Anti-GPRC5D antibody and application thereof in CAR-T

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides an anti-GPRC5D antibody and its application in CAR-T. Background Art

[0002] G protein-coupled receptor class C group 5 member D (GPRC5D) is encoded by the GPRC5D gene. Human GPRC5D is located on chromosome 12p13.3. GPRC5D consists of 3 exons with lengths of 895, 68, and 75 nucleotides respectively, separated by two introns in the middle. The lengths of the introns are 7161 and 1411 nucleotides respectively. The first exon is the largest and contains all 7 transmembrane segments (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 ubiquitously expressed in plasma cells. In normal adult tissues, the GPRC5D mRNA level is found to be relatively high in the pancreas, moderate in the kidney, small intestine, spleen, and testis, low in the lung, colon, white blood cells, prostate, and thymus, and undetectable by RT-PCR in the heart, liver, placenta, skeletal muscle, and ovary. Therefore, its limited expression in basically normal tissues makes GPRC5D a promising ideal immunotherapy target for tumors.

[0003] Although GPRC5D has been discovered for over 20 years, its endogenous ligand, signaling mechanism, physiological function, and mechanism of action under pathological conditions remain unclear. Recently, GPRC5D was identified as a distinct gene for differentiating multiple myeloma and is a promising biomarker for assessing 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 attempted to analyze the correlation between GPRC5D levels and the disease state of multiple myeloma (MM). There were no significant differences in the degree of GPRC5D protein expression among newly diagnosed MM (NDMM), relapsed / refractory (R / R) MM patients not receiving daratumumab treatment, and daratumumab-resistant (DARA-R) MM patients. Compared with the healthy control group, the GPRC5D gene expression level was significantly higher in MM patients. These findings suggest that although GPRC5D is mainly localized in plasma cells, its function does not seem to be related 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 the good preclinical efficacy of GPRC5D CAR-T cells 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, many clinical trials of BsAbs and CAR-T cell immunotherapies targeting GPRC5D emerged, opening a new chapter in the treatment of R / R MM. By 2023, the anti-GPRC5D × CD3 BsAb (talquetamab) was 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] In the past two decades, the development of chimeric antigen receptor (CAR) T cell therapy has brought great progress to the treatment of hematological 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. CAR-T cells are usually autologous, but can also be derived 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. Based on the first generation of CAR, more updated CAR constructs have incorporated co-stimulatory domains, which can enhance the persistence and anti-tumor activity of T cells (Maher J, Brentjens RJ, Gunset 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, and there are also multiple clinical trials underway to evaluate the application of CAR-T cell therapy in various diseases.

[0006] Researchers have developed CAR-T cells targeting GPRC5D. However, all anti-GPRC5D CAR T cells used clinically are second-generation CAR-T cell therapies with 4-1BB as the co-stimulatory factor, usually containing a single anti-GPRC5D scFv or bispecific scFv from human B cells, a 4-1BB (or CD28) co-stimulatory domain, and a CD3ζ signaling domain, such as the CAR-T cells disclosed in clinical trials like NCT05016778, NCT04674813, NCT05739188, NCT04555551, etc. There is still a lack of adjustment and optimization of the CAR-T structure. Although the second-generation CAR-T using 4-1BB is the most classical, the efficacy of this therapy has several limitations, 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 type of CAR-T cell therapy targeting GPRC5D to improve the tumor treatment effect, enhance the treatment targeting, reduce the side effects brought by tumor immunotherapy, and improve the safety and effectiveness of the treatment. Summary of the Invention

[0008] In the first aspect of the present invention, an anti-GPRC5D antibody is provided, wherein the antibody comprises CDR1-3 of the heavy chain variable region with amino acid sequences as shown in SEQ ID NO: 1-3 respectively, and CDR1-3 of the light chain variable region with amino acid sequences as shown in SEQ ID NO: 4-6 respectively.

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

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

[0011] The second aspect of the present invention provides a chimeric antigen receptor CAR, which comprises a signal peptide, a scFv targeting GPRC5D, a hinge region, a transmembrane region, a co-stimulatory factor, and a CD3ζ intracellular signaling domain. The scFv comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 9 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 10.

[0012] Further, the co-stimulatory factor is selected from at least one of 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 shown in SEQ ID NO: 18.

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

[0017] The third aspect of the present invention provides a CAR-T cell, which expresses the CAR or carries the nucleotide.

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

[0019] Further, the tumor is myeloma.

[0020] It is reported that GPRC5D is highly expressed in a variety of tumor cells, especially in hematological tumors, including leukemia, lymphoma, and myeloma; particularly in a variety of bone marrow tumors including multiple myeloma. Currently, a variety of antibodies or CAR-T cells targeting GPRC5D have been approved for clinical trial stage.

[0021] Beneficial effects

[0022] The present invention provides an anti-GPRC5D antibody and its application in CAR-T, with the following specific beneficial effects:

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

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

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

[0026] Figure 1 : CAR structure design;

[0027] Figure 2 : Tumor suppression effect of dual-stimulatory factor CAR-T cells;

[0028] Figure 3 : Tumor suppression effect of triple-stimulatory 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 OF THE EMBODIMENTS

[0032] 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 sources unless otherwise specified.

[0033] Example 1 Preparation of CAR-T Cells Targeting GPRC5D

[0034] 1.1 Obtaining the Anti-GPRC5D Antibody

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

[0036] Based on the hybridoma technology, mice were immunized with the target antigen, and anti-GPRC5D antibodies were screened and obtained. Through sequence determination and analysis, the antibody comprises 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. Through sequence analysis, the CDR amino acid sequences of the heavy chain variable region of the antibody are as shown in SEQ ID NOs: 1-3, and the CDR amino acid sequences of the heavy chain variable region are as shown in SEQ ID NOs: 4-6. Determined by a protein molecular interaction instrument, the antibody can bind to the target antigen with high specificity, and its kD value can reach 2.45×10 -10 M.

[0037] To reduce the heterogenicity of the antibody, facilitate subsequent in-human application, and the development of chimeric antigen receptors, and at the same time most likely maintain the affinity and specificity of the monoclonal antibody, in this application, partial sites of the framework regions of the heavy and light chains of the antibody were humanized. The amino acid sequence of the heavy chain variable region of the modified antibody 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. It was determined that the kD value of the modified antibody and the target antigen can reach 5.73×10 -9 M. Although its affinity with the target antigen has decreased, research shows that using scFv with medium affinity to construct chimeric antigen receptors can effectively reduce serious adverse reactions such as cytokine storms, thus ensuring the safety of treatment. Therefore, the antibody is suitable for subsequent CAR preparation.

[0038] 1.2 CAR Structure

[0039] In a common CAR structure, it includes an extracellular antigen-binding domain and an intracellular signal-stimulating domain. Among them, the extracellular antigen-binding domain further includes an scFv region that recognizes the target antigen, a hinge region, and a transmembrane region (TM). The anti-GPRC5D scFv used in this application is an anti-GPRC5D antibody independently developed by the applicant, including a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 9 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 10; the intracellular signal-stimulating domain usually includes a variety of T cell activation factors. Commonly, CD3ζ is used in combination with factors such as CD28, 4-1BB (i.e., CD137), OX40 (i.e., CD134), ICOS, CD27, DAP10, GITR, etc. to improve the activation effect.

[0040] In the present invention, to improve and compare the anti-tumor effects of CAR-T cells, two CAR structures were designed (as Figure 1As shown in the figure, one contains only one co-stimulatory factor, which can be selected from one of CD28, 4-1BB, OX40, ICOS, DAP10, and GITR; the other contains two co-stimulatory factors, and the combination of the co-stimulatory factors enhances the activation ability of T cells. SP in the CAR structure is a signal peptide, and its amino acid sequence is as shown in SEQ ID NO: 11; hinge is a hinge region, and its amino acid sequence is as shown in SEQ ID NO: 12; TM is a transmembrane region, and its amino acid sequence is as shown in SEQ ID NO: 13; the amino acid sequence of the CD28 co-stimulatory factor is as shown in SEQ ID NO: 14; the amino acid sequence of the 4-1BB co-stimulatory factor is as shown in SEQ ID NO: 15; the amino acid sequence of the OX40 co-stimulatory factor is as shown in SEQ ID NO: 16; other co-stimulatory factors use known sequence structures; the amino acid sequence of CD3ζ is as shown in SEQ ID NO: 17. According to Figure 1 the structure shown above to form a CAR structure. Taking the triple-stimulatory factor CAR that includes both CD28 and 4-1BB as an example, its amino acid sequence is as shown in SEQ ID NO: 18, and its nucleotide sequence is as shown in SEQ ID NO: 19.

[0041] The nucleotide sequence encoding the above CAR structure is introduced into a lentiviral vector by genetic engineering means for subsequent preparation of CAR-T cells.

[0042] 1.3 Preparation of CAR-T cells

[0043] Take the peripheral blood of healthy volunteers. Place 10 mL of blood in a heparin anticoagulant tube, add human lymphocyte separation solution (purchased from Sigma) at a ratio of 1:1, and centrifuge at 1000 g for 30 min at room temperature; slowly aspirate the middle buffy coat layer using a pipette and wash it 3 times with sterile PBS; resuspend it in RPMI 1640 medium containing 200 U / mL IL-2 and place it in a 6-well plate coated with CD3 / CD28 antibodies for culture for 48 h; collect T cells, wash them 3 times with PBS, and then adjust the cell concentration to 1×10 6 cells / mL. Take 100 μL and inoculate it into a 24-well plate, add the lentiviral vector carrying the CAR gene according to the multiplicity of infection (MOI) = 10, mix well, and culture it in an environment of 37 °C and 5% CO 2 2, and supplement fresh medium according to the culture conditions. The lentiviral vector used in this application contains the GFP fluorescent protein gene, so a fluorescence microscope can be used to observe the cell growth state and the expression of foreign genes. When the cell density reaches more than 80%, the cells are collected to obtain CAR-T cells. After detection by a fluorescence microscope, the positive proportion of CAR can reach more than 70%.

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

[0045] 2.1 Tumor suppression effect of dual-stimulatory factor CAR-T cells

[0046] The anti-tumor effect of CAR-T cells was detected using the CCK-8 reagent. Using human myeloma cell line U266 as the target cells, after culturing in vitro until the logarithmic growth phase, the cells were collected and the cell density was adjusted to 1×10 5 cells / mL. The 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 replicates in each group. The OD values of the experimental control wells (without adding CAR-T cells) and the blank control wells were set. After 48 hours of culture, 20 μL of CCK-8 reagent was added, and after incubating at 37 °C for 2 hours, the absorbance (OD value) at 450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader. The tumor inhibition rate was calculated according to the following formula: Tumor inhibition rate = (OD value of experimental control wells - OD value of CAR-T cell wells) / (OD value of experimental control wells - OD value of blank control wells) × 100%.

[0047] The results are as Figure 2 shown. CAR-T cells targeting GPRC5D can significantly inhibit myeloma cells, but there are certain differences in the activation effects of different co-stimulatory factors. Among them, the inhibitory effects of CD28, 4-1BB, and OX40 are significantly stronger than those of other co-stimulatory factors, indicating that these factors are more suitable for forming the CAR structure with the GPRC5D scFv provided in the present invention.

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

[0049] On the basis of constructing the CAR containing dual-stimulatory factors, the present invention further explored whether the combination of different co-stimulatory factors could produce a stronger anti-tumor effect. The specific detection method was the same as that in Section 2.1. The results are as Figure 3 shown. After using the triple-stimulatory factor, the anti-tumor effect changed. Among them, the combination of CD28 and 4-1BB had the strongest effect, probably because 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 invention further investigated the inhibitory effect of CAR-T cells containing CD28 and 4-1BB on tumor cells at different doses. CAR-T cells and U266 tumor cells were inoculated at effector-to-target ratios of 1:5, 1:2, 1:1, 2:1, 5:1, and 10:1, respectively. The detection method was the same as that in Section 2.1. The results are as Figure 4 shown. The anti-tumor effect of CAR-T cells showed an obvious dose-dependence, indicating that it can effectively inhibit the growth of tumor cells.

[0052] Example 3 Inhibition of tumor tissue growth by CAR-T cells targeting GPRC5D

[0053] 3.1 Animal model preparation and administration

[0054] SPF-grade NCG mice were taken. After one week of adaptive feeding, 5×10 5 U266 cells were subcutaneously injected. The growth status of the tumor tissue was observed daily. After 2 weeks, when the tumor volume reached 20 mm 3 or more, it indicated that the model establishment was successful. Twenty successfully modeled mice were randomly divided into two groups: the CAR-T group, in which 1×10 6 CAR-T cells containing CD28 and 4-1BB costimulatory factors were injected via the tail vein; the control group, in which an equal volume of normal saline was injected via the tail vein.

[0055] 3.2 Observation of animal survival period

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

[0057] 3.3 Promotion of inflammatory factor expression

[0058] Four weeks after administration, blood was collected from the tail vein. After the blood was centrifuged to obtain plasma, ELISA kits (purchased from Shanghai Enzyme-linked Biotechnology Company) were used to detect the concentrations of IL-2 and IFN-γ in the plasma. The specific steps were carried out according to the kit instructions.

[0059] The results are as Figure 6 shown. 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. Therefore, the anti-tumor effect can be further enhanced at the molecular level. However, it should also be noted that cytokine storm is the most concerning clinical side effect during CAR-T treatment. In the present invention, IL-2 and IFN-γ are still within the controllable range, ensuring the safety of this therapy.

[0060] 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 described 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. An anti-GPRC5D antibody, characterized in that The antibody comprises heavy chain variable regions CDR1-3 whose amino acid sequences are shown in SEQ ID NOs: 1-3, respectively, and light chain variable regions CDR1-3 whose amino acid sequences are shown in SEQ ID NOs: 4-6, respectively.

2. The anti-GPRC5D antibody according to claim 1, characterized in that 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.

3. The anti-GPRC5D antibody according to claim 1, characterized in that 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.

4. A chimeric antigen receptor CAR, characterized in that: The CAR includes a signal peptide, a scFv targeting GPRC5D, a hinge region, a transmembrane region, a co-stimulatory factor and a CD3ζ intracellular signal domain, and the scFv includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 9 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

10.

5. The chimeric antigen receptor CAR according to claim 4, characterized in that The co-stimulatory factor is selected from at least one of CD28, 4-1BB, OX40, ICOS, DAP10, and GITR.

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

18.

7. The chimeric antigen receptor CAR according to claim 6, characterized in that The nucleotide sequence of the CAR is shown in SEQ ID NO:

19.

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

9. Use of the antibody according to any one of claims 1 to 3, the CAR according to any one of claims 4 to 6, or the CAR-T cell according to claim 8 in the preparation of an anti-tumor drug.

10. The use according to claim 9, wherein the tumor is myeloma.

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