A chimeric antigen receptor (CAR) targeting CD37 and its use in anti-cancer treatment

Abstract

The present invention provides a chimeric antigen receptor (CAR) targeting CD37 and its use in anti-cancer treatment. The chimeric antigen receptor comprises a signal peptide, an anti-CD37 scFv, a hinge region, a transmembrane region, a co-stimulatory factor, and a CD3ζ signaling domain. The anti-CD37 scFv comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:2. The anti-CD37 scFv can effectively recognize the target antigen and has been humanized. Further, the chimeric antigen receptor further comprises an IL-21 domain. The CAR-T cells provided by the present invention can effectively inhibit the growth of tumor cells both in vivo and in vitro, providing a basis for the development of related drugs.

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 CD37 and its use in anti-cancer therapy. Background Art

[0002] Since its first approval by the US Food and Drug Administration (FDA) in 2017, chimeric antigen receptor (CAR)-T cell therapy has become a major weapon in the treatment of B-cell malignancies, including leukemia, lymphoma, and multiple myeloma (MM). To date, six products targeting CD19 or B-cell maturation antigen (BCMA) have been approved in the United States (see Cappell, K.M., and Kochenderfer J.N. Long-term outcomes following CAR T cell therapy: What we know so far. Nat. Rev. Clin. Oncol. 2023, 20: 359–371), and a large number of ongoing trials are evaluating other candidates for hematological malignancies and solid tumors (see Wang, X., and Rivière I.. Manufacturing of CAR-T cells: The assembly line. In Gene and Cellular Immunotherapy for Cancer. Ghobadi A., and DiPersio J.F., editor. Humana, Cham, Switzerland. 2022, 121–139). In addition, CD19 CAR-T cell therapy has recently been applied to the treatment of autoimmune diseases, and early data show encouraging results in patients with systemic lupus erythematosus (see Mackensen, A., Müller F., Mougiakakos D., et al. Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nat. Med. 2022, 28: 2124–2132)

[0003] Compared with B-cell malignancies, T-cell malignancies are also common hematological malignancies in clinical practice. Among them, T-cell acute lymphoblastic leukemia (T-ALL) accounts for about 25% of adult ALL and 10-15% of childhood ALL. T-ALL results from the leukemic transformation of thymic progenitor cells during T-cell development through the accumulation of genetic abnormalities. Over the past three decades, significant progress has been made in the treatment of ALL (including T-ALL), and the overall survival rate has continued to improve, especially in children under 15 years old. However, the survival rate of adult T-ALL patients is significantly lower than that of children and young people, and the prognosis of patients with relapsed / refractory diseases is poor, with a survival rate of 10-25% (see DuVall AS, Sheade J, Anderson D, et al. Updates in the management of relapsed and refractory acute lymphoblastic leukemia: an urgent plea for new treatment in being answered. JCO Oncol Pract. 2022, 18:479–487). CAR-T cell therapy has achieved considerable success in the treatment of B-cell malignancies. These advances in the treatment of various hematological malignancies have led to the exploration of CAR-T cell therapy in T-cell malignancies. However, expanding CAR-T cell therapy to T-cell malignancies is particularly challenging because of the co-expression of many cell membrane antigen targets between normal and malignant cells (see Liu J, Zhang Y, Guo R, et al. Targeted CD7 CAR T-cells for treatment of T-lymphocte leukemia and lymphoma and acute myeloid leukemia: recent advances. Fron Immunol. 2023, 14:1170968).

[0004] Therefore, the selection of anti-tumor targets is crucial for T cell tumor therapy. Common targets include CD7, CD5, etc. However, since CD7 and CD5 are expressed on normal T lymphocytes and NK cells, the unregulated CD7 expression on the T lymphocyte cell membrane will lead to autophagy. Therefore, the expression of related genes on the T cell membrane must be blocked, such as gene editing, protein blockers, and natural selection. For example, knocking out the CD7 gene using the CRISPR / CAS9 gene editing system can prevent autophagy and exert specific anti-tumor activity against malignant T leukemia cells (see Gomes-Silva D, Srinivasan M, Sharma S, et al. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Blood. 2017, 130: 285–296); Wong et al. developed anti-CD7 CAR-T cells (PCART7) in which both CD7 and CD3 expressions were depleted using a CD7 protein blocker. These cells showed strong cytotoxicity against T-ALL and T lymphoma cells. The protein blocker consists of a single-chain variable fragment and an intracellular retention domain, which anchors the target antigen in the endoplasmic reticulum and Golgi apparatus before proteolytic degradation, and can downregulate CD7 expression in T cells without CD7 gene editing (see Wong XFA, Ng J, Zheng S, et al. Development of an off-the-shelf chimeric antigen receptor (CAR)-T cell therapy for T-cell acute lymphoblastic leukemia (T-ALL) without gene editing. Blood. 2022, 140(suppl 1): 2358–2359).

[0005] Although the above method can obtain CAR-T cells that can effectively treat T-cell malignancies, it is necessary to block the expression of target proteins on the T-cell membrane in a complex manner. The preparation process is too cumbersome, the production cost is greatly increased, and it is not conducive to clinical use, resulting in a significant reduction in the therapeutic effect. Compared with CD7 and CD5, CD37 is a recently discovered therapeutic target for treating T-cell malignancies. It is a transmembrane protein of the tetraspanin superfamily. Some T-cell lymphomas express CD37 on their cell membranes. No significant fratricide-related events were observed during the use of CD37-targeted CAR-T (see Chun I, Kim KH, Chiang YH, et al. CRISPR-Cas9 knock out of CD5 enhances the antitumor activity of chimeric antigen receptor T cells. Blood. 2020, 136(suppl 1):51–52.). Therefore, it has the potential to become an effective therapeutic target for T-cell malignancies. However, there is currently little research on CD37 CAR-T cells, and only one study has entered the clinical trial stage (NCT 04136275), involving hematological malignancies, including leukemia, B-cell, and T-cell lymphomas (see Frigault MJ, Chen YB, Gallagher K, et al. Phase 1 study of CD37-directed CAR T cells in patients with relapsed or refractory CD37+ hematologic malignancies. Blood. 2021, 138(suppl 1):653).

[0006] In view of this, the present invention has developed a CD37-targeted CAR-T cell and its application in the preparation of anti-tumor drugs, which can effectively identify target cells, prevent self-attack of CAR-T cells, and effectively inhibit the growth of tumor cells.

[0007] T-cell activation is crucial for the success of the cell manufacturing process because it directly affects the efficiency of CAR transgene integration and T-cell expansion during in vitro culture. The most common method of T-cell activation is achieved through the combination of CD3 and CD28 stimulation with cytokine support. CD3 signaling (signal 1) triggers T-cell activation, while CD28 signaling provides the necessary co-stimulation (signal 2) to avoid ineffective immune cells. However, in clinical practice, it has been found that CD28 co-stimulation is difficult to produce sustained efficacy. CAR-T is rapidly metabolized and cleared in vivo, which is not conducive to the exertion of anti-tumor effects and may also lead to phenomena such as tumor recurrence, limiting the clinical application of CAR-T cells.

[0008] In view of some disadvantages and deficiencies of CAR-T targeting CD37, in the present invention, an scFv structure targeting CD37 with relatively high affinity was obtained, the CAR structure was modified, and the IL-21 domain was introduced, which can improve the anti-tumor activity of CAR-T cells, inhibit T cell exhaustion, and effectively treat tumor diseases. Summary of the Invention

[0009] In the first aspect of the present invention, there is provided a chimeric antigen receptor CAR targeting CD37, the chimeric antigen receptor comprising a signal peptide, anti-CD37 scFv, a hinge region, a transmembrane region, a co-stimulatory factor, and a CD3ζ signaling domain, wherein the anti-CD37 scFv comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:2.

[0010] Further, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:3.

[0011] Further, the co-stimulatory factor is CD28, and its amino acid sequence is as shown in SEQ ID NO:6.

[0012] Further, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:8.

[0013] Further, the chimeric antigen receptor further comprises an IL-21 domain, and the amino acid sequence of the IL-21 is as shown in SEQ ID NO:9.

[0014] Further, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:10.

[0015] In the second aspect of the present invention, there is provided a CAR-T cell expressing the chimeric antigen receptor described above.

[0016] In the third aspect of the present invention, there is provided an application of the chimeric antigen receptor described above and / or the CAR-T cell in the preparation of a drug for treating tumors.

[0017] Further, the tumor is a T cell malignancy.

[0018] Further, the tumor is T cell acute lymphoblastic leukemia T-ALL.

[0019] Advantageous Effects

[0020] The present invention provides a chimeric antigen receptor (CAR) targeting CD37 and its anti-cancer use, and the specific advantageous effects are as follows:

[0021] (1) Select CD37 as the target and construct CAR, and provide an anti-CD37 scFv with a brand-new amino acid structure, which can target and recognize the target antigen;

[0022] (2) Humanize the antigen-binding domain to reduce the inhibition of the anti-host reaction and maintain a long cell activation time;

[0023] (3) Add the IL-21 domain to the CAR structure to effectively maintain the activation state of T cells;

[0024] (4) The CAR-T cells provided by the present invention can effectively inhibit the growth of tumor cells in vivo and in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Schematic diagram of the CAR structure;

[0026] Figure 2 : In vitro tumor killing ability of CAR-T cells;

[0027] Figure 3 : Survival period of the tumor animal model;

[0028] Figure 4 : IFN-γ secretion level in the tumor animal model;

[0029] Figure 5 : IL-2 secretion level in the tumor animal model. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] In the following examples, the experimental methods are conventional methods unless otherwise specified; the reagent biological materials and detection kits can be obtained from commercial channels unless otherwise specified.

[0031] Example 1 Preparation of CAR-T Cells

[0032] 1.1 Obtain the antigen-binding domain targeting CD37

[0033] Mice were immunized with recombinant CD37 protein to screen for monoclonal antibodies targeting CD37. After obtaining the monoclonal antibodies, humanization was performed by placing the corresponding CDR regions in a humanized antibody framework to obtain a humanized antibody. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2. It was measured that this antibody has a high affinity for the target antigen, and the KD value can reach 5.16 nM. Some studies have shown that humanized scFv can optimize the lifespan of CAR-T cells after infusion and has advantages in terms of affinity, sensitivity, and specificity (see Pouya S K, Pooria S K, Roddy S O, Humanized Chimeric Antigen Receptor (CAR) T cells, J Cancer Immunol (Wilmington). 2021, 3(4):183–187.). Therefore, using humanized scFv to construct CAR-T cells in this application can improve the persistence of CAR-T cells and is beneficial for the exertion of continuous anti-tumor effects.

[0034] 1.2 CAR Structure Design

[0035] In the present invention, a second-generation CAR-T structure was used to construct a chimeric antigen receptor targeting CD37, and its structure is as Figure 1 shown in A, including a signal peptide, an scFv targeting CD37, a hinge region, a transmembrane region, CD28, and a CD3ζ domain. The amino acid sequence of the signal peptide is shown in SEQ ID NO:3, the amino acid sequence of the hinge region is shown in SEQ ID NO:4, the amino acid sequence of the transmembrane region is shown in SEQ ID NO:5, the amino acid sequence of CD28 is shown in SEQ ID NO:6, the amino acid sequence of CD3ζ is shown in SEQ ID NO:7, and the amino acid sequence of CAR-A is shown in SEQ ID NO:8.

[0036] In the present invention, CD28 was selected as a co-stimulatory factor to activate T cells. Although it has a strong T cell activation effect, there are also reports that CD28 can induce T cell apoptosis, thus affecting the persistence of CAR-T. In this regard, the present invention attempts to introduce IL-21 into the CAR structure. It has been reported that IL-21 can enhance the expansion of CAR-T cells after antigen stimulation, reduce the apoptosis level of CAR-T cells during co-culture with tumor cells, and prevent CAR-T cells from differentiating into a late memory phenotype (see Martin Pavlína P, Martin M, et al. Inducible secretion of IL-21 augments anti-tumor activity of piggyBac-manufactured chimeric antigen receptor T cells, Cytotherapy. 2020, 22(12):744-754). CARs with an additional IL-21 domain are as Figure 1 shown in B, where the amino acid sequence of IL-21 is as shown in SEQ ID NO:9, and the amino acid sequence of CAR-B is as shown in SEQ ID NO:10.

[0037] 1.3 Preparation of CAR-T

[0038] By genetic engineering means, the nucleotide sequences encoding CAR-A and CAR-B were cloned into the lentiviral backbone vectors KIRS2 / DAP12-BB and BBζ to construct lentiviral expression vectors. After sequencing and identification, the nucleic acid sequences were correct. 293T cells were cultured at 37 °C and 5% CO2 until the logarithmic growth phase. The lentiviral expression vectors carrying the CAR gene and three packaging plasmids were transfected into 293T cells by PEI. The medium was changed 6 h after transfection, and the virus suspension was collected at 24 h and 48 h respectively. The cell debris was removed by filtration through a 0.45 μm membrane, and the virus was collected by centrifugation at 4 °C and 12,000 rpm for 2 - 4 h. The precipitate was resuspended in the medium and stored in an -80 °C refrigerator.

[0039] Peripheral blood mononuclear cells were extracted by lymphocyte separation medium, and CD3+ / CD28+ T cells were sorted by magnetic beads. The T cells were cultured in a complete medium containing (10% FBS and 100 IU / mL IL-2). After 24 h, the lentivirus carrying the CAR gene was added at a ratio of MOI = 10, and the cells were cultured at 37 °C and 5% CO2. The medium was changed once every 1 - 2 days, and the cells were cultured for 7 - 8 days in total. After identification, the cells were able to stably express the target gene and were named CAR-T A and CAR-T B cells.

[0040] Example 2 Inhibition of ALL tumor cell proliferation by CAR-T cells

[0041] In this example, the killing effect of the CAR-T cells on the T-ALL cell line CCRF-CEM was detected to investigate their in vitro anti-tumor ability. CCRF-CEM cells were cultured in DMEM medium containing 10% FBS. After the cells grew to the logarithmic phase, the cells were digested, centrifuged and collected. After adjusting the cell density, they were inoculated into 96-well plates, and 1×10 4Cells were then added with CAR-T A and CAR-T B cells at effector-to-target ratios (E:T) of 1:1, 5:1, and 10:1 respectively. After culturing at 37 °C and 5% CO2 for 24 h, a LDH kit (purchased from Beyotime Biotechnology Co., Ltd.) was used to detect the tumor cell killing rate.

[0042] Cell killing rate = (OD value of experimental calibration well - OD value of effector cell spontaneous calibration well - OD value of target cell spontaneous calibration well) / (OD value of target cell maximum release calibration well - OD value of target cell spontaneous calibration well) × 100%

[0043] As shown in Figure 2, the CAR-T provided in the present invention can effectively inhibit the growth of tumor cells and shows a significant dose correlation; relatively speaking, the killing ability of CAR-T B is stronger than that of CAR-T A, indicating that the introduction of IL-21 can maintain the activated state of CAR-T cells and contribute to the continuous exertion of anti-tumor effects.

[0044] Example 3 Inhibition of tumor growth in animals by CAR-T cells

[0045] 3.1 Preparation of tumor animal model

[0046] NCG mice at 5 - 8 weeks old were taken and adaptively fed in an SPF-level environment for 1 week. During this period, CCRF-CEM cells were resuscitated and cultured. When the cell confluence reached 80 - 90%, subculture was carried out. The cells were digested with trypsin and then resuspended with sterile PBS, and the cell density was adjusted to 1×10 7 cells / mL. 100 μL of the cell suspension was subcutaneously injected into the tumor-bearing site of the mice. The tumor volume was measured daily using a vernier caliper. When the tumor volume reached 100 mm 3 it was used for subsequent experiments.

[0047] 3.2 Administration treatment and observation of animal survival rate

[0048] The successfully modeled mice were randomly divided into three groups, with 10 mice in each group, namely: CAR-T A group, intravenously injected with 1×10 6 CAR-T A cells; CAR-T B group, intravenously injected with 1×10 6 CAR-T B cells; control group: intravenously injected with an equal amount of normal saline. The animal status was observed and recorded daily, and a survival curve was plotted using GraphPad Prism 8 software.

[0049] The results are as shown in Figure 3As shown, the use of CAR-T A and CAR-T B cells for treatment can significantly extend the survival period of animals, indicating that the CAR-T cells provided by the present invention can effectively exert anti-tumor effects in vivo, and CAR-T B with the introduction of IL-21 seems to have a more significant therapeutic effect.

[0050] 3.3 Determination of cytokines in animals

[0051] Two weeks after the experimental animals were administered, blood was taken from the vein, plasma was separated by centrifugation, and the expression levels of IFN-γ and IL-2 in the plasma were detected using an ELISA kit (purchased from Abcam, USA). The specific detection method was carried out according to the kit instructions.

[0052] The results are as Figure 4 、 Figure 5 shown. During the CAR-T cell treatment process, the expression levels of cytokines such as IFN-γ and IL-2 were significantly increased, which can effectively activate immune cells in vivo to exert anti-tumor effects. Moreover, during the CAR-TB treatment process, the release level of cytokines was higher, especially in promoting the secretion of IFN-γ, which was significantly stronger than that of CAR-T A cells without the IL-21 domain.

[0053] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chimeric antigen receptor CAR targeting CD37, characterized in that: The chimeric antigen receptor includes a signal peptide, an anti CD37 scFv, a hinge region, a transmembrane region, a co-stimulatory factor and a CD3ζ signal domain, and the anti CD37 scFv includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region with 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 co-stimulatory factor is CD28, and its amino acid sequence is shown in SEQ ID NO:

6.

4. The chimeric antigen receptor according to claim 3, characterized in that The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:

8.

5. The chimeric antigen receptor according to any one of claims 1 to 3, characterized in that The chimeric antigen receptor also includes an IL-21 domain, and the amino acid sequence of the IL-21 is shown in SEQ ID NO:

9.

6. 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:

10.

7. A CAR-T cell, characterized in that: The CAR-T cell expresses the chimeric antigen receptor according to any one of claims 1-6.

8. Use of the chimeric antigen receptor according to any one of claims 1 to 6 and / or the CAR-T cell according to claim 7 in the preparation of a drug for treating tumors, wherein the tumor is T-cell acute lymphoblastic leukemia (T-ALL).

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