Baffr-targeting chimeric antigen receptor, car-t cell, and use

Abstract

Disclosed are a BAFFR-targeting chimeric antigen receptor, a CAR-T cell, and a use. The BAFFR-targeting chimeric antigen receptor comprises a signal peptide region, a BAFFR-targeting antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and a signal transduction domain, which are connected in order from the amino terminal to the carboxyl terminal, wherein the costimulatory domain comprises a CD28 costimulatory domain and a 4-1BB costimulatory domain which are connected, and the signal transduction domain is ITAM1 of CD3ζ. The CAR-T cell is constructed by means of BAFFR. By utilizing an antigen-antibody binding mechanism, autoimmune B cells or malignant proliferating B cells in patients with autoimmune diseases or some tumors can be effectively consumed, providing a new choice for the treatment of human autoimmune related diseases and some hematologic tumors.

Description

A method targeting BAFFR chimeric antigen receptor, CAR-T cells and applications

[0001] Cross-reference to related applications

[0002] This patent document claims priority and benefit to Chinese Patent Application No. 202311042556.2, filed on August 18, 2023, entitled "A Targeting BAFFR Chimeric Antigen Receptor, CAR-T Cells, and Application". The entire contents of the aforementioned patent application are incorporated herein by reference as a part of the disclosure of this patent document. Technical Field

[0003] This invention relates to the fields of molecular biology and medical technology, and in particular to a method for targeting BAFFR chimeric antigen receptors, CAR-T cells, and their applications. Background Technology

[0004] Chimeric Antigen Receptors (CARs) T-cell therapy (CAR-T) mainly utilizes genetic engineering to construct chimeric antigen receptor expression vectors and transfect T cells, enabling the T cell surface to express a chimeric antibody that can recognize target cells. By specifically recognizing the antigens on the target cell surface, it achieves the effect of specifically killing the target cells.

[0005] Compared to other immunocellular therapies, CAR-T cell technology offers several advantages. First, CAR-T cells bring themselves closer to target cells, directly and precisely attacking them. Second, because CAR-T cell killing of target cells is independent of MHC and does not require antigen presentation mechanisms for recognition, it overcomes immune escape mediated by MHC downregulation and reduced antigen presentation, resulting in more effective target cell killing. Third, the construction of the CAR gene is based on target antigens expressed by target cells; CAR-T can utilize both protein antigens and glycolipid non-protein antigens, expanding the range of target antigens. Finally, the therapeutic effect is more durable. Gene sequences that promote T cell proliferation and activation can be added to the CAR-T structure, ensuring that T cells can continue to proliferate after entering the body. CAR-T cells possess immune memory function and can survive in the body for a long time.

[0006] In recent years, this therapy has been widely used in cancer treatment, offering numerous advantages unmatched by other therapies and considered one of the most promising treatments for cancer. However, CAR-T immunotherapy has faced significant limitations in other diseases, such as autoimmune diseases, primarily due to the lack of new targets. Studies have shown that BAFF is involved in the progression of various autoimmune diseases, with significantly elevated expression levels in many. For example, BAFF is persistently highly expressed in patients with systemic lupus erythematosus, and the concentration of BAFF in the patient's serum is positively correlated with the titer of anti-dsDNA antibodies. Furthermore, BAFF expression levels are also significantly elevated in the serum of patients with Sjögren's syndrome, rheumatoid arthritis, and multiple sclerosis. BAFF can bind to three membrane receptors of the TNFR family on the surface of B cells: BAFFR, TACI, and BCMA. However, the binding of BAFF to BAFFR plays the most crucial role in the maturation of peripheral B cells. Therefore, constructing CAR-T cells targeting BAFFR would be a significant breakthrough in the treatment of autoimmune diseases and / or hematologic malignancies.

[0007] Summary of the Invention

[0008] In view of the aforementioned deficiencies or shortcomings in existing technologies, there is a need to provide a BAFFR-targeting chimeric antigen receptor, CAR-T cells, and applications. By combining phage display technology with a high-throughput expression platform, nanobodies targeting BAFFR were screened, exhibiting advantages such as high affinity, high stability, small molecular weight, low immunogenicity, and strong penetration. CAR-T cells were constructed based on the obtained BAFFR antibody sequences, specifically targeting autoimmune B cells, avoiding the immunodeficiency and infection risks associated with total B cell deletion. Furthermore, total B cell deletion may reduce patient responsiveness to vaccines, such as COVID-19 vaccines, while CAR-T cells, targeting only autoreactive B cells, can effectively avoid the low vaccine response caused by total B cell deletion, providing a new treatment option for human autoimmune diseases.

[0009] The present invention provides a BAFFR-targeting chimeric antigen receptor comprising a signal peptide region, an antigen-binding domain targeting BAFFR, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain connected sequentially from the amino terminus to the carboxyl terminus. The co-stimulatory domain includes a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain connected together, and the signal transduction domain is CD3ζITAM1.

[0010] Furthermore, the amino acid sequence of the signal peptide region is shown in SEQ ID No: 1.

[0011] Furthermore, the antigen-binding domain targeting BAFFR is a nanobody targeting BAFFR, and the amino acid sequence of the nanobody targeting BAFFR is shown in SEQ ID No: 2, or in SEQ ID No: 3, or in SEQ ID No: 4.

[0012] Furthermore, the hinge region is a CD8 hinge domain with an amino acid sequence as shown in SEQ ID No: 5;

[0013] And / or, the transmembrane domain is a CD28 transmembrane domain with an amino acid sequence as shown in SEQ ID No: 6;

[0014] And / or, the amino acid sequence of the CD28 co-stimulatory domain is shown in SEQ ID No: 7;

[0015] And / or, the amino acid sequence of the 4-1BB co-stimulatory domain is as shown in SEQ ID No: 8;

[0016] And / or, the amino acid sequence of ITM1 of CD3ζ is shown in SEQ ID No: 9.

[0017] Additionally, the present invention provides an isolated nucleic acid comprising a nucleotide sequence for expressing the above-described target BAFFR chimeric antigen receptor.

[0018] Furthermore, the isolated nucleic acid includes a nucleic acid fragment with a nucleotide sequence as shown in SEQ ID No: 10.

[0019] In addition, the present invention also provides a recombinant vector comprising the isolated nucleic acid described above.

[0020] In addition, the present invention also provides a CAR-T cell containing the isolated nucleic acid described above, or the CAR-T cell being a cell transformed by the recombinant vector described above.

[0021] In addition, the present invention also provides the application of targeting BAFFR chimeric antigen receptor in pharmaceuticals, including the application of targeting BAFFR chimeric antigen receptor as described above, isolated nucleic acid as described above, recombinant vector as described above, or CAR-T cells as described above in the preparation of pharmaceuticals for treating immune-related diseases.

[0022] Additionally, the present invention provides a pharmaceutical composition comprising an expression vector for expressing the above-described target BAFFR chimeric antigen receptor or the above-described CAR-T cells.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] (1) Currently, B cell deletion using CD19 or CD20 antibodies is the main treatment for autoimmune diseases, but the treatment effect is not ideal. On the one hand, CD19 or CD20 antibodies target all immature and mature B cells, easily causing immunodeficiency and even infection in patients. On the other hand, CD19 or CD20 antibodies may reduce the patient's responsiveness to vaccines, such as the COVID-19 vaccine. This invention constructs CAR-T cells using BAFFR, which can target autoreactive B cells, especially plasma cells, instead of deleting all B cells, thus preserving the patient's basic autoimmune capacity and vaccine response capacity.

[0025] (2) Autoantibodies exist in patients with autoimmune diseases, such as dsDNA antibodies in patients with systemic lupus erythematosus, MOG antibodies in patients with multiple sclerosis, and AQP4 antibodies in patients with neuromyelitis optica. CD20 monoclonal antibodies cannot effectively reduce the concentration of these pathogenic autoantibodies in patients (30-70%). This invention constructs CAR-T cells using BAFFR, which mainly target plasma cells that produce autoantibodies, and can effectively reduce the concentration of autoantibodies in patients.

[0026] (3) This application uses phage display technology combined with a high-throughput expression platform to screen out nanobodies targeting BAFFR, which have advantages such as high affinity, high stability, small molecular weight, low immunogenicity and strong penetration.

[0027] It should be understood that the description in the Summary of the Invention is not intended to limit the key or essential features of the embodiments of the present invention, nor is it intended to restrict the scope of the invention. Other features of the invention will become readily apparent from the following description. Attached Figure Description

[0028] Other features, objects, and advantages of the invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0029] Figure 1 is a schematic diagram of the binding activity of anti-BAFFR antibody to BAFFR measured by ELISA;

[0030] Figure 2 is a schematic diagram of the structure of CAR-T cells; where: BAFFR-CAR represents a polypeptide targeting the BAFFR chimeric antigen receptor, which includes the CD8a signal peptide region, anti-BAFFR scFv, CD8 hinge region, CD28 transmembrane region, CD28 costimulatory domain, 4-1BB costimulatory domain and CD3ζ signal transduction domain from the N-terminus to the C-terminus.

[0031] Figure 3 is a schematic diagram of the proportion of CAR plasmid transfected T cells detected by flow cytometry.

[0032] Figure 4 shows a comparison of the killing effects of BAFFR-CAR-T and control BAFFR-CAR-T on Nalm-6-Luc cells at different effector-target ratios in the embodiments.

[0033] Figure 5 shows a comparison of the secretion levels of IL-2 and IFN-γ between BAFFR-CAR-T and the control BAFFR-CAR-T at different effector-target ratios in the examples. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0035] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0036] Embodiments of the present invention provide a BAFFR-targeting chimeric antigen receptor, comprising a signal peptide region, an antigen-binding domain targeting BAFFR, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain connected sequentially from the amino terminus to the carboxyl terminus. The co-stimulatory domain includes a connected CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, and the signal transduction domain is CD3ζITAM1.

[0037] In some embodiments, the hinge region is a hinge region selected from at least one of the following proteins: CD28, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

[0038] In some embodiments, the transmembrane domain is a transmembrane region selected from at least one of the following proteins: α, β, or ζ chains of T cell receptors, CD28, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4. IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DN AM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and NKG2C.

[0039] In a preferred embodiment, the amino acid sequence of the signal peptide region is shown in SEQ ID No: 1.

[0040] SEQ ID No: 1 (amino acid sequence of the signal peptide region) is:

[0041] In a preferred embodiment, the antigen-binding domain targeting BAFFR is a BAFFR-targeting nanobody, and the amino acid sequence of the BAFFR-targeting nanobody is shown in SEQ ID No: 2, or in SEQ ID No: 3, or in SEQ ID No: 4.

[0042] Among them, SEQ ID No: 2 (the amino acid sequence of NB467-7) is:

[0043] SEQ ID No: 3 (amino acid sequence of NB467-71) is:

[0044] SEQ ID No: 4 (amino acid sequence of NB467-88) is:

[0045] In a preferred embodiment, the hinge region is a CD8 hinge domain with an amino acid sequence as shown in SEQ ID No: 5;

[0046] And / or, the transmembrane domain is the CD28 transmembrane domain with an amino acid sequence as shown in SEQ ID No: 6;

[0047] And / or, the amino acid sequence of the CD28 co-stimulatory domain is shown in SEQ ID No: 7;

[0048] And / or, the amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID No: 8;

[0049] And / or, the amino acid sequence of CD3ζ's ITAM1 is shown in SEQ ID No: 9.

[0050] Among them, SEQ ID No: 5 (CD8 hinge structure domain) is:

[0051] SEQ ID No: 6 (CD28 transmembrane domain) is:

[0052] SEQ ID No: 7 (amino acid sequence of the CD28 co-stimulatory domain) is:

[0053] SEQ ID No: 8 (amino acid sequence of the 4-1BB co-stimulatory domain) is:

[0054] SEQ ID No: 9 (the amino acid sequence of ITM1 of CD3ζ) is:

[0055] Additionally, embodiments of the present invention also provide an isolated nucleic acid comprising a nucleotide sequence for expressing the above-described target BAFFR chimeric antigen receptor.

[0056] In a preferred embodiment, the isolated nucleic acid comprises a nucleic acid fragment with a nucleotide sequence as shown in SEQ ID No: 10.

[0057] Among them, SEQ ID No: 10 is:

[0058] Furthermore, embodiments of the present invention also provide a recombinant vector comprising the isolated nucleic acid described above.

[0059] In addition, embodiments of the present invention also provide a CAR-T cell containing the isolated nucleic acid described above, or the CAR-T cell being a cell transformed by the recombinant vector described above.

[0060] Furthermore, embodiments of the present invention also provide the application of targeting BAFFR chimeric antigen receptors in pharmaceuticals, including the application of targeting BAFFR chimeric antigen receptors as described above, isolated nucleic acids as described above, recombinant vectors as described above, or CAR-T cells as described above in the preparation of pharmaceuticals for treating immune-related diseases.

[0061] Immune-related diseases include autoimmune diseases and cancer.

[0062] Among them, autoimmune diseases include neuroimmune diseases such as multiple sclerosis, neuromyelitis optica, and myasthenia gravis, as well as patients with other autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and Sjögren's syndrome.

[0063] The tumors are hematologic malignancies, such as B-cell lymphomas, including Hodgkin lymphoma and nodular lymphocytic Hodgkin lymphoma, as well as non-Hodgkin lymphomas, such as diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma / chronic lymphocytic leukemia, and mantle cell lymphoma (MCL).

[0064] Additionally, embodiments of the present invention also provide a pharmaceutical composition comprising an expression vector for expressing the above-described target BAFFR chimeric antigen receptor or the above-described CAR-T cells.

[0065] Example 1: Obtaining anti-BAFFR nanobodies via phage display

[0066] In this embodiment, multiple antibody-encoding fragments were obtained by immunizing alpacas with human BAFFR as an antigen. Candidate antibody clones were then screened using a phage library and a mammalian expression system. Specifically, the screening process for anti-BAFFR nanobodies involves the following steps.

[0067] a. Preparation of antigens

[0068] Based on the amino acid and nucleotide sequences of human BAFFR, an antigen that can effectively induce alpacas to produce specific antibodies against human BAFFR was analyzed and designed. Human IgG1 Fc was then linked to its C-terminus to obtain the modified antigen, denoted as "human BAFFR hFC antigen". Its amino acid sequence is shown in SEQ ID No: 11 and its nucleotide sequence is shown in SEQ ID No: 12.

[0069] Among them, SEQ ID No: 11 (amino acid sequence of human BAFFR hFC antigen) is:

[0070] SEQ ID No: 12 (nucleotide sequence of human BAFFR hFC antigen) is:

[0071] b. Immunized alpacas

[0072] The human BAFFR hFC antigen obtained in step a was thoroughly mixed with an equal volume of Freund's adjuvant and used for subcutaneous injection into alpacas.

[0073] Specifically, on day 1, alpacas were primed with an emulsion mixture of 500 μg of human BAFFR hFC antigen and an equal volume of Freund's complete adjuvant, and booster immunizations were administered on days 21, 42, 63, and 84 with an emulsion mixture of 250 μg of human BAFFR hFC antigen and an equal volume of Freund's complete adjuvant. Seven days after the first four immunizations, 10 ml of peripheral blood was collected from each alpaca to detect the anti-BAFFR serum titer using ELISA.

[0074] The specific steps for ELISA detection are as follows: Dilute human BAFFR his antigen to 2 μg / mL with 0.05M carbonate buffer (pH=9.6) and add 100 μL / well to the culture plate. Incubate the culture plate overnight at 4℃. Discard the coating solution and wash 3 times with PBST. Add 300 μL of 5% skim milk to each well and block at 37℃ for 1 h. Wash 3 times with PBST buffer. Add 100 μL / well of serum diluent (serial dilution starting from 1:2000) and incubate at 37℃ for 45 min. Wash 5 times with PBST, add 100 μL of goat anti-alpaca IgG (H+L)HRP (Chengdu Apak Biotechnology Co., Ltd., Cat#: S001H, Chengdu Apak Biotechnology Co., Ltd., diluted 1:1W with PBS) to each well, and incubate at 37℃ for 45 min. Wash 5 times with PBST. Add TMB colorimetric solution (100 μL / well) for color development and incubate at 37 °C for 5 min. Add stop solution to terminate the reaction (50 μL / well) and measure the optical density at 450 nm.

[0075] One week after the fifth immunization, 50 mL of peripheral blood was collected from alpacas and mononuclear cells were isolated.

[0076] c. Library Construction

[0077] RNA was extracted from the PBMCs obtained in step b, and the target gene fragment was obtained by nested PCR after reverse transcription. The target gene fragment was cloned into a eukaryotic expression vector, and the obtained expression vector was transformed into competent cells to construct the BAFFR-VHH phage display library. The specific steps are as follows.

[0078] Using the PBMC cells obtained in step b as templates, total RNA was extracted from the cells using RNAiso Plus, and PrimeScript was used to further analyze the RNA. TM The II 1st Strand cDNA Synthesis Kit reverse transcribes RNA into cDNA. The target fragment is obtained by nested PCR. The amplification system for the target fragment is shown in Table 1 below, and the amplification program is shown in Table 2 below.

[0079] Table 1. Nested PCR one-round reaction system

[0080] Table 2. Nested PCR two-round reaction system

[0081] The amplified nucleotide fragment encoding alpaca VHH was cloned into the eukaryotic expression vector pComb3XSS (Chengdu Apak Biotechnology Co., Ltd.). The resulting recombinant vector was transformed into TG1 competent cells by electroporation to obtain the VHH phage display library. To further confirm the successful construction of the BAFFR-VHH phage display library, the library was cultured on 2-YT-A plates, and 48 clones were selected from the formed colonies for sequencing.

[0082] Sequencing results showed a 100% insertion success rate for the clones. Based on the sequencing analysis of transformant number, insertion rate, and diversity, the calculated library size was 1.92 × 10⁻⁶. 9 This indicates that the obtained BAFFR-VHH phage display library has good library capacity and diversity.

[0083] d. Obtaining nanobodies

[0084] The phage display library obtained in step c was used for panning to select positive clones. The coding sequence of the selected VHH antibody was fused with the coding sequence of Human IgG1 Fc and constructed into the pTT5 (Chengdu Apak Biotechnology Co., Ltd.) expression vector for transfection into eukaryotic cells for expression and purification. Finally, anti-human BAFFR nanobody was obtained. The specific method is as follows.

[0085] Remove the SA-magnetic beads (Suzhou Navitas Technology Co., Ltd., MPHTSA-300), wash twice with PBS, and set aside. Dilute the Bio-Human BAFFR-his (Acro, BAR-H82E3) antigen with PBS to a final concentration of 5 μg / ml, and add 2×10 11 Phage libraries were incubated at 37°C for 1 hour. The incubated mixture was added to magnetic bead tubes and shaken at 4°C for 45 minutes. The supernatant was aspirated under magnetic rack conditions and washed three times with PBST and twice with PBS. 800 μL of Gly-HCl elution buffer was added and incubated at 37°C for 8 minutes to elute specifically bound phages. The elution buffer was transferred to a 1.5 mL sterile centrifuge tube and quickly neutralized with 160 μL of Tris-HCl neutralization buffer. 10 μL of the buffer was serially diluted, the titer was determined, and the panning recovery rate was calculated. The remaining eluent was mixed and amplified and purified for the next round of affinity panning.

[0086] After two rounds of screening, monoclonal phage supernatant was identified by ELISA after the second round of screening. Bio-Human BAFFR-his antigen was immobilized in 96-well ELISA plates at a concentration of 2 μg / ml * 100 μl and incubated overnight at 4°C. The coating solution was discarded, and the plates were washed three times with PBST. 300 μL of 5% skim milk was added to each well, and the plates were blocked at 37°C for 1 h. The plates were washed three times with PBST buffer, and 100 μL of a two-fold diluted phage supernatant was added to each well, incubating at 37°C for 45 min. The plates were washed five times with PBST, and 100 μL of mouse anti-M13 antibody HRP (Chengdu Apak Biotechnology Co., Ltd.) was added to each well, incubating at 37°C for 45 min. The plates were washed five times with PBST. 100 μL of TMB chromogenic solution was added to each well, and the plates were incubated at 37°C for 5 min.

[0087] The reaction was terminated by adding 50 μL / well of 1M HCl stop solution, and the optical density was detected at 450 nm. Positive clones (OD450>1) were screened based on the detection results. Human IgG1 Fc fused with the positive clones was constructed into the pTT5 plasmid, and the corresponding plasmid was transfected into HEK293T mammalian cells. Expression was performed for 7 days in a shaker at 37℃ and 5% CO2. After culture, the cell supernatant was collected, and three target antibodies were isolated and purified using Protein A affinity packing material (Suzhou Navitas Technology Co., Ltd., 17010-050100), named NB467-7, NB467-71, and NB467-88, respectively.

[0088] The VHH amino acid and nucleotide sequences of nanobodies NB467-7, NB467-71, and NB467-88 are shown below:

[0089] SEQ ID No: 2 (amino acid sequence of NB467-7) is:

[0090] SEQ ID No: 3 (amino acid sequence of NB467-71) is:

[0091] SEQ ID No: 4 (amino acid sequence of NB467-88) is:

[0092] SEQ ID No: 18 (nucleotide sequence of NB467-7) is:

[0093] SEQ ID No: 19 (nucleotide sequence of NB467-71) is:

[0094] SEQ ID No: 20 (nucleotide sequence of NB467-88) is:

[0095] Example 2: Binding of anti-BAFFR nanobody to 293T cells overexpressing BAFFR

[0096] The binding affinity kinetics of the three anti-BAFFR nanobodies NB467-7, NB467-71 and NB467-88 obtained in Example 1 were characterized by flow cytometry.

[0097] Specifically, HEK-293T-BAFFR cells were collected from cell culture flasks, washed twice with FACS buffer, and centrifuged. The cells were resuspended in an appropriate volume of flow cytometry staining solution to achieve a final cell concentration of 2 × 10⁻⁶ cells per tube. 5 Cells / mL.

[0098] The antibodies NB467-7, NB467-71, and NB467-88 generated in Example 1, as well as the control antibody, were diluted to 200 nM. 50 μl of the antibody solution was added to a test tube containing HEK-293T-BAFFR cells. The mixture was thoroughly mixed and incubated at 4°C for 60 minutes. After incubation, the cells were washed once with FACS buffer.

[0099] Dilute the anti-human FC-647 secondary antibody (Jackson, 109-605-003) according to the manufacturer's instructions, and add 100 μl of the dilution buffer to the sample tube. After mixing thoroughly, incubate at 4°C for 60 minutes. After incubation, wash the cells three times with FACS buffer, and resuspend the cell pellet obtained by centrifugation in 200 μl of PBS. Analyze the cells using a flow cytometer (Sony, SA3800). The results are shown in Table 3 and Figure 1.

[0100] Table 3. Binding of nanobodies to 293T cells overexpressing BAFF (ECG) 50 )

[0101] Figure 1 shows the mean fluorescence intensity (MFI) values ​​of each antibody, confirming that the three nanobodies of this invention have stronger binding affinity to 293T cells overexpressing human BAFFR compared to the control antibody. Table 3 shows the EC values ​​of the nanobodies of this invention. 50 value.

[0102] Example 3: Construction of BAFFR-CAR-T cells

[0103] I. Construction of CAR Lentiviral Expression Vector

[0104] After optimizing the codons of the obtained BAFFR antibody sequence, a chimeric antigen receptor lentiviral expression vector was constructed by synthesizing gene fragments from the relevant hinge region, transmembrane region, and intracellular signaling region. The constructed vector was selected as the PCDH lentiviral vector (pCDH-EF1a), as shown in Figure 2. After confirming the inserted sequence was correct via Sanger sequencing, lentiviral packaging was performed.

[0105] II. CAR Lentiviral Packaging

[0106] 1) Prepare a 15cm cell culture dish and seed 1×10⁶ cells. 7 293T cells were added to complete culture medium (DMEM high glucose, 10% FBS) and incubated overnight at 37°C in a 5% CO2 incubator.

[0107] 2) Remove LVTransm, lentiviral expression plasmid (CAR plasmid), and lentiviral packaging plasmid mix from the freezer. After thawing at room temperature, thoroughly mix them by pipetting. Remove the PBS buffer and warm it to room temperature. Add 2 mL of PBS to one well of a 6-well plate, and add 20 μg of lentiviral expression plasmid and 30 μL of lentiviral packaging plasmid mix. After thoroughly mixing by pipetting, add 150 μL of LVTransm and immediately mix by pipetting. Let it stand at room temperature for 10 minutes.

[0108] 3) Add the DNA / LVTransm complex dropwise into a 15cm culture dish, gently shake the dish to mix thoroughly. Place the culture dish in a 37℃, 5% CO2 incubator and incubate for 6-8 hours. Then remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium.

[0109] 4) After continuous incubation for 48 hours, collect the virus-containing culture supernatant from the culture dish, filter it through a 0.45 μm filter membrane, transfer it to a centrifuge tube, balance the mixture, and centrifuge at 40,000 × g and 4 °C for 1.5 hours. After centrifugation, carefully aspirate the liquid from the centrifuge tube in a biosafety cabinet, add 1 mL of PBS buffer to resuspend the precipitate, and store the virus at -80 °C.

[0110] III. Isolation of peripheral blood T cells

[0111] 1) Transfer the anticoagulated blood sample to a 50mL sterile centrifuge tube, tighten the cap, and centrifuge at 800×g for 20 minutes.

[0112] 2) After centrifugation, remove the centrifuge tube from the centrifuge, avoiding vigorous shaking or inverting the centrifuge tube. Remove the upper light yellow serum layer, and then add an equal volume of PBS to the lower peripheral blood cell layer. Gently invert the tube to mix.

[0113] 3) Take out the lymphocyte separation solution and invert it several times to mix thoroughly. Add 20 mL of lymphocyte separation solution to a 50 mL centrifuge tube, and then carefully add the diluted blood sample from step 2 to the upper layer of the lymphocyte separation reagent along the tube wall using a pipette, avoiding mixing of the separation reagent and the blood sample. Centrifuge at 800×g for 20 minutes.

[0114] 4) After centrifugation, gently remove the centrifuge tube and transfer the white mononuclear cell layer in the middle to a new sterile centrifuge tube. Add an equal volume of physiological saline and mix gently. Centrifuge at 800×g for 5 minutes. After centrifugation, remove the supernatant and wash the PBMCs once more to adjust the cell density to 5×10⁻⁶ cells / mL. 7 1 cell / mL, transferred to 2ml cell cryopreservation tubes, and cryopreserved at 1ml / tube.

[0115] IV. CAR-T Cell Preparation

[0116] 1) Wash CD3 / CD28 Dynabeads twice with PBS.

[0117] 2) Add an appropriate amount of Dynabeads to PBMC, mix gently, and incubate at room temperature for 20 minutes.

[0118] 3) Insert a 2mL cell cryopreservation tube into the magnetic pole, let it stand at room temperature for 1 minute, keeping the cryopreservation tube inserted into the hole of the magnetic pole, gently invert it, and pour out the liquid in the tube.

[0119] 4) Remove the cell cryovial from the magnetic pole, add an appropriate amount of X-Vivo 15 medium (containing 200 IU / mL IL2, 10 ng / mL IL7, and 5 ng / mL IL15), resuspend the cell and bead mixture using a pipette, and adjust the cell density to 0.5–1 × 10⁻⁶ cells / mL. 6 1 cell / mL, transferred to 6-well plates.

[0120] 5) After incubating the cells at 37°C and 5% CO2 for 48 hours, adjust the cell density to 1×10⁻⁶. 6 per mL.

[0121] 6) Take the lentivirus out of the -80℃ ultra-low temperature freezer and thaw it quickly.

[0122] 7) Inject the prepared T cells (1×10⁻⁶) 6 Add 2 ml of the culture dish (cells / ml) to a final concentration of 6 μg / mL, add polybrene to a final concentration of 6 μg / mL, add 300 μL of lentivirus, gently pipette to mix thoroughly, seal the culture dish with sealing film, and centrifuge at 800×g at room temperature for 1 hour.

[0123] 8) After centrifugation, continue culturing for 24 hours and change the medium for the T cells.

[0124] 9) After culturing for another 24 hours, the CAR-T cell positivity rate was detected by flow cytometry using EGFR antibody incubation.

[0125] 10) Continue culturing the remaining CAR-T cells, gently pipetting the beads / cell clusters in the system daily until they are completely separated. When the cell density is greater than 1×10⁻⁶, continue culturing. 6 When the cell density reaches 0.5-0.7 × 10⁶ cells / mL, add X-Vivo 15 medium (containing 200 IU / mL IL-2, 10 ng / mL IL-7, and 5 ng / mL IL-15) to adjust the cell density to 0.5-0.7 × 10⁶ cells / mL. 6 per mL.

[0126] Example 4: Detection of CAR-T cells

[0127] I. Detection of CAR positivity rate

[0128] 1) Centrifuge at 500×g for 5 minutes and collect the cell pellet (prepare 2 tubes of cells, 5×10⁶ cells / mL). 5 (cells / tube).

[0129] 2) Wash the cell pellet three times with PBS buffer containing 0.5% BSA, and centrifuge at 500×g for 5 minutes each time.

[0130] 3) Resuspend the cell pellet in 100 μL of diluted EGFR antibody (1 μg / tube) and incubate at room temperature for 60 minutes. Centrifuge at 500×g for 5 minutes and remove the supernatant (keep one tube of cells as a control; only add PBS containing 0.5% BSA in this step).

[0131] 4) Wash the cell pellet three times with PBS buffer containing 0.5% BSA, centrifuging at 500×g for 5 minutes each time.

[0132] 5) Resuspend the cell pellet in 100 μL of diluted PE anti-human IgG (1:500 dilution) (incubate all cells with secondary antibody) and incubate in the dark for 45 minutes. Centrifuge at 500×g for 5 minutes and collect the cell pellet.

[0133] 6) Wash the cell pellet three times with PBS buffer containing 0.5% BSA, centrifuging at 500×g for 5 minutes each time.

[0134] 7) Finally, the cell pellet was resuspended in 400 μL PBS and analyzed by flow cytometry. The specific results are shown in Figure 3.

[0135] Figure 3 shows that the CAR positivity rate was greater than 30% when using EGFR antibody detection, indicating that CAR-T in vitro killing detection can be performed. The positivity rates of NB467-7 CAR-T were 53.22%, NB467-77 CAR-T were 69.65%, and NB467-88 CAR-T were 51.93%.

[0136] II. Lysis of target cells by CAR-T cells

[0137] 1) Resuspend the target cells Nalm-6-Luc in complete culture medium (RPMI 1640 + 10% FBS) and adjust the cell density to 2 × 10⁶ cells / year. 5 To obtain target cells, take a new 96-well plate and seed it with 100 μL of target cells per well. Add 100 μL of sterile water to each of the unused wells around the perimeter of the 96-well plate to prevent water evaporation from the central wells. Place the plate in a 5% CO2, 37°C incubator overnight.

[0138] 2) Collect the prepared CAR-T cells by centrifugation and resuspend them in 1640 medium with 10% FBS. Remove the 96-well plate from the incubator, completely aspirate the medium from the wells, gently wash the cells once with sterile PBS, and then add CAR-T cells according to different E / T ratios (1:1, 2.5:1, 5:1, 10:1), bringing the final volume to 200 μL / well. Maxilysis involves seeding the same number of target cells but without adding CAR-T cells. Place the plate in a 5% CO2, 37°C incubator and incubate for 18 hours.

[0139] 3) After culture, remove the well plate from the incubator, centrifuge to collect the supernatant, and store it at -80℃ for ELISA detection of IL2 and IFN-γ expression. Adding Bright-Glo™ to the cells and detecting Luciferase activity reflects the lytic ability of recombinant CAR-T cells to target cells.

[0140] 4) Formula for calculating the percentage of target cell lysis:

[0141] 5) See Figure 4 for the specific results.

[0142] Figure 4 shows that when Nalm-6-Luc target cells were co-cultured with CAR-T cells at effector-to-target ratios of 1:1, 2.5:1, 5:1, and 10:1, NB467-7 CAR-T, NB467-71 CAR-T, and NB467-88 CAR-T cells exhibited strong killing activity against Nalm-6-Luc compared to blank T cells.

[0143] III. Detection of CAR-T cytokine secretion levels

[0144] The supernatant from the co-culture was collected, and 100 μL of the stock solution was used for the detection of IL2 and IFN-γ. The secretion of IL2 and IFN-γ was detected according to the instructions of the IL2 and IFN-γ ELISA kits. The specific results are shown in Figure 5.

[0145] Figure 5 shows that, compared with blank T cells, NB467-7 CAR-T, NB467-71 CAR-T and NB467-88 CAR-T cells showed significantly enhanced IL-2 and IFN-γ secretion after stimulation with Nalm-6-Luc cells.

[0146] This invention provides a CAR-T cell targeting BAFFR, primarily targeting autoreactive B cells, offering a new treatment option for autoimmune diseases and certain hematologic malignancies.

[0147] In the description of this specification, the terms "one embodiment," "some embodiments," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0148] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for targeting BAFFR chimeric antigen receptors, characterized in that, It includes a signal peptide region, an antigen-binding domain targeting BAFFR, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain, which are sequentially connected from the amino terminus to the carboxyl terminus. The co-stimulatory domain includes a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain connected together. The signal transduction domain is CD3ζITAM1.

2. The targeted BAFFR chimeric antigen receptor according to claim 1, characterized in that, The amino acid sequence of the signal peptide region is shown in SEQ ID No:

1.

3. The targeted BAFFR chimeric antigen receptor according to claim 1, characterized in that, The antigen-binding domain targeting BAFFR is a nanobody targeting BAFFR, and the amino acid sequence of the nanobody targeting BAFFR is shown in SEQ ID No: 2, or in SEQ ID No: 3, or in SEQ ID No:

4.

4. The targeted BAFFR chimeric antigen receptor according to claim 1, characterized in that, The hinge region is a CD8 hinge domain with an amino acid sequence as shown in SEQ ID No: 5; And / or, the transmembrane domain is a CD28 transmembrane domain with an amino acid sequence as shown in SEQ ID No: 6; And / or, the amino acid sequence of the CD28 co-stimulatory domain is shown in SEQ ID No: 7; And / or, the amino acid sequence of the 4-1BB co-stimulatory domain is as shown in SEQ ID No: 8; And / or, the amino acid sequence of ITM1 of CD3ζ is shown in SEQ ID No:

9.

5. An isolated nucleic acid, characterized in that, The isolated nucleic acid includes a nucleotide sequence for expressing the BAFFR chimeric antigen receptor as described in any one of claims 1-4.

6. The isolated nucleic acid according to claim 5, characterized in that, The isolated nucleic acid includes a nucleic acid fragment with a nucleotide sequence as shown in SEQ ID No:

10.

7. A recombinant vector, characterized in that, The recombinant vector comprises the isolated nucleic acid as described in any one of claims 5 or 6.

8. A CAR-T cell, characterized in that, The CAR-T cells contain the isolated nucleic acid as described in claim 5 or 6, or the CAR-T cells are cells transformed by the recombinant vector as described in claim 7.

9. The application of a drug targeting the BAFFR chimeric antigen receptor, characterized in that, The use of the targeted BAFFR chimeric antigen receptor as described in any one of claims 1-4, the isolated nucleic acid as described in claim 5 or 6, the recombinant vector as described in claim 7, or the CAR-T cell as described in claim 8 in the preparation of a medicament for treating immune-related diseases.

10. A pharmaceutical composition, characterized in that, This includes expression vectors for expressing the BAFFR chimeric antigen receptor as described in any one of claims 1-4 or CAR-T cells as described in claim 8.

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