A chimeric antigen receptor (CAR) targeting BCMA and its anticancer use

Abstract

The present invention provides a chimeric antigen receptor (CAR) targeting BCMA and its anticancer use, wherein the chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region comprises a signal peptide, an anti BCMA scFv and a hinge region, wherein the intracellular region comprises a co-stimulatory factor and a CD3ζ signal domain, and wherein the anti BCMA scFv comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 1 and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 2. The BCMA-targeted antigen binding domain has a unique amino acid structure and can specifically bind to the target antigen, and the CAR-T cells can effectively inhibit tumor cell proliferation in vivo and in vitro, and prolong the survival of experimental animals.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a chimeric antigen receptor (CAR) targeting BCMA and its anti-cancer use. Background Art

[0002] Multiple myeloma (MM) is a plasma cell malignancy characterized by clonal proliferation of malignant plasma cells in the bone marrow, accompanied by overproduction of monoclonal immunoglobulins (called M proteins) and subsequent end-organ damage, accounting for approximately 10% of hematological malignancies. With the deepening of the understanding of the pathogenesis of MM and the application of new therapeutic drugs, such as proteasome inhibitors (such as bortezomib, ixazomib and carfilzomib), immunomodulatory drugs (such as thalidomide, lenalidomide and pomalidomide), monoclonal antibodies (such as daratumumab, ixatuximab and elotuzumab) and selective nuclear export inhibitors (such as selinexor), the survival outcomes of MM patients have been greatly improved (see Facon T, Kumar S, Plesner T, Orlowski RZ, Moreau P, Bahlis N, et al..Daratumumab plus lenalidomide and dexamethasone for untreated myeloma. N Engl J Med (2019) 380 (22): 2104–15.). However, almost all patients with multiple myeloma will eventually relapse, especially those with extramedullary disease (EMD) or high-risk cytogenetic abnormalities. In addition, under the selective pressure of treatment, MM cells often undergo clonal evolution, which may lead to disease progression and resistance to conventional treatments. Therefore, new therapeutic approaches are urgently needed to treat R / R multiple myeloma.

[0003] In recent years, chimeric antigen receptor T (CAR-T) cell therapy has become a promising immunotherapy and has profoundly changed the treatment landscape for hematological malignancies. To generate CAR-T cells that can specifically recognize tumor surface antigens, T cells from patients or healthy donors are genetically modified to have a specific tumor-targeting receptor, which is called a chimeric antigen receptor (CAR). The CAR structure contains a single-chain variable fragment (scFv) that can specifically recognize tumor surface antigens without the need for MHC-restricted antigen presentation. Similar to effector T cells, CAR-T cells can also mediate tumor killing in a variety of ways, including secretion of cytotoxic granules containing perforin and granzymes, production of proinflammatory cytokines such as IFN-γ and TNF-α, and activation of the Fas / Fas ligand (Fas / FasL) pathway. Currently, B cell maturation antigen (BCMA) is the most successful target of CAR-T cell therapy in MM. Anti-BCMA CAR-T cell therapy has achieved unprecedented remission in R / R MM patients, bringing new hope to R / R MM patients (see Xu J, Chen LJ, Yang SS, Sun Y, Wu W, Liu YF, et al.. Exploratory trial of a biepitopic CAR T-targeting b cell maturation antigen in relapsed / refractory multiple myeloma. Proc Natl Acad Sci USA (2019) 116 (19): 9543–51; Wang D, Wang J, Hu G, Wang W, Xiao Y, Cai H, et al.. A phase 1 study of a novel fully human BCMA-targeting CAR (CT103A) in patients with relapsed / refractory multiple myeloma.Blood (2021) 137(21):2890–901). Currently, two anti-BCMACAR-T cell products, idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel), have been approved by the US Food and Drug Administration (FDA) for the treatment of R / R MM.In recent years, with the development of CAR-T cell clinical trials, CAR-T-related adverse events have gradually been recognized and are generally controllable, such as cytokine release syndrome, CAR-T cell-related encephalopathy syndrome, hematopoiesis, infection, etc. In particular, due to the humoral immune deficiency of MM patients, as well as B cell aplasia caused by lymphocyte depletion chemotherapy and anti-BCMACAR-T cell therapy, patients are extremely susceptible to bacterial infection. Therefore, for these immunocompromised patients, immunoglobulin supplementation and preventive anti-infection treatment are extremely necessary. To this end, researchers explore effective improvement strategies from different angles.

[0004] On the one hand, overcome antigen escape. Most MM patients will still relapse after receiving anti-BCMA C-T cell therapy. One of the main mechanisms is antigen downregulation or antigen loss under treatment pressure (see Gazeau N, Beauvais D, Yakoub-Agha I, Mitra S, Campbell TB, Facon T, et al.. Effective anti-BCMA retreatment in multiple myeloma. Blood Adv (2021) 5 (15): 3016–20). Therefore, targeting different surface antigens is an effective strategy to prevent antigen negative escape. Multiple alternative targets are being discovered, including CD138, CD38, CD19, GPRC5D, SLAMF7 (CS1), APRIL, TACI, CD229, CD56, MUC1, NKG2D ligand, integrin β7, κ light chain, FcRH5, CCR10 and CD44v6 (see Radhakrishnan SV, Luetkens T, Scherer SD, Davis P, Vander MauseER, Olson ML, et al.. CD229 CAR T cells eliminate multiple myeloma and tumor propagating cells without fratricide. Nat Commun (2020) 11 (1): 798; Hosen N, Matsunaga Y, Hasegawa K, Matsuno H, Nakamura Y, Makita M, et al.. The activated conformation of integrin β7 is a novel multiple myeloma-specific target for CART cell therapy.Nat Med (2017) 23(12):1436–43), and it can be combined with BCMA to form a bispecific antibody or bispecific CAR-T.

[0005] On the other hand, CAR-T cell exhaustion can be prevented. MM patients only achieve short-term clinical remission after anti-BCMACAR-T cell therapy, partly due to CAR-T cell exhaustion, which manifests as poor persistence and dysfunction of CAR-T cells. It is currently believed that CAR-T cell exhaustion involves multiple factors, including continuous antigen stimulation and immunosuppressive tumor microenvironment, as well as impaired T cell function caused by previous anti-myeloma treatment. There are many potential strategies to improve CAR-T cell dysfunction, such as optimizing CAR-T cell structure, utilizing early memory T cells, and inhibiting intracellular exhaustion-related signals through genetic modification or inhibitors (see McLellan AD, Ali Hosseini Rad SM. Chimeric antigen receptor T cell persistence and memory cell formation. Immunol Cell Biol (2019) 97 (7): 664–74)

[0006] In view of some shortcomings and deficiencies of CAR-T targeting BCMA, the present invention obtains a scFv structure targeting BCMA with higher affinity, and transforms the CAR structure, which can improve the anti-tumor activity of CAR-T cells, inhibit T cell exhaustion, and effectively treat tumor diseases. Summary of the invention

[0007] The first aspect of the present invention provides a chimeric antigen receptor CAR targeting BCMA, wherein the chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region comprises a signal peptide, an anti BCMA scFv and a hinge region, wherein the intracellular region comprises a co-stimulatory factor and a CD3ζ signaling domain, and the anti BCMA scFv comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:1 and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:2.

[0008] Furthermore, the amino acid sequence of the signal peptide is shown in SEQ ID NO:3.

[0009] Furthermore, the hinge region and transmembrane region are selected from the CD8 extracellular region and transmembrane region, and their amino acid sequences are shown in SEQ ID NO:4.

[0010] Furthermore, the co-stimulatory factor is selected from at least one of CD28, 4-1BB, ICOS, and OX40.

[0011] Furthermore, the co-stimulatory factors are OX40 and CD28.

[0012] Furthermore, the amino acid sequence of CD28 is shown in SEQ ID NO:5.

[0013] Furthermore, the amino acid sequence of OX40 is shown in SEQ ID NO:6.

[0014] Furthermore, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:9.

[0015] Currently, CD28, 4-1BB, ICOS and OX40 are the most commonly used co-stimulatory molecules in CAR-T cell production. CD28 co-stimulation can trigger strong activation of T cells, and thus may accelerate CAR-T cell exhaustion; on the contrary, 4-1BB co-stimulation can promote the expansion of stem cell memory T cells and improve CAR-T cell exhaustion (see Long AH, Haso WM, Shern JF, Wanhainen KM, Murgai M, Ingaramo M, et al.. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med (2015) 21 (6): 581–90). ICOS is a member of the CD28 family. The combination of ICOS and 4-1BB co-stimulation can significantly improve the persistence of CAR-T cells (see Guedan S, Posey AD, Jr, Shaw C, Wing A, Da T, Patel PR, et al.. Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight (2018) 3 (1): e96976). As a member of the TNF-R superfamily, OX40 has the ability to promote T cell proliferation and memory formation. Recent studies have shown that compared with 4-1BB-mediated BCMA-targeted CAR-T cells, OX40-mediated BCMA-targeted CAR-T cells exhibit stronger proliferation ability and more persistent anti-tumor activity under repeated BCMA stimulation (see Tan J, Jia Y, Zhou M, Fu C, Tuhin IJ, Ye J, et al.. Chimeric antigen receptors containing the OX40 signalling domain enhance the persistence of T cells even under repeated stimulation with multiple myeloma target cells. J Hematol Oncol (2022) 15 (1): 39). In addition, the fully humanized CAR structure can reduce the immunogenicity of anti-BCMA CAR-T cells and avoid immune rejection mediated by the host immune system.More importantly, a phase 1 clinical trial demonstrated that R / R MM patients who relapsed after receiving mouse anti-BCMA CAR-T cell therapy can also obtain clinical responses from fully humanized anti-BCMA CAR-T cells (see Wang D, Wang J, Hu G, Wang W, Xiao Y, Cai H, et al.. A phase 1 study of a novelfully human BCMA-targeting CAR (CT103A) in patients with relapsed / refractory multiple myeloma. Blood (2021) 137 (21): 2890–901).

[0016] A second aspect of the present invention provides a CAR-T cell targeting BCMA, wherein the CAR-T cell expresses the chimeric antigen receptor.

[0017] The third aspect of the present invention provides a use of the chimeric antigen receptor and / or the CAR-T cell in the preparation of a drug for treating tumors.

[0018] Furthermore, the tumor is myeloma.

[0019] Beneficial Effects

[0020] The present invention provides a chimeric antigen receptor (CAR) targeting BCMA and its anti-cancer use, with the following specific beneficial effects:

[0021] (1) Providing a BCMA-targeted antigen-binding domain with a unique amino acid structure that can specifically bind to the target antigen;

[0022] (2) They attempted to construct CARs using different co-stimulatory factors and obtained CARs that could effectively kill tumor cells and secrete high levels of cytokines;

[0023] (3) The CAR-T cells can effectively inhibit tumor cell proliferation in vivo and in vitro and prolong the survival of experimental animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Schematic diagram of CAR structure;

[0025] Figure 2 : CAR-T cell tumor killing ability;

[0026] Figure 3 : CAR-T cells promote perforin secretion;

[0027] Figure 4 : CAR-T cells promote the secretion of granzyme B;

[0028] Figure 5 :CAR-T cells promote IFN-γ secretion.

[0029] Figure 6 : Animal survival period. DETAILED DESCRIPTION

[0030] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits are all commercially available unless otherwise specified.

[0031] Example 1 Structure of CAR

[0032] 1.1 Acquisition of BCMA-targeted antigen-binding domain

[0033] The present invention obtains a monoclonal antibody targeting BCMA through hybridoma technology. After sequence determination and bioinformatics analysis, the amino acid sequence of the light chain variable region is determined as shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is determined as shown in SEQ ID NO: 2. Further, the present invention investigates the affinity of the antibody to the target antigen. After determination, its Kd value can reach 6.31nM, which can effectively recognize the target antigen. Since the antibody can bind to the BCMA antigen with high specificity, the present invention uses its scFv region as the antigen binding domain in the chimeric antigen receptor.

[0034] 1.2 CAR structural design

[0035] BCMA is a common target in CAR-T cells. Most of the CAR-T cells targeting BCMA that have been marketed or entered the clinical trial stage are second-generation CAR-T, that is, CAR-T with CD28 or 4-1BB as co-stimulatory factors. However, existing CAR-T cells are prone to T cell exhaustion, resulting in poor therapeutic effects. To this end, the present invention attempts to use different T cell co-stimulatory factors to obtain better anti-tumor effects.

[0036] To this end, various CAR structures are designed in the present invention, such as Figure 1As shown, including traditional second-generation CARs such as CAR-01 and CAR-02; also including third-generation CARs with dual co-stimulatory factors, such as CAR-03, CAR-04, CAR-05, etc., hoping to inhibit T cell exhaustion by selecting different co-stimulatory factors. In the CAR structure, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 3; the hinge region and the transmembrane region are selected from the extracellular region and the transmembrane region of CD8, and its amino acid sequence is shown in SEQ ID NO: 4; the amino acid sequence of CD28 is shown in SEQ ID NO: 5, the amino acid sequence of OX40 is shown in SEQ ID NO: 6, the amino acid sequence of 4-1BB is shown in SEQ ID NO: 7, and the amino acid sequence of ICOS is shown in SEQ ID NO: 8. Taking CAR04 as an example, its amino acid sequence is shown in SEQ ID NO: 9, and the nucleotide sequence is shown in SEQ ID NO: 10.

[0037] Example 2 Preparation of CAR-T cells

[0038] Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy volunteers and cultured in RPMI1640 medium containing 100 mL / L FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. All cells were cultured in an incubator at 37°C and 5% CO2 until the logarithmic growth phase. 6 / well, T cells were plated in a 24-well plate; CD3 / CD28 magnetic beads were taken out, and magnetic beads were added at a ratio of T cells: CD3 / CD28 magnetic beads of 1:1. Before adding the magnetic beads, they were washed 3 times with serum-free T cell culture medium to screen CD3+CD28+T cells; after obtaining the target T cells, they were placed in complete culture medium containing 50 IU / mL of IL-2 and incubated for 24 hours to activate the T cells.

[0039] The nucleotide sequence encoding the CAR was amplified by PCR and ligated to the retroviral vector MSCV (purchased from Jiangsu Miaoling Biotechnology Co., Ltd.) by enzyme cutting, and then transfected into DH5α competent cells, inoculated into 100mL LB liquid culture medium, and cultured at 37°C and 180rpm for 12-16h. The bacteria were collected by centrifugation, and the plasmid was extracted. The sequence was correct after retrieval.

[0040] 20 μg of the constructed CAR-carrying MSCV plasmid and 5 μg of the retroviral packaging helper plasmid were mixed evenly and added to 460 μL Opti-MEM basal medium. Then 40 μL Lipofectamine 3000 was added, mixed evenly and dripped into a 6-well plate inoculated with platE cells. The cells were cultured at 37°C and 5% CO2 for 6 h, the cell supernatant was discarded, and DMEM basal medium was added. After culturing for 48 h, the supernatant was removed by centrifugation at 2500g and 4°C for 10 min. The supernatant was concentrated to obtain a retroviral concentrate. The titer of the concentrate was determined to be 1.31×10 8 pfu / mL or more. The retrovirus was added to the activated T cells at an MOI of 10, and incubated at 37°C and 5% CO2 for 12-24h. After measurement, highly expressed CAR was obtained, and the corresponding cells were recorded as CAR-T 01, CAR-T 02, CAR-T 03, CAR-T 04 and CAR-T 05.

[0041] Example 3 CAR-T kills tumor cell growth

[0042] In this section, the human myeloma cell line MM1.S was used as the research object to investigate the anti-tumor effect of CAR-T cells.

[0043] 3.1CAR-T inhibits tumor cell growth

[0044] After the tumor cells and CAR-T cells were cultured to the logarithmic growth phase, they were inoculated in a 96-well plate at an effector-target ratio of 5:1 and cultured in a 37°C, 5% CO2 incubator. The tumor killing ability was detected by LDH method at 12h, 24h, 36h and 48h after culture. The specific detection method includes: centrifugation to remove the supernatant, adding 50μL / well of the detection mixture, mixing evenly and incubating at room temperature in the dark for 30min; adding 50μL / well of the stop solution, shaking and mixing thoroughly, using a spectrophotometer to detect the absorbance at 492nm, and calculating the cell killing rate = (experimental calibration well OD value-effect cell spontaneous calibration well OD value-target cell spontaneous calibration well OD value) / (target cell maximum release calibration well OD value-target cell spontaneous calibration well OD value) × 100%.

[0045] The results are as follows Figure 2As shown in the figure, the anti-tumor effect of CAR-T01 with 4-1BB as a co-stimulatory factor is stronger than that of CAR-T02 with CD28 as a co-stimulatory factor, but the anti-tumor effect of CAR-T02 is stronger in the early stage, indicating that although CD28 can strongly activate T cells, it also causes premature exhaustion of T cells, making it difficult to exert sustained and effective tumor killing power. Compared with the second-generation CAR-T, the anti-tumor effect of the third-generation CAR-T with dual co-stimulatory factors has been significantly enhanced, among which CAR-T04 and CAR-T05 have the strongest effect, which may be due to the fact that OX40 and ICOS have a certain repair ability for T cells, allowing the CAR-T cells to continue to play a role.

[0046] 3.2CAR-T cells promote the secretion of perforin and granzymes

[0047] After CAR-T cells and tumor cells were incubated for 48 h, the supernatant was collected by centrifugation, dispensed into 1.5 mL EP tubes, and stored at -80°C. The concentrations of perforin and granzyme B in the supernatant were detected using an ELISA kit purchased from Beijing Xinbosheng Company. The specific steps were carried out according to the kit instructions.

[0048] Perforin and granzyme B are important cell-killing factors that play an important role in tumor immunotherapy and can mediate the anti-tumor response of various immune cells such as T cells, NK cells, CAR-T cells, CAR-NK cells, TCR-T cells, etc. CAR-T cells in the present invention can promote the secretion of perforin and granzyme B, such as Figure 3 , 4 As shown, CAR-T cell incubation increased the expression levels of perforin and granzyme B. In terms of perforin, CAR-T04 had the highest secretion level, followed by CAR-T03 and CAR-T05; in terms of granzyme B, CAR-T04 and CAR-T05 had the highest secretion level, followed by CAR-T03; and the overall situation of the third-generation CAR-T was better than that of the second-generation CAR-T cells.

[0049] 3.3CAR-T cells promote IFN-γ secretion

[0050] IFN-γ is an important immune factor and cytokine in the body, which can activate and recruit immune cells and regulate the secretion of other cytokines. In this section, ELISA kit (purchased from Abcam) was used to detect the level of IFN-γ in the supernatant, and the method was the same as in Section 3.2. Figure 5As shown in the figure, CAR-T cells can significantly increase the expression level of IFN-γ, and CAR-T04 and CAR-T05 have the strongest promoting effect, with no significant difference between the two, followed by CAR-T03, while CAR-T02 and CAR-T01 have relatively low expression levels.

[0051] Example 4 CAR-T treatment of tumor animal model

[0052] The second-generation CAR-T with 4-1BB as a co-stimulatory factor is a classic CAR-T cell structure, while the third-generation CAR-T with CD28 and OX40 as co-stimulatory factors has the best effect in the cell experiment of the present invention. Therefore, the present invention uses the above two CAR-T cells as therapeutic agents to carry out animal experiments to further verify the in vivo anti-tumor effect.

[0053] 4.1 Animal model preparation

[0054] Take 4-week-old SPF male NOD / SCID mice, weighing 18-20 g, and feed them for 1 week to adapt to the environment. Take MM1.S cells in the logarithmic growth phase, digest them with trypsin, collect the cells, resuspend them in sterile PBS, count them, and adjust the cell density to 2×10 7 / mL, and 100μL of cell suspension was injected subcutaneously on the back of the left upper limb of mice to establish the model. After 14 days of modeling, the tumor volume of mice reached more than 80-100mm3, indicating that the model was successfully established.

[0055] Thirty tumor-bearing mice were randomly divided into three groups, with 10 mice in each group: CAR-T01 group, intravenously injected with 1×10 7 CAR-T01 cells; CAR-T01 group, 1×10 7 CAR-T04 cells; CAR-T04 group, 1×10 7 CAR-T04 cells; CAR-T01 group, 1×10 7 CAR-T04 cells; control group, intravenous injection of normal saline.

[0056] 4.2 Animal survival

[0057] The mice's spirit and vitality were observed every day, and their survival status was recorded. The observation lasted for 30 days, and the animal survival curve was drawn. Figure 6As shown, compared with the control group, CAR-T01 and CAR-T04 can significantly prolong the survival of animals, and CAR-T04 seems to be more effective in in vivo experiments, while CAR-T01 can also effectively inhibit tumor growth, indicating that the second-generation and third-generation CAR-T cells constructed using the scFv targeting BCMA provided in the present invention can effectively inhibit tumor growth in vivo, providing a basis for the development of corresponding tumor therapeutic drugs.

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chimeric antigen receptor CAR targeting BCMA, characterized in that: The chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region comprises a signal peptide, an anti BCMA scFv and a hinge region, the intracellular region comprises a co-stimulatory factor and a CD3ζ signaling domain, and the anti BCMA scFv comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 1 and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:

2.

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

3.

3. The chimeric antigen receptor according to claim 1, characterized in that The hinge region and transmembrane region are selected from the CD8 extracellular region and transmembrane region, and the amino acid sequence thereof is shown in SEQ ID NO:

4.

4. The chimeric antigen receptor according to claim 1, characterized in that The co-stimulatory factor is selected from at least one of CD28, 4-1BB, ICOS, and OX40.

5. The chimeric antigen receptor according to claim 4, characterized in that The co-stimulatory factors are OX40 and CD28.

6. The chimeric antigen receptor according to claim 4, characterized in that The amino acid sequence of CD28 is shown in SEQ ID NO:

5.

7. The chimeric antigen receptor according to claim 4, characterized in that The amino acid sequence of OX40 is shown in SEQ ID NO:

6.

8. The chimeric antigen receptor according to claim 5, characterized in that The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:

9.

9. A CAR-T cell targeting BCMA, characterized in that: The CAR-T cell expresses the chimeric antigen receptor according to any one of claims 1-8.

10. Use of the chimeric antigen receptor according to any one of claims 1 to 8 and / or the CAR-T cell according to claim 9 in the preparation of a drug for treating a tumor, wherein the tumor is myeloma.

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