Chimeric antigen receptors with cd81 costimulatory domains and uses thereof

Abstract

The application discloses a chimeric antigen receptor with a CD81 costimulatory domain and application thereof. The chimeric antigen receptor with the CD81 costimulatory domain comprises, from N-terminus to C-terminus: (i) an extracellular antigen binding domain; (ii) a transmembrane domain; (iii) a costimulatory domain, wherein the costimulatory domain is a CD81 costimulatory domain; and (iv) a signaling domain. The application connects the C-terminal intracellular domain of the CD81 molecule as the costimulatory domain of the CAR with the signaling domain, successfully constructs a novel CD19-CAR-T cell (81z) with the CD81 as the costimulatory signal. Compared with the existing CD28 or 4-1BB costimulatory domain CAR-T, the CAR-T cell with the CD81 costimulatory domain obviously reduces the release of toxic cytokines after tumor cell stimulation, reduces the release of mononuclear cell inflammatory factors IL-1beta and IL-6 mediated, and has good targeted killing CD19 + tumor cell ability.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a chimeric antigen receptor having a CD81 co-stimulatory domain and its uses. Background Technology

[0002] Chimeric antigen receptor-modified T cell (CAR-T) therapy has shown excellent efficacy in treating B-cell malignancies. However, with the increasing clinical application of CAR-T, adverse reactions have become increasingly recognized and concerning, with cytokine release syndrome (CRS) being the most common. While glucocorticoids and tocilizumab can effectively alleviate symptoms in mild to moderate CRS, treatment options are limited and the prognosis is poor for severe CRS. Therefore, researching how to reduce the incidence of CRS at its root and improve the safety of CAR-T therapy is a challenging problem that has attracted much attention and urgently needs to be solved in the field of cell therapy.

[0003] All marketed CAR-T products utilize second-generation CAR structures, with intracellular co-stimulatory domains of CD28 or 4-1BB. Clinical research data shows that the incidence of CRS associated with CD28 co-stimulatory domain CAR-T therapy is higher than that of 4-1BB co-stimulatory domain CAR-T, which may be related to their T cell phenotype and cytokine secretion capacity. The CD28 co-stimulatory domain promotes the differentiation of CAR-T cells into effector memory T cells, resulting in stronger cytokine release and killing ability; while the 4-1BB co-stimulatory domain mainly induces CAR-T cells to differentiate into central memory T cells, resulting in more durable anti-tumor ability. The higher the degree of T cell differentiation, the stronger the cytokine secretion capacity, but the weaker the self-renewal and survival capacity. Studies have shown that low-differentiation stem cell-like memory CAR-T cells can not only improve efficacy but also reduce CRS. Therefore, exploring new co-stimulatory domains to promote the formation of even lower-differentiation CAR-T cells may fundamentally reduce the occurrence of CRS. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a chimeric antigen receptor with a CD81 co-stimulatory domain and its application. The inventors have discovered that using the intracellular domain of the C-terminal CD81 molecule as the co-stimulatory domain of CAR, compared with CD28 or 4-1BB co-stimulatory domain CAR-T, the CD81 co-stimulatory domain can promote the formation of stem cell-like memory CAR-T, which can both reduce the occurrence of CRS and maintain its anti-tumor activity.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] Firstly, a chimeric antigen receptor having a CD81 co-stimulatory domain, comprising, from the N-terminus to the C-terminus:

[0007] (i) Extracellular antigen-binding domain;

[0008] (ii) Transmembrane structural domains;

[0009] (iii) a co-stimulatory domain, wherein the co-stimulatory domain is a CD81 co-stimulatory domain; and

[0010] (iv) Signal conduction structural domain;

[0011] The amino acid sequence of the CD81 co-stimulatory domain is shown in SEQ ID NO.7;

[0012] The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.11.

[0013] Furthermore, the chimeric antigen receptor also includes a signal peptide, a cleavage peptide, and / or a tag protein.

[0014] Preferably, the chimeric antigen receptor sequentially comprises: a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, a CD81 co-stimulatory domain, and a signal transduction domain. More preferably, the chimeric antigen receptor sequentially comprises: a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, a CD81 co-stimulatory domain, a signal transduction domain, a cleavage peptide, and a tag protein.

[0015] Furthermore, the signal peptide is selected from at least one of the following signal peptides: CD8α, CD80, CD86, IgG1, and IgG4;

[0016] The extracellular antigen-binding domain can be any extracellular antigen-binding domain epitope, preferably but not limited to FMC63 scFv targeting CD19;

[0017] The hinge region is selected from at least one of the hinge regions of the following molecules: CD8α, CD28, DAP12, IgG1, and IgG4;

[0018] The transmembrane domain is selected from at least one of the transmembrane domains of the following molecules: CD8α, CD28, NKG2D, 2B4, DNAM1, CD3ζ;

[0019] The tag protein is selected from at least one of NGFR, EGFP, and mCherry.

[0020] In a second aspect, a nucleotide encoding a chimeric antigen receptor having a CD81 co-stimulatory domain, the nucleotide sequence of which is shown in SEQ ID NO.16.

[0021] Thirdly, a vector expressing a chimeric antigen receptor having a CD81 co-stimulatory domain, said vector comprising the aforementioned nucleotides.

[0022] Preferably, the vector includes a plasmid vector, a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector. The retroviral vector can be replaced by introducing autologous / allogeneic / γδ T / NK / iPSC-derived cells via a lentiviral vector, a non-viral vector, or mRNA.

[0023] Fourthly, a cell expressing a chimeric antigen receptor having a CD81 co-stimulatory domain, said cell comprising the chimeric antigen receptor described above, the nucleotide described above, or the carrier described above.

[0024] Preferably, the cells include T cells, NK cells, NKT cells, γδT cells, or macrophages.

[0025] Fifthly, the application of the aforementioned chimeric antigen receptor, the aforementioned nucleotide, the aforementioned carrier, or the aforementioned cell in the preparation of drugs for treating tumors.

[0026] The tumor includes hematologic malignancies or solid tumors; preferably, the solid tumor includes any one of lung cancer, brain cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, liver cancer, kidney cancer, stomach cancer, cervical cancer, head and neck cancer, ovarian cancer, testicular cancer, pituitary cancer, esophageal cancer, skin cancer, pancreatic cancer, and bone cancer; preferably, the hematologic malignancy includes leukemia, myeloma, or lymphoma.

[0027] Sixthly, the application of chimeric antigen receptors in the preparation of drugs to reduce CAR-T therapy-induced cytokine release syndrome. The aforementioned CD81 co-stimulatory signaling promotes the differentiation of CAR-T cells into stem memory-like cells and reduces cytokine secretion, which can fundamentally reduce the occurrence of CAR-T cell-related CRS, demonstrating good anti-tumor effects while having lower toxicity.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention connects the C-terminal intracellular domain of the CD81 molecule as a co-stimulatory domain to the signal transduction domain of a CAR, successfully constructing a novel CD19-CAR-T cell (81z) with CD81 as the co-stimulatory signal. Compared with existing CD28 or 4-1BB co-stimulatory domain CAR-T cells, CAR-T cells with the CD81 co-stimulatory domain show significantly reduced release of toxic cytokines after tumor cell stimulation, reduced release of monocyte inflammatory factors IL-1β and IL-6, and good targeted killing of CD19. +Tumor cell activity. To further clarify the safety of 81z CAR-T, high-tumor-burden humanized mice were constructed to simulate cytokine release syndrome (CRS). Leukemia mice treated with CD81 co-stimulated CAR-T did not exhibit severe CRS responses such as weight loss or elevated IL-6 levels. The mice showed lower CRS-related mortality and demonstrated good therapeutic efficacy, significantly prolonging the survival time of leukemia mice. Therefore, the novel CAR-T with CD81 co-stimulatory signaling exhibits good efficacy and safety. Attached Figure Description

[0030] To more clearly illustrate the solutions in this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 A schematic diagram of the CAR structure associated with the co-stimulatory domains of CD81, CD28, and 4-1BB.

[0032] Figure 2 Positive rate of CAR-T cells prepared from CARs associated with CD81, CD28, and 4-1BB co-stimulatory domains;

[0033] Figure 3 The expression of CD107a, IFN-γ, Granzyme B and TNF-α in 81z, 28z and BBz CAR-T cells after stimulation by CD19 positive tumor cells;

[0034] Figure 4 The antitumor effects of 81z, 28z, and BBz CAR-T cells co-cultured with CD19+Nalm6, Raji, and CD19-K562 cells at different effector-target ratios for 4 hours were evaluated.

[0035] Figure 5 The efficacy of 81z, 28z, and BBz CAR-T cells in treating leukemia mice;

[0036] Figure 6 The release levels of IL-1β and IL-6 after co-culturing 81z, 28z, and BBz CAR-T cells with CD19+ Nalm6 cells and THP1 monocytes;

[0037] Figure 7 The figure shows the effect of 81z, 28z, and BBz CAR-T cells on CRS in humanized mice. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0039] As used in this invention, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0040] The term "comprises" or "comprising" as used in this invention means "including, but not limited to," "including." This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "substantially consisting of." In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of."

[0041] As used in this invention, the terms "optional," "any," "arbitrary," or "any one" mean that the event or circumstance described below may, but does not have to, occur, including the occurrence or non-occurrence of such event or circumstance. For example, "optionally includes one antibody heavy chain variable region" means that the antibody heavy chain variable region of a particular sequence may, but does not have to, be present.

[0042] As used in this invention, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0043] The term "and / or" as used in this invention should be understood to mean any one of the options or any combination of two or more of the options.

[0044] Example 1: CD19-CAR sequence (81z) with CD81 co-stimulatory domain

[0045] The chimeric antigen receptor CAR molecular structure provided in this embodiment is as follows: Figure 1As shown, the CAR structure mainly consists of an IgG leader region (amino acid sequence as shown in SEQ ID NO:1), an FMC63 VL (amino acid sequence as shown in SEQ ID NO:2), a linker (amino acid sequence as shown in SEQ ID NO:3), an FMC63 VH (amino acid sequence as shown in SEQ ID NO:4), an IgG4 hinge (amino acid sequence as shown in SEQ ID NO:5), a CD28 transmembrane region (TM) (amino acid sequence as shown in SEQ ID NO:6), a CD81 co-stimulatory domain (amino acid sequence as shown in SEQ ID NO:7), a CD3zeta domain (amino acid sequence as shown in SEQ ID NO:8), a P2A cleavage peptide (amino acid sequence as shown in SEQ ID NO:9), and a reporter gene NGFR (amino acid sequence as shown in SEQ ID NO:10), forming a chimeric antigen receptor 81z (SEQ ID NO:11) containing a CD81 co-stimulatory domain. In addition, this embodiment also designed a CAR containing a CD28 co-stimulatory domain (SEQ ID NO:12) and a CAR containing a 4-1BB co-stimulatory domain (SEQ ID NO:13) as control groups, which are 28z (SEQ ID NO:14) and BBz (SEQ ID NO:15), respectively.

[0046] The amino acid sequence of the IgG leader region: MEFGLSWLFLVAILKGVQCSR (SEQ ID NO:1);

[0047] The amino acid sequence of FMC63 VL:

[0048] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLELKR(SEQ ID NO:2);

[0049] The amino acid sequence of the linker:

[0050] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3);

[0051] The amino acid sequence of FMC63 VH:

[0052] EVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVTVSS(SEQ ID NO:4);

[0053] The amino acid sequence of the IgG4 hinge region:

[0054] ESKYGPPCPPCP(SEQ ID NO:5);

[0055] The amino acid sequence of the transmembrane region of CD28:

[0056] FWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:6);

[0057] The amino acid sequence of the CD81 co-stimulatory domain:

[0058] VLCCGIRNSSVY (SEQ ID NO:7);

[0059] The amino acid sequence of the CD3zeta domain:

[0060] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 8);

[0061] The amino acid sequence of the cleaved peptide P2A:

[0062] ATNFSLLKQAGDVEENPGP(SEQ ID NO:9);

[0063] The amino acid sequence of the reporter gene NGFR:

[0064] MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNS(SEQ ID NO:10);

[0065] Amino acid sequence of 81z:

[0066] MEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVVLCCGIRNSSVYRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNS(SEQ ID NO:11);

[0067] Amino acid sequence of the CD28 co-stimulatory domain:

[0068] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:12);

[0069] Amino acid sequence of 28z:

[0070] MEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNS(SEQ ID NO:14);

[0071] Amino acid sequence of the 4-1BB costimulatory domain:

[0072] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:13);

[0073] Amino acid sequence of BBz:

[0074] (SEQ ID NO:15).

[0075] Example 2: Packaging of Retroviruses

[0076] DNA encoding 81z, 28z, and BBz was synthesized from the whole genome, and subcloned into the retroviral vector pSFG after double digestion with Nco I and Mlu I. The vectors were then transformed into competent DH5α cells, and single clones were selected for plasmid extraction. After enzyme digestion and identification, the clones were sent for sequencing to further confirm the CAR sequence.

[0077] The vector plasmid containing the CAR structure, along with the packaging plasmids PegPam3 and RDF, were mixed at a mass ratio of 3.75:3.75:2.5 μg. Then, 30 μL of P3000 and 1 mL of Opti-MEM were added. In another centrifuge tube, 30 μL of transfection reagent Lipo3000 and 1 mL of Opti-MEM were mixed and allowed to stand for 5 minutes. The Opti-MEM mixture containing Lipo3000 was added dropwise to the plasmid DNA mixture while shaking to mix, and allowed to stand at room temperature for 20 minutes. Finally, the above mixture was added dropwise to a pre-layered 293T cell culture dish (10 cm), gently shaken to mix, and incubated for 48 and 72 hours. The viral supernatant was collected after centrifugation to remove cell debris, filtered through a 0.45 μm syringe filter, aliquoted into 15 mL centrifuge tubes, and stored at -80°C for later use.

[0078] Nucleotide sequence encoding 81z:

[0079]

[0080] Example 3: Preparation of CAR-T cells with CD81 co-stimulatory domain

[0081] (1) Extraction of human peripheral blood mononuclear cells (PBMCs)

[0082] Fresh PBMC isolation and cryopreservation: Take the white membrane layer of PBMCs, dilute them 1:1 with PBS, carefully add a blood sample to the surface of the separation liquid, centrifuge at 800g for 20 minutes at 22℃, with an ascending speed of 1 and a descending speed of 0; after centrifugation, the centrifuge tube will separate into 4 layers from top to bottom: the first layer is the plasma layer, the second layer is the ring-shaped milky white lymphocyte layer, the third layer is the clear layer, and the fourth layer is the red blood cell layer; take the white ring layer, wash twice with more than 3 times the volume of PBS, centrifuge at 150g for 10 minutes, and discard the supernatant; count the cells after a 1:1 dilution of trypan blue, and cryopreserve in FBS-DMSO cryopreservation solution (9:1) for 2×10 7 / Tube.

[0083] Resuscitation of cryopreserved PBMCs: Remove PBMCs from the liquid nitrogen tank and incubate at 37°C for 1 minute; add 9 mL of RPMI-1640 culture medium to a 15 mL centrifuge tube, add PBMC cryopreservation solution dropwise, centrifuge at 300 g for 5 minutes, and count the cells after 1:1 dilution with trypan blue before centrifugation; discard the supernatant and break up the cell clumps by vibratory tapping.

[0084] (2) T cell activation (Day 0)

[0085] Following the instructions of the T Cell TransAct kit from Miltenyi Biotechnology (Germany), Anti-CD3 / CD28 activated PBMCs were added with magnetic beads and cultured in Excell's OptiVitro serum-free T cell medium, supplemented with 5% human AB serum (GeminiBio) (complete T cell culture medium) and 100 IU / mL recombinant human IL-2 (PeproTech).

[0086] (3) Retrovirus transduction of T cells (Day 2)

[0087] CAR gene transduction can be performed 48 hours after T cell activation. The cells can be coated with RetroNectin at 4°C one day in advance, or at 37°C for 2 hours. Prepare the appropriate volume of 7μg / mL RetroNectin solution with PBS and coat the 24-well plate. Seal the edge of the well with Parafilm and coat overnight at 4°C. After the RetroNectin solution is recovered, freeze it at -20°C. It can be used twice.

[0088] Remove the viral solution half an hour before the scheduled time, thaw at room temperature, and mix the 48-hour and 72-hour viral solutions at a 1:1 ratio. Discard the RetroNectin solution coating in the 24-well plate, and quickly add 2 mL of retrovirus containing 81 oz, 28 oz, or BB oz to each well. Seal the edges with Parafilm and centrifuge at 32°C for 2000g × 90 minutes. During centrifugation, collect and count the activated T cells, centrifuge at 300g × 5 minutes, and culture in OptiVitro T cell complete medium (containing 100 IU / mL IL-2). Resuspend the cells at 0.1 × 10⁻⁶. 6 / mL. Gently aspirate the virus solution, then gently add 2mL of T cells (0.2×10⁹ / mL) to each well. 6 / hole, two holes are operated each time to prevent the holes from drying out, and a non-transduction control group is set up at the same time.

[0089] After adding cells to the well plate, centrifuge at 300g for 5 minutes to allow the cells to reach the bottom for better contact with the virus. Three days after T cell transduction with CAR virus, count the cells and collect 0.2 × 10⁻⁶ cells. 6 The CAR expression rate of cells was detected by flow cytometry. Figure 2 The remaining cells were centrifuged at 300g for 5 minutes and then expanded using OptiVitro T cells containing IL-2 in a 37°C 5% CO2 incubator.

[0090] Example 4: 81z CAR-T cells secrete lower levels of cytotoxic factors

[0091] CD107a expression on the membrane is considered a marker of activated cytotoxic lymphocytes, including CD8. + T cells and NK cells. Flow cytometry analysis was used to assess the direct correlation between CD107a expression and T cell cytotoxic activity and cytokine secretion.

[0092] Effector cells such as 81z CAR-T cells, 28z CAR-T cells, and BBz CAR-T cells were respectively inoculated with CD19 + Nalm6 target cells were cultured in 96-well U-shaped plates containing cytokine-free medium at a 5:1 ratio, with CD107a antibody added. After 4 hours of culture, cells were collected by centrifugation, and extracellular surface staining was performed using antibodies against human CD3 and NGFR. After staining at room temperature for 20 minutes, the cells were washed once with PBS. Cells were then fixed and permeabilized using BD Fixation and Permeabilization Solution according to the manufacturer's instructions. Intracellular staining was then performed using antibodies against human IFN-γ, TNF-α, and Granzyme B. Finally, CD3+ was analyzed by flow cytometry.+ NGFR + Expression of CD107a, IFN-γ, TNF-α and Granzyme B in T cells.

[0093] The results are as follows Figure 3 As shown, after stimulation by target cells, the expression levels of CD107a, IFN-γ, TNF-α, and Granzyme B in 81z CAR-T cells were significantly lower than those in 28z CAR-T cells and BBz CAR-T cells, suggesting that 81z CAR-T cells have weaker T-cell cytotoxic activity and cytokine release capacity; CD81 co-spiking domain CAR-T cells have weaker activity and the ability to secrete antitumor factors IFN-γ, TNF-α, and Granzyme B.

[0094] Example 5: 81z CAR-T cells target and kill CD19 + Tumor cells

[0095] Tumor cell killing efficiency was evaluated using a luciferase-based cytotoxicity assay. First, CD19-positive Burikit lymphoma cells (Raji), human B-lymphoblastic leukemia cells (Nalm6), and CD19-negative human chronic myeloid leukemia cells (K562) expressing the luciferase reporter gene Luciferase were used as target cells. These cells were mixed with effector cells (81z CAR-T cells, 28z CAR-T cells, BBz CAR-T cells, and NT (unmodified)-T cells) at different effector-to-target cell ratios of 8:1, 4:1, 2:1, 1:1, 0.5:1, and 0.25:1) in 96-well plates containing cytokine-free medium. After 6 hours of co-culturing, the reporter gene substrate Luciferin was added, and the release of biofluorescence (RLU) was then detected. Using target cells cultured alone as a control group, the target cell lysis rate (% Lysis) was calculated as: [(RLU of control wells - RLU of test wells) / RLU of control wells] × 100%.

[0096] The results are as follows Figure 4 As shown, regardless of the effector-to-target ratio, 81z CAR-T cells can effectively kill CD19-positive lymphoma cells Raji and human B-lymphoblastic leukemia cells Nalm6, and are significantly higher than NT-T cells. However, the killing efficiency against CD19-negative human chronic myeloid leukemia cells K562 is not significantly different from that of NT-T cells, and this targeted killing ability is comparable to that of 28z CAR-T and BBz CAR-T cells.

[0097] Example 6: 81z CAR-T cells can effectively eliminate leukemia in mice.

[0098] Nalm6 cells expressing Luciferase (5 × 10⁻⁶) 5 ) was injected into severely immunodeficient (NSG) mice via the tail vein, and 5 × 10⁵ mice were injected via the tail vein 4 days later. 6 81z CAR-T cells, 28z CAR-T cells, and BBz CAR-T cells were injected into NSG mice with tumor burden. After continuous observation for 24 days, the substrate Luciferin (15 mg / mL) was injected into the mice intraperitoneally at a dose of 10 μL / g of mouse body weight. After waiting for 5 minutes, the tumor burden in vivo was detected by an in vivo imaging system.

[0099] The results are as follows Figure 5 As shown, leukemia progressed rapidly in untreated (PBS) mice, while leukemia was controlled in mice treated with 81z CAR-T cells, 28z CAR-T cells, and BBz CAR-T cells, with 81z CAR-T cells demonstrating good therapeutic effects.

[0100] Example 7: Decreased IL-1β and IL-6 release from monocytes mediated by CD81 co-stimulatory domain CAR-T cells

[0101] First, PAM was used to induce THP1 monocytes to differentiate into monocytes. Then, 81z CAR-T cells, 28z CAR-T cells, and BBz CAR-T cells were co-cultured with CD19 positive tumor cells Nalm6 for 24 hours. The supernatant was then used to destimulate the monocytes for 24 hours. The release of inflammatory factors IL-1β and IL-6 was detected by ELISA.

[0102] The results are as follows Figure 6 As shown, CAR-T cells themselves can release a small amount of IL-6. After stimulating monocytes with the supernatant of CAR-T cells and tumor cells, the levels of IL-1β and IL-6 were significantly upregulated. However, the release of IL-1β and IL-6 in monocytes mediated by 81z CAR-T was significantly lower than that of 28z CAR-T and BBz CAR-T cells.

[0103] Example 8: 81z CAR-T cell therapy can effectively reduce CRS response in humanized mice.

[0104] Human-made CD34 + Cell preparation: Human umbilical cord blood was collected and density gradient centrifuged to obtain mononuclear cells (CBMCs). Cells were counted in increments of 10-1. 8Add 300 μL of MACS buffer to each CBMC, then add 100 μL of FCR Blocking Reagent and CD34 magnetic beads, mix well, and incubate at 4°C for 30 minutes. After incubation, repeat every 10 minutes. 8 Wash each CBMC with 5 mL of MACS buffer, centrifuge at 300 g for 10 minutes, and discard the supernatant. Every 10 8 One CBMC was resuspended in 500 μL of MACS buffer. Before sorting, an LS sorting column and a 15 mL centrifuge tube were installed. The sorting column was rinsed with 3 mL of MACS buffer, and then cell suspension incubated with antibody was added. The sorting column was rinsed three times with 3 mL of MACS buffer. The sorting column was removed from the magnetic rack, 5 mL of MACS buffer was added, and the liquid was collected in a 15 mL centrifuge tube using the stopcock. This is CD34. + Hematopoietic stem cells (HSCs). Take 10. 5 Each positively selected cell was stained with CD34 antibody, and the CD34 level was determined by flow cytometry. + The proportion of HSC is over 95%.

[0105] Establishment of the HSC humanized immunodeficient mouse model: Female NOD.Cg-Prkdc^scid Il2rg^tm1Wjl / SzJ (NSG) mice aged 4-6 weeks and weighing 18-22g were selected and housed in an SPF-grade barrier environment with a 12-hour light / dark cycle, and were allowed free access to 60 Co-irradiated sterilized feed and acidified water. All procedures were approved by the institution's laboratory animal ethics committee. Four hours before transplantation, mice were given a single whole-body gamma irradiation at a dose of 1.5 Gy (dose rate 0.5 Gy / min). 250 μL of cell suspension was slowly injected via the tail vein using a 1 mL insulin syringe, with each mouse receiving 1 × 10-1 cells. 5 CD34 + Cells were transplanted on Day 0. At weeks 4, 8, and 12 post-transplantation, 100 μL of blood was collected via the medial canthal venous plexus and anticoagulated with EDTA. After erythrocyte lysis, the following antibody combination was used for flow cytometry analysis: anti-human CD45-APC (HI30), anti-human CD3-BV421 (OKT3), and anti-human CD19-FITC (HIB19). Data was collected using a BD Fortessa X20 and analyzed using FlowJo v10. Successful humanization criteria: peripheral blood human CD45... + Cells ≥20%, and no decreasing trend in two consecutive flow cytometry analyses.

[0106] Safety study of CD81 co-stimulatory domain CAR-T therapy in CRS mouse model: Humanized mice at week 12 were injected via tail vein with Nalm6 leukemia cells expressing CD19 (1×10⁻⁶). 6 One week later, 1×10⁻⁶ was injected via the tail vein. 7 81z CAR-T cells, 28z CAR-T cells, BBz CAR-T cells, and unmodified T cells (NT-T) were introduced into tumor-burden-humanized NSG mice. The mice's body weight, body temperature changes, and the release of CRS-related cytokines such as IL-6 and IFN-γ were closely monitored. Flow cytometry was used to detect CAR-T engraftment and Nalm6 cell residue in peripheral blood, spleen, liver, and bone marrow. The survival status of the mice was closely monitored.

[0107] The results are as follows Figure 7 As shown, on day 13 post-treatment, tumor cells remained in the bone marrow of mice in the untransduced T-cell therapy group, while tumor cells were almost completely eliminated from the bone marrow of mice in the CAR-T cell therapy group. Serum levels of CRS-related inflammatory factors IL-6 and IFN-γ in the 81z CAR-T cell therapy group were significantly lower than those in the 28z CAR-T cell and BBz CAR-T cell therapy groups, and the survival time of mice in the 81z CAR-T cell therapy group was significantly longer than that of mice in the 28z and BBz CAR-T cell therapy groups.

[0108] The above detailed embodiments have provided a clear description of the present invention, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0109] Obviously, the embodiments described above are only some embodiments of this application, not all embodiments. The accompanying drawings show preferred embodiments of this application, but do not limit the patent scope of this application. This application can be implemented in many different forms; rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this application's specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this application.

Claims

1. A chimeric antigen receptor with a CD81 co-stimulatory domain that can reduce the occurrence of CRS, characterized in that, The co-stimulatory domain of the chimeric antigen receptor is a CD81 co-stimulatory domain; the amino acid sequence of the CD81 co-stimulatory domain is shown in SEQ ID NO.7; the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.

11.

2. A chimeric antigen receptor with a CD81 co-stimulatory domain according to claim 1, characterized in that, The chimeric antigen receptor also includes a signal peptide, a cleavage peptide, and / or a tag protein.

3. A chimeric antigen receptor with a CD81 co-stimulatory domain according to claim 2, characterized in that, The signal peptide is selected from at least one of the following signal peptides: CD8α, CD80, CD86, IgG1, and IgG4.

4. A chimeric antigen receptor with a CD81 co-stimulatory domain according to claim 2, characterized in that, The transmembrane domain is selected from at least one of the transmembrane domains of the following molecules: CD8α, CD28, NKG2D, 2B4, DNAM1, and CD3ζ.

5. A chimeric antigen receptor with a CD81 co-stimulatory domain according to claim 2, characterized in that, The tag protein is selected from at least one of NGFR, EGFP, and mCherry.

6. A nucleotide encoding a chimeric antigen receptor having a CD81 co-stimulatory domain, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

16.

7. A vector expressing a chimeric antigen receptor having a CD81 co-stimulatory domain, characterized in that, The carrier comprises the nucleotides of claim 6.

8. A cell expressing a chimeric antigen receptor having a CD81 co-stimulatory domain, characterized in that, The cell comprises the chimeric antigen receptor of any one of claims 1-5, the nucleotide of claim 6, or the vector of claim 7.

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