A chimeric antigen receptor secreting IL10 targeting BAFFR, CAR-T cell and application thereof

Abstract

The present invention discloses a chimeric antigen receptor for secreting IL10 targeting BAFFR, CAR-T cells and applications. The chimeric antigen receptor for secreting IL10 targeting BAFFR includes a signal peptide region, an antigen binding domain targeting BAFFR, a hinge region, a transmembrane domain, a co-stimulatory domain, a signal transduction domain and human IL10 connected in sequence 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 ITAM1 of CD3ζ. The present invention can specifically target autoimmune B cells, avoiding immunodeficiency caused by full B cell deletion and infection of patients. The secretion of IL10 on CAR-T cells will reduce the exhaustion rate of CAR-T cells in vivo, thereby playing a long-term inhibitory effect on the disease process.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and medical technology, and in particular to a chimeric antigen receptor that secretes IL10 and targets BAFFR, a CAR-T cell and an application thereof. Background Art

[0002] Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable clinical success in the treatment of B-cell malignancies. However, CAR-T immunotherapy is greatly limited in other diseases such as autoimmune diseases. Some patients experience insufficient efficacy, relapse, and drug resistance after CAR-T treatment. The main reasons for these limitations are the loss of CAR-T cell antigens on the surface of disease cells and the functional impairment caused by the exhaustion of CAR-T cells themselves.

[0003] Studies have shown that BAFF expression levels are significantly increased in many autoimmune diseases. For example, BAFF is continuously 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 (anti-dsDNAantibodies). In addition, BAFF expression levels are also significantly increased 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, namely BAFFR, TACI, and BCMA, but the combination of BAFF and BAFFR plays the most important role in the maturation of peripheral B cells. For this reason, constructing CAR-T cells targeting BAFFR will be an important breakthrough in the treatment of autoimmune diseases and\or blood-type tumors.

[0004] Emerging evidence suggests that CAR-T cells cultured in medium supplemented with cytokines, such as engineered interleukin-2 (IL-2) or IL-15, exhibit improved metabolic fitness and enhanced antitumor immunity in vivo when transferred to therapeutic tumor-bearing mice. This may be due to the fact that these cytokines increase oxidative phosphorylation in CAR-T cells in a mitochondrial pyruvate carrier-dependent manner, promoting the proliferation and effector function of CAR-T cells. At the same time, loading CAR-T cells with cytokines such as interleukins may induce stem cell-like memory responses in lymphoid organs, thereby providing sustained resistance to disease reinvasion. Therefore, regulating the metabolism of CAR-T cells in vivo is a novel and promising therapeutic strategy to combat T cell exhaustion-related dysfunction and induce effective and long-term maintenance of immune function. Summary of the invention

[0005] In view of the above-mentioned defects or deficiencies in the prior art, it is expected to provide a chimeric antigen receptor, CAR-T cell and application that secretes IL10 and targets BAFFR. Through phage display technology combined with a high-throughput expression platform, nanoantibodies targeting BAFFR were screened, which have the advantages of high affinity, high stability, small molecular weight, low immunogenicity and strong permeability. CAR-T cells were constructed based on the obtained BAFFR antibody sequence, which can specifically target autoimmune B cells and avoid immune deficiency and infection of patients caused by full B cell deletion. On the other hand, full B cell deletion may reduce the patient's responsiveness to vaccines, such as the COVID-19 vaccine, while CAR-T cells only target self-reactive B cells, which can effectively avoid the phenomenon of low vaccine response caused by full B cell deletion, and provide a new option for the treatment of human autoimmune-related diseases. Due to the secretion of IL10 on CAR-T cells, the exhaustion rate of CAR-T cells in the body will be reduced, thereby playing a long-term inhibitory effect on the disease process.

[0006] The present invention provides a chimeric antigen receptor for secreting IL10 and targeting BAFFR, comprising a signal peptide region, an antigen binding domain targeting BAFFR, a hinge region, a transmembrane domain, a co-stimulatory domain, a signal transduction domain and human IL10, which are sequentially connected from the amino terminus to the carboxyl terminus; the co-stimulatory domain comprises a connected CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, and the signal transduction domain is ITAM1 of CD3ζ; the antigen binding domain targeting BAFFR is a nano antibody targeting BAFFR, and the amino acid sequence of the nano antibody targeting BAFFR is shown in SEQ ID No: 2.

[0007] Furthermore, the amino acid sequence of the signal peptide region is shown in SEQ ID No: 1.

[0008] Further, the hinge region is a CD8 hinge domain having an amino acid sequence as shown in SEQ ID No: 3;

[0009] And / or, the transmembrane domain is a CD28 transmembrane domain having an amino acid sequence as shown in SEQ ID No: 4;

[0010] and / or, the amino acid sequence of the CD28 costimulatory domain is as shown in SEQ ID No: 5;

[0011] and / or, the amino acid sequence of the 4-1BB costimulatory domain is as shown in SEQ ID No: 6;

[0012] And / or, the amino acid sequence of ITAM1 of CD3ζ is shown in SEQ ID No:7.

[0013] Furthermore, the amino acid sequence of human IL10 is shown in SEQ ID No: 8.

[0014] In addition, the present invention also provides an isolated nucleic acid, which comprises a nucleotide sequence for expressing the above-mentioned secreted IL10 targeting BAFFR chimeric antigen receptor; the isolated nucleic acid comprises a nucleic acid fragment having a nucleotide sequence as shown in SEQ ID No:9.

[0015] In addition, the present invention also provides a recombinant vector, which comprises the above-mentioned isolated nucleic acid.

[0016] In addition, the present invention also provides a CAR-T cell, wherein the CAR-T cell contains the above-mentioned isolated nucleic acid, or the CAR-T cell is a cell transformed by the above-mentioned recombinant vector.

[0017] In addition, the present invention also provides a use of the above-mentioned IL10-secreting BAFFR-targeting chimeric antigen receptor, the above-mentioned isolated nucleic acid, the above-mentioned recombinant vector or the above-mentioned CAR-T cell in the preparation of a drug for treating immune-related diseases.

[0018] In addition, the present invention also provides a pharmaceutical composition, comprising an expression vector for expressing the above-mentioned IL10-secreting BAFFR-targeting chimeric antigen receptor or the above-mentioned CAR-T cell.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] (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 will target all immature and mature B cells, which can easily cause immunodeficiency or even infection in patients. On the other hand, CD19 or CD20 antibodies may reduce patients' responsiveness to vaccines, such as COVID-19 vaccines. The present invention uses antibodies that bind to BAFFR with high specificity to target autoreactive B cells, especially plasma cells, rather than deleting all B cells, thereby retaining the patient's basic autoimmune ability and vaccine response ability. In addition, the antibodies used in the present invention have higher affinity than existing control antibodies, which can effectively reduce the amount of antibodies used and more effectively consume autoimmune B cells or malignant proliferating B cells in patients with autoimmune diseases and some tumors.

[0021] (2) The CAR-T cells constructed with the BAFFR antibody used in the present invention have good specific lysis ability for target cells expressing BAFFR, and compared with the existing control BAFFR CAR-T cells, their lysis ability is improved by about 20%, and the released cytokines are also improved. Using a smaller dose than the control BAFFR CAR-T cells, the same therapeutic effect can be achieved, reducing the possibility of side effects caused by CAR-T cell therapy itself;

[0022] (3) The secretion of human IL10 cytokine further enhances the lysis ability of CAR-T cells on target cells, and the released cytokines also increase significantly; in addition, the secretion of human IL10 cytokine reduces the exhaustion rate of CAR-T cells, thereby producing long-acting CAR-T cells in the body, which can effectively reduce the concentration of autoantibodies in the patient's body for a long time and reduce the frequency of CAR-T use, providing a new option for the treatment of human autoimmune diseases and some tumors.

[0023] It should be understood that the contents described in the summary of the invention are not intended to limit the key or important features of the embodiments of the present invention, nor are they intended to limit the scope of the present invention. Other features of the present invention will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0025] Figure 1 Schematic diagram of ELISA to determine the binding activity of anti-BAFFR antibody to BAFFR;

[0026] Figure 2 Schematic diagram of the structure of CAR-T cells;

[0027] Wherein: BAFFR-CAR secreting human IL10 means a BAFFR chimeric antigen receptor polypeptide secreting IL10 targeting, which includes a CD8a signal peptide region, an anti-BAFFR scFv, a CD8 hinge region, a CD28 transmembrane region, a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, a CD3ζ signal transduction domain, and a human IL10 region from the N-terminus to the C-terminus;

[0028] Figure 3 Schematic diagram of the proportion of T cells transfected with CAR plasmid detected by flow cytometry;

[0029] Figure 4 This is a comparison of the killing effects of BAFFR-IL10-CAR-T in the example and the control BAFFR-CAR-T on Nalm-6 cells at different effector-target ratios;

[0030] Figure 5 This is a comparison chart of the secretion levels of IL-10 and IFN-γ of BAFFR-IL10-CAR-T and control BAFFR-CAR-T at different effector-target ratios in the example. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It should also be noted that, for ease of description, only the parts related to the invention are shown in the accompanying drawings.

[0032] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0033] Please refer to Figure 2 An embodiment of the present invention provides a chimeric antigen receptor that secretes IL10 and targets BAFFR, comprising a signal peptide region, an antigen binding domain targeting BAFFR, a hinge region, a transmembrane domain, a co-stimulatory domain, a signal transduction domain and an IL10 region, which are sequentially connected 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 ITAM1 of CD3ζ.

[0034] In some embodiments, the hinge region is at least one hinge region selected from the group consisting of CD28, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

[0035] In some embodiments, the transmembrane domain is at least one transmembrane region selected from the group consisting of the α, β or ζ chain of a T cell receptor, 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.

[0036] In a preferred embodiment, the amino acid sequence of the signal peptide region is shown as SEQ ID No: 1.

[0037] SEQ ID No: 1 (amino acid sequence of the signal peptide region) is:

[0038] MALPVTALLLPLALLLHAARP;

[0039] In a preferred embodiment, 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.

[0040] Among them, SEQ ID No: 2 (the amino acid sequence of NB467-35) is:

[0041] QVQLVESGGGLVQPGGSLRLSCAASGFSLEYYAIGWFRQTPGKEREAISCIDSPAKSTYYADSVKGRFTISRDNSKNTVYLQMNSLKAEDTAVYYCAADVYYRGSYLYRSCNPTESKYWGQGTQVTVSS;

[0042] In a preferred embodiment, the hinge region is a CD8 hinge domain having an amino acid sequence as shown in SEQ ID No: 3;

[0043] and / or, the transmembrane domain is a CD28 transmembrane domain having an amino acid sequence as shown in SEQ ID No: 4;

[0044] and / or, the amino acid sequence of the CD28 costimulatory domain is shown in SEQ ID No: 5;

[0045] and / or, the amino acid sequence of the 4-1BB costimulatory domain is as shown in SEQ ID No: 6;

[0046] And / or, the amino acid sequence of ITAM1 of CD3ζ is shown in SEQ ID No:7.

[0047] Among them, SEQ ID No: 3 (amino acid sequence of CD8 hinge domain) is:

[0048] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

[0049] SEQ ID No: 4 (amino acid sequence of CD28 transmembrane domain) is:

[0050] FWVLVVVGGVLACYSLLVTVAFIIFWV;

[0051] SEQ ID No: 5 (amino acid sequence of CD28 co-stimulatory domain) is:

[0052] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS;

[0053] SEQ ID No: 6 (amino acid sequence of the 4-1BB co-stimulatory domain) is:

[0054] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

[0055] SEQ ID No: 7 (amino acid sequence of ITAM1 of CD3ζ) is:

[0056] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0057] In a preferred embodiment, the amino acid sequence of human IL10 (interleukin 10) cytokine is shown in SEQ ID No:8.

[0058] SEQ ID No: 8 (amino acid sequence of human IL10) is:

[0059] MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0060] In addition, an embodiment of the present invention further provides an isolated nucleic acid, which includes a nucleotide sequence for expressing the above-mentioned BAFFR-targeted chimeric antigen receptor that secretes IL10; the isolated nucleic acid includes a nucleic acid fragment with a nucleotide sequence as shown in SEQ ID No:9.

[0061] Wherein, SEQ ID No: 9 is:

[0062] ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC

[0063] CAGGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCTCTGGCTTCAGCCTCGAGTACTATGCCATCGGCTGGTTCAGACAGACCCCTGGCAAAGAGAGAGAGGCCATCAGCTGCATCGACAGCCCTGCCAAGAGCACCTACTACGCCGATAGCGTGA AGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCGTGTACCTGCGATGAACAGCCTGAAGGCCGAGGACACCGCCGTGTACTATTGTGCCGCTGACGTGTACTACCGGGGCAGCTACCTGTACAGAAGCTGCAACCCTACCGAGAGCAAGTACTGGGGCCAGGGCACACAAGTGACCGTGTCATCT

[0064] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAAGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0065] TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG

[0066] AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCGCCGCCTACCGGAGC

[0067] AAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG

[0068] CGCGTGAAGTTCAGCAGATCAGCCGATGCTCCTGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA

[0069] GAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCC

[0070] .

[0071] In addition, an embodiment of the present invention further provides a recombinant vector, which includes the above-mentioned isolated nucleic acid.

[0072] In addition, an embodiment of the present invention further provides a CAR-T cell, which contains the above-mentioned isolated nucleic acid, or the CAR-T cell is a cell transformed by the above-mentioned recombinant vector.

[0073] In addition, embodiments of the present invention further provide a use of the chimeric antigen receptor targeting BAFFR as described above, the isolated nucleic acid as described above, the recombinant vector as described above, or the CAR-T cell as described above in the preparation of a drug for treating immune-related diseases.

[0074] Immune-related diseases are autoimmune diseases or tumors.

[0075] Among them, the autoimmune disease is selected from patients with nervous system immune diseases such as multiple sclerosis, neuromyelitis optica, myasthenia gravis, etc., or other autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, etc.

[0076] The tumors are B-cell lymphomas, such as Hodgkin lymphoma and nodular lymphocyte-predominant Hodgkin lymphoma, and 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).

[0077] In addition, an embodiment of the present invention further provides a pharmaceutical composition, comprising an expression vector for expressing the above-mentioned chimeric antigen receptor targeting BAFFR or the above-mentioned CAR-T cell.

[0078] Example 1: Obtaining anti-BAFFR nanobodies by phage display

[0079] In this example, a variety of antibody coding fragments were obtained by immunizing alpacas with human BAFFR as an antigen, and then candidate antibody clones were screened through phage libraries and mammalian expression systems. Specifically, the specific steps of the screening process of anti-BAFFR nanoantibodies are as follows.

[0080] a. Preparation of Antigen

[0081] Based on the amino acid sequence and nucleotide sequence of human BAFFR, an antigen that can effectively induce alpacas to produce specific antibodies against human BAFFR was analyzed and designed, and Human IgG1 Fc was connected to its C-terminus to obtain a modified antigen, recorded as "human BAFFR hFC antigen", whose amino acid sequence is shown in SEQ ID No: 10, and the nucleotide sequence is shown in SEQ ID No: 11.

[0082] Among them, SEQ ID No: 10 (amino acid sequence of human BAFFR hFC antigen) is:

[0083] MGWSCIILFLVATATGVHSSLRGRDAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQESVGAGAGEAALPLPGLEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0084] SEQ ID No: 11 (nucleotide sequence of human BAFFR hFC antigen) is:

[0085] ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTCGCTACCGCCACAGGCGTCCACTCCTCCCTGAGGGGCAGAGACGCCCCCGCTCCAACACCTTGCGTGCCTGCCGAGTGCTTCGATCTGCTGGTGAGGCACTGCGTGGCCTGCGGCCTGCTGAGGACCCCTAGGCCTAAGCCCGCCGGCGCTAGCAGCCCAGCTCCTAGAACAGCCCTGCAGCCCCAGGAGAGCGTGGGCGCTGGAGCTGGAGAGGCCGCTCTGCCCCTGCCTGGCCTGGAACCAAAATCTTGTGACAAAACCCACACATGCCCACCTTGTCCCGCCCCTGAACTGCTGGGCGGACCTTCTGTCTTTCTGTTCCCCCCCAAACCCAAGGATACACTGATGATCTCTAGAACCCCCGAGGTCACATGTGTCGTCGTGGATGTGTCCCATGAGGACCCTGAAGTGAAATTCAACTGGTACGTGGACGGAGTGGAAGTCCATAACGCCAAAACCAAACCACGAGAGGAACAGTACAATAGCACATATCGAGTCGTGTCTGTGCTGACTGTGCTGCATCAGGATTGGCTGAACGGCAAGGAATACAAATGTAAAGTGTCCAATAAGGCACTGCCCGCTCCTATTGAAAAAACAATTTCAAAGGCAAAAGGCCAGCCTCGTGAACCTCAGGTGTACACACTGCCACCTTCTCGGGAGGAAATGACCAAAAACCAGGTGTCACTGACCTGTCTGGTCAAGGGCTTTTACCCTTCCGATATTGCTGTCGAGTGGGAGAGTAACGGCCAGCCCGAAAACAACTACAAAACCACCCCTCCTGTGCTGGATTCCGATGGCTCATTCTTCCTGTACTCTAAACTGACCGTGGATAAGAGTCGCTGGCAGCAGGGCAATGTGTTTTCTTGCTCCGTGATGCATGAGGCACTGCACAACCACTACACCCAGAAATCCCTGTCACTGTCTCCCGGAAAATAA。

[0086] b. Immunization of alpacas

[0087] The human BAFFR hFC antigen obtained in step a is completely mixed with an equal volume of Freund's adjuvant and used for subcutaneous injection into alpacas.

[0088] Specifically, on day 1, alpacas were primed with an emulsified mixture of 500 μg of human BAFFR hFC antigen and an equal volume of Freund's complete adjuvant, and boosted with an emulsified mixture of 250 μg of human BAFFR hFC antigen and an equal volume of Freund's complete adjuvant four times on days 21, 42, 63, and 84. Seven days after the first four immunizations, 10 ml of peripheral blood was collected from the alpacas to detect the anti-BAFFR serum titer in the blood by ELISA.

[0089] The specific steps of 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. Coat the culture plate overnight at 4°C. Discard the coating solution and wash 3 times with PBST. Add 300 μL of 5% skim milk to each well and block at 37°C for 1 hour. Wash 3 times with PBST buffer. Add 100 μL / well of serum dilution (starting from 1:2000 dilution) and incubate at 37°C for 45 minutes. 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°C for 45 minutes. Wash the plate 5 times with PBST. TMB color development solution was added for color development (100 μL / well) and incubated for 5 min at 37° C. Stop solution was added to terminate the reaction (50 μL / well) and the optical density was measured at 450 nm.

[0090] One week after the fifth immunization, 50 mL of peripheral blood was collected from the alpacas and mononuclear cells were isolated.

[0091] c. Library construction

[0092] Extract the RNA of PBMC obtained in step b, and obtain the target gene fragment by nested PCR after reverse transcription. Clone the target gene fragment into a eukaryotic expression vector, and transform the obtained expression vector into competent cells to construct a BAFFR-VHH phage display library. The specific steps are as follows.

[0093] Using the PBMC cells screened in step b as a template, RNAiso Plus was used to extract total cell RNA, and the RNA was reverse transcribed into cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit. The target fragment was obtained by nested PCR. The amplification system of the target fragment is shown in Table 1 below, and the amplification procedure is shown in Table 2 below.

[0094] Table 1 Nested PCR one-round reaction system

[0095]

[0096] Table 2 Nested PCR second round reaction system

[0097]

[0098] The amplified nucleotide fragment encoding alpaca VHH was cloned into the eukaryotic expression vector pComb3XSS (Chengdu Apak Biotechnology Co., Ltd.). The generated recombinant vector was transformed into TG1 competent cells by electroporation to obtain the VHH phage display library. In order to further identify whether the BAFFR-VHH phage display library was successfully constructed, the library was cultured on a 2-YT-A plate, and 48 clones were selected from the formed colonies for sequencing.

[0099] The sequencing results showed that the successful insertion rate of the clones was 100%. Based on the number of transformants, library insertion rate, and diversity sequencing analysis results, the library capacity was calculated to be 1.92×10 9 , indicating that the library capacity and diversity of the obtained BAFFR-VHH phage display library are good.

[0100] d. Acquisition of Nanobodies

[0101] The phage display library obtained in step c was used for panning and positive clones were selected. The coding sequence of the selected VHH antibody was fused with the Human IgG1 Fc coding sequence and constructed into the pTT5 (Chengdu Apark Biotechnology Co., Ltd.) expression vector to transfect eukaryotic cells for expression and purification. Finally, anti-human BAFFR nano antibodies were obtained. The specific method is as follows.

[0102] The SA-magnetic beads (MPHTSA-300, Suzhou Nawei Technology Co., Ltd.) were removed and washed twice with PBS for later use; the Bio-Human BAFFR-his (Acro, BAR-H82E3) antigen was diluted with PBS to a final concentration of 5 μg / ml, and 2×10 11Phage library, incubate at 37℃ for 1h; add the incubated mixture to the magnetic bead tube and shake at 4℃ for 45min; aspirate the supernatant under the action of the magnetic stand, wash with PBST 3 times and PBS 2 times; add 800μL Gly-HCl eluent, incubate at 37℃ for 8min to elute the specifically bound phage; transfer the eluate to a 1.5mL sterile centrifuge tube and quickly neutralize with 160μL Tris-HCl neutralization buffer; take 10μL for gradient dilution, determine the titer, calculate the panning recovery rate, and mix the remaining eluate for amplification and purification for the next round of affinity panning.

[0103] After two rounds of screening, monoclonal phage supernatant ELISA was performed after the second round of screening. Bio-HumanBAFFR-his antigen was fixed in a 96-well ELISA plate at 2μg / ml*100μl and incubated overnight at 4℃. Discard the coating solution and wash 3 times with PBST. Add 300μL of 5% skim milk to each well and block at 37℃ for 1h. Wash 3 times with PBST buffer, add two-fold diluted phage supernatant at 100μL / well, and incubate at 37℃ for 45min. Wash 5 times with PBST, add mouse anti-M13 antibody HRP (mouse anti-M13 antibody HRP, Chengdu Apak Biotechnology Co., Ltd.) to each well at 100μL / well, and incubate at 37℃ for 45min. Wash the plate 5 times with PBST. Add TMB colorimetric solution at 100μL / well for color development and incubate at 37℃ for 5min.

[0104] The reaction was terminated by adding 1M HCl stop solution at 50 μL / well, and the optical density was detected at a wavelength of 450 nm. The positive clones (OD450>1) were screened by the detection results. Human IgG1 Fc fused with the positive clone was constructed into the pTT5 plasmid, and the corresponding plasmid was transfected into mammalian cells HEK293T and expressed in a shaker at 37°C and 5% carbon dioxide for 7 days. After culture, the cell supernatant was collected and purified using Protein A affinity filler (Suzhou Nawei Technology Co., Ltd., 17010-050100) to obtain the target antibody, which was named NB467-35.

[0105] The VHH amino acid sequence and nucleotide sequence of Nanobody NB467-35 are shown below:

[0106] SEQ ID No: 2 (amino acid sequence of NB467-35) is:

[0107] QVQLVESGGGLVQPGGSLRLSCAASGFSLEYYAIGWFRQTPGKEREAISCIDSPAKSTYYADSVKGRFTISRDNSKNTVYLQMNSLKAEDTAVYYCAADVYYRGSYLYRSCNPTESKYWGQGTQVTVSS;

[0108] SEQ ID No: 17 (nucleotide sequence of NB467-35) is:

[0109] CAGGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCTCTGGCTTCAGCCTCGAGTACTATGCCATCGGCTGGTTCAGACAGACCCCTGGCAAAGAGAGAGAGGCCATCAGCTGCATCGACAGCCCTGCCAAGAGCACCTACTACGCCGATAGCGTGAA GGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGGCCGAGGACACCGCCGTGTACTATTGTGCCGCTGACGTGTACTACCGGGGCAGCTACCTGTACAGAAGCTGCAACCCTACCGAGAGCAAGTACTGGGGCCAGGGCACACAAGTGACCGTGTCATCT.

[0110] Example 2: Binding of anti-BAFFR nanobodies to 293T cells overexpressing BAFFR

[0111] The anti-BAFFR nanobody NB467-35 obtained in Example 1 was subjected to characterization of binding affinity kinetics by flow cytometry.

[0112] 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 a final concentration of 2 × 10 cells per tube. 5 cells / mL.

[0113] The antibody NB467-35 produced in Example 1 and the control antibody were diluted to 100 nM respectively. 50 μl of the antibody solution was added to the test tube with HEK-293T-BAFFR cells. Mix well and incubate at 4°C for 60 minutes. After incubation, wash the cells once with FACS buffer.

[0114] Anti-human FC-647 secondary antibody (Jackson, 109-605-003) was diluted according to the instructions, and 100 μl of secondary antibody dilution was added to the sample tube. After mixing, incubate at 4°C for 60 minutes. After incubation, wash the cells 3 times with FACS buffer, resuspend the cell pellet obtained by centrifugation in 200 μl PBS, and detect it with a flow cytometer (Sony, SA3800). The test results are shown in Table 3 and Figure 1 .

[0115] Table 3. Binding of NB467-35 and control NB467-7 to 293T cells overexpressing BAFFR (EC 50 )

[0116]

[0117] Figure 1 The mean fluorescence intensity (MFI) values ​​of each antibody are shown in Table 3, which confirms that the nanobody NB467-35 of the present invention has a stronger binding ability to 293T cells overexpressing human BAFFR than the control antibody NB467-7. 50 value.

[0118] Example 3: Construction of BAFFR-CAR-T cells

[0119] 1. Construction of CAR lentiviral expression vector

[0120] After optimizing the codons of the obtained BAFFR antibody sequence information, the chimeric antigen receptor lentiviral expression vector was constructed by synthesis with the relevant hinge region, transmembrane region and intracellular signal region gene fragments and the human IL10 gene. The constructed vector was selected as the PCDH lentiviral vector (pCDH-EF1a), such as Figure 2 After the insertion sequence was confirmed to be correct by Sanger sequencing, lentiviral packaging was performed.

[0121] CAR Lentivirus Packaging

[0122] 1) Prepare a 15 cm cell culture dish and seed 1×10 7 293T cells were added with complete culture medium (DMEM high glucose, 10% FBS) and placed in a 37°C, 5% CO2 incubator for overnight culture.

[0123] 2) Take out LVTransm, lentiviral expression plasmid (CAR plasmid), and lentiviral packaging plasmid mix from the refrigerator. After thawing at room temperature, pipette up and down to mix thoroughly. Take out PBS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 20μg lentiviral expression plasmid and 30μL lentiviral packaging plasmid Mix respectively, pipette up and down to mix thoroughly, add 150μL LVTransm, pipette up and down to mix immediately, and let stand at room temperature for 10 minutes.

[0124] 3) Add the above DNA / LVTransm complex dropwise into a 15 cm culture dish, gently shake the dish and mix thoroughly. Place the dish in a 37°C, 5% CO2 incubator. After 6-8 hours of culture, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium.

[0125] 4) After continuous culture for 48 hours, collect the supernatant of the culture medium containing the virus in the culture dish, filter it with a 0.45μm filter membrane, transfer it to a centrifuge tube, balance it, and centrifuge it at 40,000×g and 4℃ for 1.5 hours. After the centrifugation, carefully remove the liquid in the centrifuge tube in the biosafety cabinet, add 1mL PBS buffer to resuspend the precipitate, and store the virus at -80℃.

[0126] 3. Isolation of peripheral blood T cells

[0127] 1) Transfer the anticoagulated blood sample into a 50 mL sterile centrifuge tube, tighten the cap, and centrifuge at 800 × g for 20 minutes.

[0128] 2) After centrifugation, remove the centrifuge tube from the centrifuge, avoid shaking or inverting the tube violently, remove the upper light yellow serum layer, then add an equal volume of PBS to the lower peripheral blood cell layer, and gently invert it upside down to mix.

[0129] 3) Take out the lymphocyte separation solution and mix it thoroughly by inverting it several times. Add 20 mL of lymphocyte separation solution to a 50 mL centrifuge tube, and then use a pipette to carefully add the diluted blood sample in step 2 to the upper layer of the lymphocyte separation reagent along the tube wall in equal volumes to avoid mixing the separation reagent and the blood sample, and centrifuge at 800×g for 20 minutes.

[0130] 4) After centrifugation, gently remove the centrifuge tube, aspirate the white mononuclear cell layer in the middle into a new sterile centrifuge tube, add an equal volume of physiological saline and gently mix, centrifuge at 800×g for 5 minutes, remove the supernatant, repeat the washing of PBMC, count, and prepare to sort T cells. The sorting kit is Human Pan T Cell Isolation Kit.

[0131] 5) Binding biotin antibody: After counting, centrifuge the cells to the bottom of the tube, discard all PBS, and 7 Add 40 μL of sterile sorting buffer (PBS, pH 7.2, 0.5% BSA, 2 Mm EDTA) and 10 μL Pan T Cell Biotin-Antibody Cocktail to the cells, mix gently with a 200 μL pipette, try to avoid bubbles, and let it stand at 4°C for 5 min.

[0132] 6) Magnetic bead binding: Take out the cell antibody mixture from 4℃ and 7 Add 30 μL of sterile sorting buffer (PBS, pH 7.2, 0.5% BSA, 2 Mm EDTA) and 20 μL of Pan T Cell MicroBead Cocktail to the cells, mix gently with a 200 μL pipette, try to avoid bubbles, and let it stand at 4°C for 10 min.

[0133] 7) LS column washing: Take an LS column and place it on the purple adapter magnet, add 3 mL of sterile buffer for sorting to wash the column, take out the cells that have been bound to the magnetic beads from 4°C, and add 0.5-1 mL of buffer.

[0134] 8) Sorting: Connect a sterile 15 mL centrifuge tube under the LS column, add the magnetic bead-cell mixture to the LS column, wait for the upper liquid to flow out, add 5 mL of buffer to the LS column, and collect it in the same centrifuge tube. Centrifuge at 300×g, 90% up and 90% down for 5 minutes to collect T cells, resuspend the cells in X-VIVO medium, and count them.

[0135] 9) T cell stimulation: Kit is Gibco's Bynabeads Human T-Activator CD3 / CD28, 4×10 7 100 μg of magnetic beads per ml, take the same amount of magnetic beads as T cells, put them into a sterile flow tube, place it on a magnet dedicated to the flow tube, remove the original buffer of the magnetic beads, wash them once with buffer, wash them once with X-Vivo 15 culture medium (containing 200 IU / mL IL2, 10 ng / mL IL7, 5 ng / mL IL15), mix them evenly with the cells, put them into a 24-well plate, and culture them in a cell culture incubator.

[0136] 4. CAR-T Cell Preparation

[0137] 1) Adjust the cell density to 1×10 6 Take out the lentivirus from the -80℃ ultra-low temperature freezer and thaw it quickly.

[0138] 2. Prepared T cells (1×10 6 cells / ml), a total of 2 ml, add polybrene to a final concentration of 6 μg / mL, add 300 μL of lentivirus, gently pipette to mix thoroughly, seal the culture vessel with sealing film, and centrifuge at 800 × g at room temperature for 1 hour.

[0139] 3) After centrifugation, continue culturing for 24 hours and replace the medium for T cells.

[0140] 4) After culturing for 24 hours, the positive rate of CAR-T cells was detected by flow cytometry by incubating BAFFR antigen.

[0141] 5) Continue to culture the remaining CAR-T cells, gently blow the beads / cell clusters in the system every day until they are completely separated. When the cell density is greater than 1×106 cells / mL, add X-Vivo 15 medium (containing 200 IU / mL IL-2, 10 ng / mL IL-7, 5 ng / mL IL-15) to adjust the cell density to 0.5-0.7×106 cells / mL.

[0142] Example 4: Detection of CAR-T cells

[0143] 1. Detection of CAR Positive Rate

[0144] 1) Centrifuge at 500×g for 5 minutes and collect the cell pellet (prepare 2 tubes of cells, 5×10 5 cells / tube).

[0145] 2) Wash the cell pellet three times with PBS buffer containing 0.5% BSA and centrifuge at 500×g for 5 minutes each time.

[0146] 3) Resuspend the cell pellet with 100 μL of diluted BAFFR-His antigen protein (0.5 μg / mL) and incubate at room temperature for 60 minutes. Centrifuge at 500×g for 5 minutes and discard the supernatant (keep one tube of cells as a control, and only add PBS containing 0.5% BSA in this step).

[0147] 4) Wash the cell pellet three times with PBS buffer containing 0.5% BSA and centrifuge at 500×g for 5 minutes each time.

[0148] 5) Resuspend the cell pellet (all cells are incubated with secondary antibody) with 100 μL diluted FITC anti His Tag antibody (1:500 dilution) and incubate for 30 minutes in the dark. Centrifuge at 500×g for 5 minutes to collect the cell pellet.

[0149] 6) Wash the cell pellet three times with PBS buffer containing 0.5% BSA and centrifuge at 500×g for 5 minutes each time.

[0150] 7) Finally, resuspend the cell pellet with 400 μL PBS and perform flow cytometry analysis. Figure 3 .

[0151] Figure 3 The results showed that the CAR positivity rate was greater than 30% when tested with BAFFR antigen, and CAR-T in vitro killing detection could be performed. The positivity rate of NB467-7 control CAR-T was 46.1%, the positivity rate of NB467-35 CAR-T was 44.0%, and the positivity rate of NB467-35-IL10 CAR-T was 49.1%.

[0152] 2. Lysis of target cells by CAR-T cells

[0153] 1) Label the target cell Nalm-6 with CFSE (CFSE must be labeled with PBS, 5 μM working concentration, 1×10 7 Use 1 mL of culture medium to resuspend and label each cell. If the number of cells is lower than this, reduce the concentration of CFSE working solution accordingly. Incubate for 15 min, add 5 times the volume of 20% FBS complete culture medium to terminate the reaction, centrifuge, discard the supernatant, wash 2-3 times with PBS, add 2 mL of culture medium and incubate in the incubator for 10 min) and resuspend in complete culture medium (RPMI1640+10% FBS).

[0154] 2) Adjust the cell density of CFSE-labeled target cells Nalm-6 to 2×10 5 / mL, take a new 96-well plate and inoculate the target cells at 100μL / well. Add 100μL of sterile water to each of the unused wells around the 96-well plate to prevent the water in the middle experimental well from evaporating.

[0155] 3) 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 in the wells, gently wash the cells with sterile PBS, and then add CAR-T cells according to different E / T ratios (1:2, 1:1, 2.5:1, 5:1), and make up the final volume to 200 μL / well; reserve the wells without CAR-T cells as blank controls, place the well plate in a 5% CO2, 37°C incubator, and culture for 24 hours.

[0156] 4) After the culture is completed, the plate is removed from the incubator, the supernatant is collected by centrifugation and stored in a -80°C refrigerator for Elisa detection of IL10 and IFN-γ secretion. 7-AAD is added to the cells to detect the activity of CFSE-positive cells to reflect the lysis ability of recombinant CAR-T cells on target cells.

[0157] Target cell lysis percentage = (number of 7-AAD-positive cells in the experimental group − number of 7-AAD-positive cells in the control group) / number of CFSE-positive cells*100%;

[0158] Experimental group: a mixed group of target cells and CAR-T cells in different proportions;

[0159] Control group: target cell group without CAR-T cells.

[0160] 5) See the specific results Figure 4 .

[0161] Figure 4 The results showed that the target cells Nalm-6-Luc were co-cultured with CAR-T cells at effector-target ratios E / T=1:2, 1:1, 2.5:1, and 5:1. Compared with the blank T cells and the Mock group, the NB467-7 control CAR-T, NB467-35 CAR-T, and NB467-35-IL10 CAR-T cells all had killing activity against Nalm-6. Due to the advantage of antibody affinity, compared with the NB467-7 control CAR-T, the NB467-35 CAR-T showed better killing ability. At the same time, the killing ability of NB467-35-IL10 CAR-T cells was significantly higher than that of NB467-35 CAR-T.

[0162] 3. Detection of CAR-T cell factor secretion level

[0163] The supernatant of the co-culture was removed, and 50 μL of the stock solution was taken for the detection of human IL10 and IFN-γ. The secretion of human IL10 and IFN-γ was detected according to the operating instructions of the ELISA detection kits for IL10 and IFN-γ. The specific results are shown in Figure 5 .

[0164] Figure 5The results showed that compared with the blank T and Mock groups, the IFN-γ secretion of NB467-7 control CAR-T, NB467-35 CAR-T and NB467-35-IL10 CAR-T cells was significantly enhanced after being stimulated by Nalm-6 cells, among which the secretion amount of NB467-35 CAR-T was higher than that of NB467-7 control CAR-T, and the secretion ability of NB467-35-IL10 CAR-T was higher than that of NB467-35CAR-T; compared with the blank T, Mock groups, NB467-7 CAR-T and NB467-35 CAR-T, only NB467-35-IL10CAR-T had the ability to secrete IL10.

[0165] The present invention provides a CAR-T cell that secretes IL10 against BAFFR, which mainly targets autoreactive B cells and provides a new option for the treatment of autoimmune diseases and certain blood tumors.

[0166] In the description of this specification, the description of the terms "one embodiment", "some embodiments", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0167] The above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A chimeric antigen receptor that secretes IL10 and targets BAFFR, 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, a signal transduction domain and human IL10, which are sequentially connected 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 ITAM1 of CD3ζ; 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.

2. The secreted IL10 targeting 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 secreted IL10 targeting BAFFR chimeric antigen receptor according to claim 1, characterized in that: The hinge region is a CD8 hinge domain having an amino acid sequence as shown in SEQ ID No: 3; And / or, the transmembrane domain is a CD28 transmembrane domain having an amino acid sequence as shown in SEQ ID No: 4; and / or, the amino acid sequence of the CD28 costimulatory domain is as shown in SEQ ID No: 5; and / or, the amino acid sequence of the 4-1BB costimulatory domain is as shown in SEQ ID No: 6; And / or, the amino acid sequence of ITAM1 of CD3ζ is shown in SEQ ID No:

7.

4. The secreted IL10 targeting BAFFR chimeric antigen receptor according to claim 1, characterized in that: The amino acid sequence of human IL10 is shown in SEQ ID No:

8.

5. An isolated nucleic acid, characterized in that The isolated nucleic acid comprises a nucleotide sequence for expressing the secreted IL10-targeted BAFFR chimeric antigen receptor according to any one of claims 1 to 4; the isolated nucleic acid comprises a nucleic acid fragment with a nucleotide sequence as shown in SEQ ID No:

9.

6. A recombinant vector, characterized in that: The recombinant vector comprises the isolated nucleic acid according to claim 5.

7. A CAR-T cell, characterized in that: The CAR-T cell contains the isolated nucleic acid according to claim 5, or the CAR-T cell is a cell transformed by the recombinant vector according to claim 6.

8. A use of the IL10-secreting BAFFR-targeting chimeric antigen receptor according to any one of claims 1 to 4, the isolated nucleic acid according to claim 5, the recombinant vector according to claim 6, or the CAR-T cell according to claim 7 in the preparation of a drug for treating an immune-related disease; the immune-related disease is an autoimmune disease and a tumor; The autoimmune disease is selected from multiple sclerosis, neuromyelitis optica, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, and Sjögren's syndrome; The tumor is a B-cell lymphoma.

9. A pharmaceutical composition, characterized in that It comprises an expression vector for expressing the IL10-secreting BAFFR-targeting chimeric antigen receptor as described in any one of claims 1 to 4 or the CAR-T cell as described in claim 7.

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