Anti-gprc5d antibodies and their use in car-t

Abstract

The application provides an anti-GPRC5D antibody and application thereof in CAR-T, and a corresponding CAR-T cell is developed based on the antibody, an intracellular stimulation domain in a chimeric antigen receptor is adjusted and optimized, an activation domain capable of efficiently activating the CAR-T cell is screened and obtained, tumor cells can be effectively killed, and the survival period of animals is prolonged.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology research and development, and specifically provides an anti-GPRC5D antibody and application thereof in CAR-T. BACKGROUND

[0002] G protein-coupled receptor class C group 5 member D (GPRC5D) is encoded by the GPRC5D gene, and human GPRC5D is located on chromosome 12p13.3. GPRC5D is composed of 3 exons with lengths of 895, 68 and 75 nucleotides, respectively, and is separated by two introns with lengths of 7161 and 1411 nucleotides, respectively. The first exon is the largest and contains all 7 transmembrane fragments (see H, Jensen AA, Sheppard PO, Brodin B, Krogsgaard-Larsen P, O'Hara P. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim Biophys Acta. 2001; 1518(3): 237-48). GPRC5D is almost universally expressed in plasma cells, and in normal adult tissues, GPRC5D mRNA levels are found to be high in the pancreas, moderate in the kidney, small intestine, spleen and testis, low in the lung, colon, leukocytes, prostate and thymus, and undetectable by RT-PCR in the heart, liver, placenta, skeletal muscle and ovary, thus its limited expression in basic normal tissues makes GPRC5D a promising ideal immunotherapy target for tumors.

[0003] Although GPRC5D has been discovered for more than 20 years, its endogenous ligand, signaling mechanism, physiological function, and mechanism of action under pathological conditions are still unclear. Recently, GPRC5D was identified as a significant gene for differentiating myeloma and is a promising biomarker for evaluating MM cell burden (see Cohen Y, Gutwein O, Garach-Jehoshua O, et al. GPRC5D is a promising marker for monitoring the tumor load and to target multiple myeloma cells. Hematology. 2013; 18(6):348-51). Researchers tried to analyze the correlation between GPRC5D level and multiple myeloma (MM) disease status, and there was no significant difference in the degree of GPRC5D protein expression among newly diagnosed MM (NDMM), R / R MM not receiving daratumumab treatment, and daratumumab-refractory (DARA-R) MM patients. Compared with the healthy control group, the GPRC5D gene expression level of MM patients was significantly higher, and these findings indicated that although GPRC5D is mainly located in plasma cells, its function seems to be unrelated to plasma cell biology (see Verkleij C, Broekmans M, Duin M, et al. Preclinical activity and determinants of response of the GPRC5DxCD3 bispecific antibody talquetamab in multiple myeloma. Blood Adv. 2021; 5(8):2196-215).

[0004] Currently, GPRC5D has become a promising target for immunotherapy against MM. In 2019, Eric et al. identified GPRC5D as an immunotherapy target and demonstrated that GPRC5D CAR-T cells have good preclinical efficacy in the treatment of MM (see Smith EL, Harrington K, Staehr M, et al. GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells. Sci Transl Med. 2019; 11(485): eaau7746). Subsequently, there have been many clinical trials of BsAb and CAR-T cell immunotherapy targeting GPRC5D, opening up a new chapter for the treatment of R / R MM. By 2023, anti-GPRC5DxCD3 BsAb (talquetamab) has been approved by the FDA, becoming the first immunotherapy product targeting GPRC5D (see Kodama T, Kochi Y, Nakai W, et al. Anti-GPRC5D / CD3 bispecific T-cell-redirecting antibody for the treatment of multiple myeloma. Mol Cancer Ther. 2019; 18(9): 1555-64).

[0005] Over the past two decades, the development of chimeric antigen receptor (CAR) T cell therapy has brought great progress in the treatment of hematologic malignancies (see Almasbak H, Aarvak T, Vemuri MC. CAR T cell therapy: a game changer in cancer treatment. J Immunol Res. 2016;2016:5474602). CAR-T cell therapy uses engineered T cells to target overexpressed tumor cell surface antigens, and CAR-T cells are usually autologous, but can also come from allogeneic donors. T cells are engineered to express CAR, a synthetic receptor protein, enabling T cells to target specific antigens, thus constituting the first generation of CAR. On the basis of the first generation of CAR, the updated generation of CAR constructs has integrated costimulatory domains, which can improve the persistence and antitumor activity of T cells (Maher J, Brentjens RJ, Gunnet G, et al. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta / CD28 receptor. Nat Biotechnol. 2002;20:70-5.). Currently, the US FDA has approved six CAR-T cell therapies for use, while there are also a number of ongoing clinical trials to evaluate the application of CAR-T cell therapy in various diseases.

[0006] Researchers have developed CAR-T cells targeting GPRC5D, but all anti-GPRC5D CAR T cells used in clinical use are second-generation CAR-T cell therapies with 4-1BB as a costimulatory factor, usually containing a single anti-GPRC5D scFv or bispecific scFv from human B cells, a 4-1BB (or CD28) costimulatory domain and a CD3 zeta signal domain, such as the CAR-T cells disclosed in clinical experiments NCT05016778, NCT04674813, NCT05739188, NCT04555551, etc. There is a lack of adjustment and optimization of CAR-T structure. The second-generation CAR-T using 4-1BB is the most classic, but this therapy has several limitations in efficacy, including on-target / off-tumor targeting, antigen escape and CAR-T cell dysfunction, etc. (see Mauro Castellarin, Caroline Sands, Tong Da, et al. A rational mouse model to detect on-target, off-tumor CAR T cell toxicity. JCI Insight. 2020; 5(14): e136012)

[0007] In summary, it is necessary to develop a new CAR-T cell therapy targeting GPRC5D in order to improve tumor treatment effect, improve treatment targeting, reduce side effects of tumor immunotherapy, and improve safety and effectiveness of treatment. SUMMARY

[0008] The first aspect of the present application provides an anti-GPRC5D antibody, which comprises heavy chain variable region CDR1-3 with amino acid sequences shown in SEQ ID NO: 1-3, respectively, and light chain variable region CDR1-3 with amino acid sequences shown in SEQ ID NO: 4-6, respectively.

[0009] Further, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0010] Further, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10.

[0011] The second aspect of the present application provides a chimeric antigen receptor (CAR) comprising a signal peptide, a scFv targeting GPRC5D, a hinge region, a transmembrane region, a co-stimulatory factor and a CD3 zeta intracellular signaling domain, wherein the scFv comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 10.

[0012] Further, the co-stimulatory factor is at least one selected from CD28, 4-1BB, OX40, ICOS, DAP10 and GITR.

[0013] Further, the co-stimulatory factor is CD28, 4-1BB or OX40.

[0014] Further, the co-stimulatory factor is CD28 and 4-1BB.

[0015] Further, the amino acid sequence of the CAR is as set forth in SEQ ID NO: 18.

[0016] Further, the nucleotide sequence of the CAR is as set forth in SEQ ID NO: 19.

[0017] The third aspect of the present application provides a CAR-T cell expressing the CAR or carrying the nucleotide.

[0018] The fourth aspect of the present application provides use of the CAR or the CAR-T cell in the preparation of an anti-tumor drug.

[0019] Further, the tumor is myeloma.

[0020] GPRC5D is reported to be highly expressed in various tumor cells, especially in hematological tumors, including leukemia, lymphoma and myeloma; in particular, in various myeloma tumors including multiple myeloma, and currently various antibodies or CAR-T cells targeting GPRC5D have been approved to enter the clinical trial stage.

[0021] Advantages

[0022] The present application provides an anti-GPRC5D antibody and its use in CAR-T, which has the following advantages:

[0023] 1. A novel anti-GPRC5D antibody is provided, which can specifically bind to the target antigen; and part of the sites of the antibody are humanized to reduce heterologous reactions;

[0024] 2. Based on the anti-GPRC5D antibody, the corresponding CAR-T cells were prepared, and the design of the stimulation domain was optimized to obtain CAR-T cells that can be efficiently activated;

[0025] 3. The CAR-T cells can effectively inhibit tumor cell proliferation and increase the expression level of immune factors. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 : CAR structure design;

[0027] Figure 2 : Tumor inhibition effect of double-stimulation factor CAR-T cells;

[0028] Figure 3 : Tumor inhibition effect of triple-stimulation factor CAR-T cells;

[0029] Figure 4 : Dose dependence of CAR-T cells;

[0030] Figure 5 : Animal survival curve;

[0031] Figure 6 : Expression level of immune factors in plasma. DETAILED DESCRIPTION

[0032] The experimental methods described in the following examples are all conventional methods unless otherwise specified. The reagents, biological materials, and detection kits described are all commercially available unless otherwise specified.

[0033] Example 1 Preparation of CAR-T cells targeting GPRC5D

[0034] 1.1 Obtaining of GPRC5D antibody

[0035] The sequence information of human GPRC5D (GenBank NP_061124) was obtained by using bioinformatics website (https: / / www.ncbi.nlm.nih.gov / ), and the nucleotide sequence encoding the protein was introduced into an E. coli expression vector to express and obtain the target antigen GPRC5D protein.

[0036] Based on the hybridoma technology, the mouse is immunized with the target antigen, and the GPRC5D antibody is screened and obtained. The sequence determination and analysis show that the antibody includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8. The heavy chain variable region CDR amino acid sequence of the antibody obtained by sequence analysis is as shown in SEQ ID NO: 1-3, and the heavy chain variable region CDR amino acid sequence is as shown in SEQ ID NO: 4-6. The protein molecule interaction instrument shows that the antibody can be combined with the target antigen with high specificity, and the kD value can reach 2.45*10 -10 M.

[0037] In order to reduce the heterogeneity of the antibody, facilitate the subsequent application in the human body, and develop the chimeric antigen receptor, while it is very likely to maintain the affinity and specificity of the monoclonal antibody, the frame region part of the antibody light and heavy chain is humanized in the application. The amino acid sequence of the modified antibody heavy chain variable region is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 10. The determination shows that the kD value of the modified antibody and the target antigen can reach 5.73*10 -9 M. Although the affinity of the antibody and the target antigen is reduced, studies show that the use of scFv with moderate affinity to construct a chimeric antigen receptor can effectively reduce the serious adverse reactions such as immune factor storm, so as to ensure the safety of treatment, and therefore the antibody is suitable for the preparation of the subsequent CAR

[0038] 1.2 CAR structure

[0039] In the common CAR structure, the extracellular antigen binding domain and the intracellular signal stimulation domain are included, wherein the extracellular antigen binding domain further includes the scFv region, the hinge region and the transmembrane region (TM) for recognizing the target antigen. The scFv for targeting GPRC5D used in the application is the anti-GPRC5D antibody developed by the applicant, including the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 9 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 10. The intracellular signal stimulation domain usually includes various T cell activation factors, and in common cases, CD3 zeta is used in combination with CD28, 4-1BB (i.e. CD137), OX40 (i.e. CD134), ICOS, CD27, DAP10, GITR and other factors, so as to improve the activation effect.

[0040] In order to improve and compare the anti-tumor effect of CAR-T cells, two kinds of CAR structures (such as Figure 1The CAR structure is composed of the structure shown in the figure. Taking the triple stimulator CAR including CD28 and 4-1BB as an example, the amino acid sequence is shown in SEQ ID NO: 18, and the nucleotide sequence is shown in SEQ ID NO: 19. Figure 1 The CAR structure is composed of the structure shown in the figure. Taking the triple stimulator CAR including CD28 and 4-1BB as an example, the amino acid sequence is shown in SEQ ID NO: 18, and the nucleotide sequence is shown in SEQ ID NO: 19.

[0041] The nucleotide sequence encoding the CAR structure described above is introduced into a lentivirus vector by genetic engineering means, so as to facilitate the subsequent preparation of CAR-T cells.

[0042] 1.3 Preparation of CAR-T cells

[0043] Take the peripheral blood of a healthy volunteer, and place 10 mL of blood in a heparin anticoagulation tube. Add human lymphocyte separation medium (purchased from Sigma Company) at a ratio of 1:1, and centrifuge at 1000 g for 30 min at room temperature. Slowly aspirate the middle white membrane layer using a pipette, and wash 3 times with sterile PBS. Resuspend with RPMI 1640 medium containing 200 U / mL IL-2, and place in a 6-well plate coated with CD3 / CD28 antibody for culture for 48 h. Collect the T cells, wash 3 times with PBS, and then adjust the cell concentration to 1×10 6 The lentivirus vector used in the present application contains a GFP fluorescent protein gene, so the growth state of the cells and the expression of the exogenous gene can be observed using a fluorescence microscope. When the cell density reaches more than 80%, the cells are collected to obtain CAR-T cells. Fluorescence microscope detection shows that the CAR positive proportion can reach more than 70%.

[0044] Example 2 CAR-T cells targeting GPRC5D inhibit tumor cell proliferation

[0045] 2.1 Tumor inhibition effect of double-stimulatory factor CAR-T cells

[0046] The anti-tumor effect of CAR-T cells was detected using CCK-8 reagent. Human myeloma cells U266 were used as target cells, which were cultured in vitro to the logarithmic growth phase, then collected and adjusted to a cell density of 1 x 10 5 6 / mL; CAR-T cells were cultured as effector cells, and co-cultured in a 96-well plate at an effector-to-target ratio of 10:1, with 3 replicate wells in each group, and setting the OD values of experimental control wells (without adding CAR-T cells) and blank control wells. After 48 h of culture, 20 μL of CCK-8 reagent was added, and the absorbance (OD value) at 450 nm was detected using an enzyme-labeled instrument after 2 h of incubation at 37°C. The tumor inhibition rate was calculated according to the following formula: (experimental control well OD value - CAR-T cell well OD value) / (experimental control well OD value - blank control well OD value) x 100%.

[0047] The results, as shown in Figure 2 , CAR-T cells targeting GPRC5D can produce a significant inhibitory effect on myeloma cells, but the activation effect of different co-stimulatory factors has some differences, among which CD28, 4-1BB and OX40 have a significantly stronger inhibitory effect than other co-stimulatory factors, indicating that these factors are more suitable for forming a CAR structure with the GPRC5D scFv provided in the present application.

[0048] 2.2 Tumor inhibition effect of triple-stimulatory factor CAR-T cells

[0049] Based on the construction of CAR containing double-stimulatory factors, the present application further explores whether the combination of different co-stimulatory factors can produce a stronger anti-tumor effect. The specific detection method is the same as section 2.1, and the results, as shown in Figure 3 , the anti-tumor effect has changed after using triple-stimulatory factors, among which the combination of CD28 and 4-1BB has the strongest effect, which may be due to the fact that CD28 can improve the activation degree of T cells, while 4-1BB can delay the persistence of T cells, and the two can produce a synergistic effect.

[0050] 2.3 Dose-dependent study of triple-stimulatory factor CAR-T cells

[0051] To study the inhibitory effect of CAR-T cells on tumor cells, the present application further investigates the inhibitory effect of CAR-T cells containing CD28 and 4-1BB on tumor cells at different doses, and CAR-T cells and U266 tumor cells are inoculated at an effector-to-target ratio of 1:5, 1:2, 1:1, 2:1, 5:1, and 10:1, respectively, and the detection method is the same as in Section 2.1. The results are shown in Figure 4 Figure 2, which shows that the anti-tumor effect of CAR-T cells is dose-dependent, indicating that they can effectively inhibit tumor cell growth.

[0052] Example 3: CAR-T cells targeting GPRC5D inhibit tumor tissue growth

[0053] 3.1 Preparation of animal models and administration

[0054] SPF NCG mice were taken, and after one week of adaptive feeding, 5x10 5 U266 cells were subcutaneously injected, the growth status of tumor tissue was observed daily, and after 2 weeks, the tumor volume reached 20mm 3 Above, indicating that the modeling was successful. Twenty successfully modeled mice were randomly divided into two groups, namely: CAR-T group, 1x10 6 CAR-T cells containing CD28 and 4-1BB costimulatory factors were injected into the tail vein; control group, the same amount of normal saline was injected into the tail vein.

[0055] 3.2 Observation of animal survival period

[0056] The survival status of experimental animals was observed and recorded daily, and the survival curve was plotted. As shown in Figure 5 Figure 3, treatment with CAR-T cells can significantly prolong the survival period of animals and inhibit tumor growth in vivo.

[0057] 3.3 Promote the expression of inflammatory factors

[0058] After 4 weeks of administration, blood was taken from the tail vein, and after centrifugation of the blood to obtain plasma, the concentration of IL-2 and IFN-γ in the plasma was detected using an ELISA kit (purchased from Shanghai Zymed Biological Technology Co., Ltd.), and the specific steps were performed according to the kit instructions.

[0059] The results are shown in Figure 6 Figure 4, which shows that after treatment with CAR-T cells, the expression levels of IL-2 and IFN-γ in vivo increased significantly, and the above cytokines are important anti-tumor factors, so the anti-tumor effect can be further strengthened at the molecular level; however, it should also be noted that the immune factor storm is the most concerned clinical side effect during CAR-T treatment, and in the present application, IL-2 and IFN-γ are still within a controllable range, ensuring the safety of the therapy.

[0060] The above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; 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 application.

Claims

1. An anti-GPRC5D antibody, characterized in that, The antibody comprises heavy chain variable region CDR1-3 with amino acid sequences shown as SEQ ID NO: 1-3 respectively, and light chain variable region CDR1-3 with amino acid sequences shown as SEQ ID NO: 4-6 respectively.

2. The anti-GPRC5D antibody of claim 1, wherein, The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

8.

3. The anti-GPRC5D antibody of claim 1, wherein The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

10.

4. A chimeric antigen receptor (CAR) comprising, The CAR comprises a signal peptide, a scFv targeting GPRC5D, a hinge region, a transmembrane region, a costimulatory factor and a CD3 zeta intracellular signaling domain, the scFv comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO: 9 and a light chain variable region with an amino acid sequence shown as SEQ ID NO:

10.

5. The chimeric antigen receptor CAR of claim 4, wherein, The costimulatory factor is selected from at least one of CD28, 4-1BB, OX40, ICOS, DAP10, GITR.

6. The chimeric antigen receptor CAR of claim 5, wherein, The amino acid sequence of the CAR is shown as SEQ ID NO:

18.

7. The chimeric antigen receptor CAR of claim 6, wherein, The nucleotide sequence of the CAR is shown as SEQ ID NO:

19.

8. A CAR-T cell, wherein the cell expresses the CAR of any one of claims 4-6 or carries the nucleotide of claim 7.

9. Use of the antibody of any one of claims 1-3, the CAR of any one of claims 4-6 or the CAR-T cell of claim 8 in the preparation of an antitumor drug, wherein the tumor is myeloma.

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