCAR2 amino acid sequence:

[0072]

[0073] 10. CD70-BCMA-DualstimCAR amino acid sequence:

[0074]

[0075] 11. Amino acid sequence of CD70 VHH HCDR1

[0076] GFTLDYYA

[0077] 12. Amino acid sequence of CD70 VHH HCDR2

[0078] ISSSSDG

[0079] 13. Amino acid sequence of CD70 VHH HCDR3

[0080] CSTDVLTSCRSDRYLEV

[0081] 14. Amino acid sequence of CD70 VHH

[0082] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAVGWFRQAPGKEREGVSCISSSSDGSTYYVDSVLGRFTISRNNAENTVYLQMNSLKPEDTAVYYCSTDVLTSCRSDRYLEVWGQGTLVTVSG

[0083] 15. Amino acid sequence of BCMA HCDR1

[0084] GFTFSSYA

[0085] 16. Amino acid sequence of BCMA HCDR2

[0086] ISGSGGST

[0087] 17. Amino acid sequence of BCMA HCDR3

[0088] AKVLMVYAVNWFDP

[0089] 18. Amino acid sequence of BCMA LCDR1

[0090] QSVSSSY

[0091] 19. Amino acid sequence of BCMA LCDR2

[0092] GAS

[0093] 20. Amino acid sequence of BCMA LCDR3

[0094] QHYGSSPPGYT

[0095] 21. Amino acid sequence of BCMA scFv

[0096] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPPGYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLMVYAVNWFDPWGQGTLVTVSA

Claims

1. A dual-targeting chimeric antigen receptor targeting CD70 and BCMA, characterized in that, The dual-target chimeric antigen receptor comprises an anti-CD70 VHH antibody and an anti-BCMA antibody or its antigen-binding fragment.

2. The dual-targeting chimeric antigen receptor according to claim 1, characterized in that, The anti-CD70 VHH antibody comprises HCDR1-3 as shown in SEQ ID NO:11-13, and the anti-BCMA antibody or its antigen-binding fragment comprises HCDR1-3 as shown in SEQ ID NO:15-17 and LCDR1-3 as shown in SEQ ID NO:18-20; preferably, the anti-CD70 VHH antibody has the amino acid sequence shown in SEQ ID NO:14, and the anti-BCMA antibody has the amino acid sequence shown in SEQ ID NO:

21.

3. The dual-targeting chimeric antigen receptor according to claim 1, characterized in that, The dual-targeting chimeric antigen receptor comprises a first CAR and a second CAR. The first CAR comprises an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular co-stimulatory domain targeting CD70. The second CAR comprises an extracellular antigen-binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signal transduction domain targeting BCMA. Preferably, a self-cleaving peptide is present between the first CAR and the second CAR. More preferably, the self-cleaving peptide is selected from P2A, T2A, E2A, and F2A.

4. The dual-targeting chimeric antigen receptor according to claim 3, characterized in that, The hinge region is selected from the CD8α hinge region, CD8 hinge region, CD28 hinge region, IgD hinge region, IgG1 Fc CH2CH3 hinge region and IgG4 Fc CH2CH3 hinge region; The transmembrane domain is selected from the transmembrane domains of the following proteins: CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, and the α, β, or ζ chain of the T cell receptor; The intracellular co-stimulatory domain is derived from at least one of the following intracellular domains: 4-1BB, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, and 41BBL. The intracellular signal transduction domain is derived from at least one of the intracellular domains CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

5. The dual-targeting chimeric antigen receptor according to claim 1, characterized in that, The dual-target chimeric antigen receptor has an amino acid sequence as described in any of SEQ ID NO:1-5.

6. A nucleic acid molecule having a sequence selected from any of the following: (1) The coding sequence of the dual-targeting chimeric antigen receptor according to any one of claims 1-5; (2)(1) complementary sequences.

7. A nucleic acid construct comprising the nucleic acid molecule of claim 6, preferably, the nucleic acid construct being a cloning vector, an expression vector, or an integration vector.

8. A host cell, selected from: (1) Expressing and / or secreting the dual-targeting chimeric antigen receptor as described in any one of claims 1-5; (2) Contains the nucleic acid molecule as described in claim 6; and / or (3) Contains the nucleic acid construct according to claim 7. Preferably, the host cell is an immune effector cell, more preferably a T cell.

9. A pharmaceutical composition comprising the dual-targeting chimeric antigen receptor of any one of claims 1-5, the nucleic acid molecule of claim 6, the nucleic acid construct of claim 7, or the host cell of claim 8, and pharmaceutically acceptable excipients.

10. Use of the chimeric antigen receptor of any one of claims 1-5, the nucleic acid molecule of claim 6, the nucleic acid construct of claim 7, or the host cell of claim 8 in the preparation of activated immune cells, or in the preparation of a medicament for the prevention or treatment of diseases or conditions related to CD70 or BCMA expression. Preferably, the disease or condition is selected from one or more of the following: multiple myeloma, acute myeloid leukemia, non-Hodgkin's lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, renal cell carcinoma, pancreatic cancer, breast cancer, and glioblastoma.

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