CN119080947A

CN119080947A - A chimeric antigen receptor (CAR) targeting CD37 and its anti-cancer use

Info

Publication number: CN119080947A
Application number: CN202411209636.7A
Authority: CN
Inventors: 戴宇航; 杨建华
Original assignee: Beijing Kaiji Biotechnology Co ltd
Current assignee: Mount Huangshan Yongsheng Tribal Stem Cell Technology Co ltd
Priority date: 2024-08-30
Filing date: 2024-08-30
Publication date: 2024-12-06
Anticipated expiration: 2044-08-30
Also published as: CN119080947B

Abstract

The invention provides a Chimeric Antigen Receptor (CAR) targeting CD37 and anticancer application thereof, wherein the chimeric antigen receptor comprises a signal peptide, an anti-CD 37scFv, a hinge region, a transmembrane region, a costimulatory factor and a CD3 zeta signal domain, the anti-CD 37scFv comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 1 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 2, and the anti-CD 37scFv can effectively recognize a target antigen and is subjected to humanized transformation. Further, the chimeric antigen receptor also includes an IL-21 domain. The CAR-T cell provided by the invention can effectively inhibit the growth of tumor cells in vivo and in vitro, and provides a basis for the development of related medicaments.

Description

CD 37-targeted Chimeric Antigen Receptor (CAR) and anticancer application thereof

Technical Field

The invention belongs to the field of biotechnology research and development, and particularly provides a Chimeric Antigen Receptor (CAR) targeting CD37 and an anticancer application thereof.

Background

Chimeric Antigen Receptor (CAR) -T cell therapies have been the primary weapon of treatment of B cell malignancies, including leukemia, lymphoma and Multiple Myeloma (MM), since the first approval by the united states Food and Drug Administration (FDA) in 2017. Six products against CD19 or B Cell Maturation Antigen (BCMA) have been approved to date 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 number of ongoing trials are evaluating other candidate drugs against 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.,andDiPersio J.F.,editor.Humana,Cham,Switzerland.2022,121–139). furthermore, CD19 CAR-T cell therapy has recently been applied to treat autoimmune diseases, 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)

Compared to B-cell malignancies, T-cell malignancies are also clinically common hematological tumors, in which T-cell acute lymphoblastic leukemia (T-ALL) accounts for about 25% of adult ALL, accounting for 10-15% of pediatric ALL. T-ALL is derived from leukemic transformation of thymic progenitor cells during T cell development by accumulation of genetic abnormalities. In the last three decades, significant advances have been made in ALL (including T-ALL) therapies, with an overall increase in survival rates that continue to increase, especially in children under 15 years of age, whereas adult T-ALL patients have significantly lower survival rates than children and young adults, with poor prognosis for patients with recurrent/refractory disease, with survival rates 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 inbeing answered.JCO Oncol Pract.2022,18:479–487).CAR-T cell therapies for considerable success in the treatment of B-cell malignancies, these advances in the treatment of various hematological malignancies have led to the search for CAR-T cell therapies in T cell malignancies, whereas expanding CAR-T cell therapies to T cell malignancies is particularly challenging because of the co-expression of many cellular 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).

Therefore, the selection of anti-tumor targets is critical for T cell tumor therapy. Common targets include CD7, CD5, etc., but since CD7, CD5 are expressed on normal T lymphocytes and NK cells, uninhibited expression of CD7 on T lymphocyte membranes will lead to self-phase killing, and thus the expression of related genes on T cell membranes must be blocked, such as gene editing, protein blockers, natural selection, etc. For example, knocking out CD7 gene using CRISPR/CAS9 gene editing system can prevent autogenous killing and exert specific antitumor activity on 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 for development of CD7 CAR-T cells (PCART 7) depleted in both CD7 and CD3 expression using CD7 protein blockers consisting of single-chain variable fragments and intracellular retention domains which down-regulate CD7 expression in T cells without CD7 gene editing (see CD7 gene editing Wong XFA,Ng J,Zheng S,et al.Development of an off-the-Sh elf 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).

Although CAR-T cells capable of effectively treating T cell malignancies can be obtained in the above manner, complex blocking of expression of target proteins on T cell membranes is required, the preparation process is too complicated, the production cost is greatly increased, clinical use is not facilitated, and the therapeutic effect is greatly compromised. CD37 is a recently newly discovered therapeutic target for the treatment of T cell malignancies compared to CD7 and CD5, which is a transmembrane protein of the four transmembrane protein superfamily, and part of the T cell lymphomas express CD37 on their cell membranes, and CD 37-targeted CAR-T does not observe significant autopsy-related events during use (see Chun I,Kim KH,Chiang YH,et al.CRISPR-Cas9 knock out ofCD5 enhances the antitumor activity of chiemeric antigen receptor T cells.Blood.2020,136(suppl 1):51–52.), and thus it is hopefully an effective T cell malignancy therapeutic target; but currently there is less research into CAT-T cells of CD37, only one study has entered the clinical laboratory stage (NCT 04136275), involving hematological malignancies including leukemia, B-cells and T cell lymphomas (see Frigault MJ,Chen YB,Gallagher K,et al.Phase 1study ofCD37-directed CAR T cells in patients with relapsed or refractory CD37+hematologic malignancies.Blood.2021,138(suppl1):653).

In view of the above, the invention develops a CD 37-targeted CAR-T cell and application thereof in preparing anti-tumor drugs, which can effectively identify target cells, prevent the CAR-T cell from self-phase attack and effectively inhibit the growth of tumor cells.

T cell activation is critical to the success of the cell manufacturing process, as 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 by stimulation of binding cytokine support by CD3 and CD 28. CD3 signaling (signal 1) triggers T cell activation, while CD28 signaling provides the necessary co-stimulation (signal 2) to avoid immune cell inefficiency. However, in clinical practice, it is found that the co-stimulation of CD28 is difficult to produce a sustained curative effect, and the CAR-T is rapidly metabolized and cleared in vivo, which is unfavorable for the exertion of anti-tumor effect, and also can cause tumor recurrence and other phenomena, thus limiting the clinical application of the CAR-T cells.

In view of the disadvantages and shortcomings of the CD 37-targeted CAR-T, the invention obtains the CD 37-targeted scFv structure with higher affinity, modifies the CAR structure, introduces the IL-21 structural domain, can improve the anti-tumor activity of the CAR-T cells, inhibit T cell failure and effectively treat tumor diseases.

Disclosure of Invention

In a first aspect, the invention provides a CD 37-targeting chimeric antigen receptor CAR, which comprises a signal peptide, an anti-CD 37 scFv, a hinge region, a transmembrane region, a co-stimulatory factor and a CD3 zeta signal domain, wherein the anti-CD 37 scFv comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID No. 1 and a light chain variable region having the amino acid sequence shown in SEQ ID No. 2.

Furthermore, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 3.

Furthermore, the co-stimulatory factor is CD28, and the amino acid sequence of the co-stimulatory factor is shown as SEQ ID NO. 6.

Furthermore, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 8.

Furthermore, the chimeric antigen receptor also comprises an IL-21 structural domain, and the amino acid sequence of the IL-21 is shown as SEQ ID NO. 9.

Furthermore, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 10.

In a second aspect the invention provides a CAR-T cell expressing said chimeric antigen receptor.

In a third aspect, the invention provides the use of the chimeric antigen receptor and/or the CAR-T cell in the manufacture of a medicament for the treatment of a tumour.

Further, the tumor is a T cell malignancy.

Further, the tumor is T-ALL (T-cell acute lymphoblastic leukemia).

Advantageous effects

The invention provides a Chimeric Antigen Receptor (CAR) targeting CD37 and anticancer application thereof, which have the following specific beneficial effects:

(1) Selecting CD37 as a target to construct CAR, providing anti CD37 scFv with a brand new amino acid structure, and being capable of targeting and recognizing the target antigen;

(2) The antigen binding domain is subjected to humanized transformation, so that the inhibition of anti-host reaction is reduced, and longer cell activation time can be maintained;

(3) The IL-21 structural domain is added in the CAR structure, so that the activation state of T cells can be effectively maintained;

(4) The CAR-T cell provided by the invention can effectively inhibit the growth of tumor cells in vivo and in vitro.

Drawings

FIG. 1 is a schematic diagram of CAR structure;

FIG. 2 in vitro tumor killing capacity of CAR-T cells;

FIG. 3 survival of tumor animal models;

FIG. 4 IFN-gamma secretion levels in tumor animal models;

FIG. 5 IL-2 secretion levels in tumor animal models.

Detailed Description

The experimental methods described in the examples below, if not specified, are all conventional methods, and the reagent biomaterials and the detection kits, if not specified, are all commercially available.

Example 1 preparation of CAR-T cells

1.1 Obtaining a CD 37-targeting antigen binding Domain

Immunizing a mouse with recombinant CD37 protein, screening a monoclonal antibody targeting CD37, performing humanized transformation after obtaining the monoclonal antibody, and placing a corresponding CDR region in a humanized antibody frame to obtain a humanized antibody, wherein the amino acid sequence of a heavy chain variable region of the antibody is shown as SEQ ID NO. 1, and the amino acid sequence of a light chain variable region of the antibody is shown as SEQ ID NO. 2. The antibody has higher affinity with target antigen, and KD value can reach 5.16nM. Studies show that humanized scFv can optimize the service life of CAR-T cells after infusion, and has advantages in 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, the humanized scFv is used for constructing the CAR-T cells, so that the persistence of the CAR-T cells can be improved, and the continuous anti-tumor effect can be exerted.

1.2 Structural design of CAR

The invention utilizes a second generation CAR-T structure to construct a chimeric antigen receptor targeting CD37, the structure of which is shown in figure 1A and comprises a signal peptide, scFv targeting CD37, a hinge region, a transmembrane region, CD28 and a CD3 zeta domain, wherein 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 CD3 zeta is shown in SEQ ID NO. 7, and the amino acid sequence of CAR-A is shown in SEQ ID NO. 8.

In the invention, CD28 is selected as a co-stimulatory factor to activate T cells, and although the co-stimulatory factor has strong T cell activation effect, the CD28 is also reported to induce T cell apoptosis, thereby affecting the persistence of CAR-T. In contrast, in the present invention, an attempt was made to introduce IL-21 into the CAR structure, and it was reported that IL-21 could enhance the expansion of CART cells after antigen stimulation, reduce the apoptosis level of CART cells during co-culture with tumor cells, and prevent the differentiation of CART cells to the late memory phenotype (seePavlína P,Martin M,et al.Inducible secretion of IL-21augments anti-tumor activity of piggyBac-manufactured chimeric antigen receptor T cells,Cytotherapy.2020,22(12):744-754). The CAR for increasing the domain of IL-21 is shown in FIG. 1B, wherein the amino acid sequence of IL-21 is shown in SEQ ID NO. 9, and the amino acid sequence of CAR-B is shown in SEQ ID NO. 10.

1.3CAR-T preparation

Through genetic engineering means, the nucleotide sequences encoding the CAR-A and the CAR-B are cloned into lentiviral skeleton carriers KIRS/DAP 12-BB and BBζ, lentiviral expression vectors are constructed, and the nucleic acid sequences are correct through sequencing identification. 293T cells were cultured under conditions of 37℃and 5% CO2 to a logarithmic growth phase, 293T cells were transfected with a lentiviral expression vector carrying a CAR gene and 3 packaging plasmids by PEI, the transfection was performed for 6 hours, virus suspensions were collected at 24 hours and 48 hours, cell debris was removed by filtration through a 0.45 μm membrane, viruses were collected by centrifugation at 4℃and 12000rpm for 2-4 hours, and the pellet was resuspended in a medium and stored in a-80℃refrigerator.

Peripheral blood mononuclear cells are extracted through lymphocyte separation liquid, CD3+/CD28+ T cells are sorted by magnetic beads, the T cells are cultured in a complete culture medium containing 10% FBS and 100IU/mL IL-2, after 24 hours, lentivirus carrying the CAR gene is added according to the MOI=10 proportion, and the culture is carried out under the conditions of 37 ℃ and 5% CO2, the liquid is changed for 1 time every 1-2 days, and the culture is carried out for 7-8 days. The cells were identified as being capable of stably expressing the gene of interest, designated CAR-T A and CAR-T B cells.

Example 2CAR-T cells inhibit ALL tumor cell proliferation

The killing effect of the CAR-T cells on the T-ALL cell line CCRF-CEM is detected in the embodiment, and the in-vitro antitumor capability of the CAR-T cells is examined. CCRF-CEM cells were cultured with DMEM medium containing 10% fbs, after the cells were grown to the logarithmic phase, the cells were digested, collected by centrifugation, inoculated into 96-well plates after cell density adjustment, inoculated with 1×10 ⁴ cells per well, and then CAR-T A and CAR-T B cells were added at an effective target ratio (E; T) of 1:1,1:5 and 1:10, respectively, and tumor cell killing rates were measured using LDH kit (purchased from peyunnan biotechnology ltd) after culturing for 24 hours at 37 ℃ with 5% co ₂.

Cell killing rate = (experimental calibration well OD value-effector 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%

The result shows that the CAR-T provided by the invention can effectively inhibit the growth of tumor cells and shows remarkable dose correlation, and the killing capacity of the CAR-T B is relatively higher than that of the CAR-T A, so that the IL-21 can maintain the activation state of the CAR-T cells, and the development of continuous anti-tumor effect is facilitated.

Example 3CAR-T cells inhibit tumor growth in animals

3.1 Preparation of tumor animal models

NCG mice of 5-8 weeks of age were acclimatized in an SPF-grade environment for 1 week. Recovering and culturing CCRF-CEM cells, subculturing when the cell fusion degree reaches 80-90%, digesting the cells with pancreatin, and then re-suspending with sterile PBS, and adjusting the cell density to 1×10 ⁷/mL. 100 μl of the cell suspension was subcutaneously injected into tumor bearing sites of mice, and tumor volumes were measured daily using vernier calipers and used for the subsequent experiments when tumor volumes reached 100mm ³.

3.2 Administration treatment and Observation of survival of animals

The mice with successful modeling are randomly divided into three groups, 10 mice in each group are respectively CAR-T A groups, 1X 10 ⁶ CAR-T A cells are intravenously injected, CAR-T B groups, 1X 10 ⁶ CAR-T B cells are intravenously injected, and the control group is an equivalent physiological saline solution. Animal status was observed daily and recorded and survival curves were plotted using GRAPHPAD PRISM software.

The results are shown in figure 3, and the treatment with the CAR-T A and the CAR-T B cells can significantly prolong the survival time of animals, which shows that the CAR-T cells provided by the invention can effectively play an anti-tumor role in vivo, and the CAR-T B introduced with IL-21 seems to have more significant treatment effect.

3.3 Determination of cytokines in animals

After 2 weeks of dosing, the experimental animals were bled intravenously, the plasma was centrifuged, and the IFN-. Gamma.and IL-2 expression levels in the plasma were detected using ELISA kits (purchased from Abcam Corp. USA), and specific detection methods were performed according to the kit instructions.

The results are shown in fig. 4 and 5, and in the treatment process of the CAR-T cells, the expression level of cytokines such as IFN-gamma, IL-2 and the like is obviously improved, so that immune cells in the body can be effectively activated to play an anti-tumor role. Moreover, the release levels of cytokines during CAR-T B treatment were higher, especially in promoting IFN- γ secretion, significantly stronger than CAR-T A cells that did not contain the IL-21 domain.

The embodiments are only used to illustrate the technical scheme of the present invention, but not to limit the technical scheme, and although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the technical scheme described in the foregoing embodiments may be modified or some or all technical features may be equivalently replaced, and the modification or replacement does not deviate the essence of the corresponding technical scheme from the scope of the technical scheme of the embodiments of the present invention.

Claims

1. A chimeric antigen receptor CAR targeting CD37, characterized in that 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 signal domain, and 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.

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, whose 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 further comprises 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 to 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.

9. The use according to claim 10, characterized in that the tumor is a T-cell malignant tumor.

10. The use according to claim 10, characterized in that the tumor is T-cell acute lymphoblastic leukemia T-ALL.