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Abstract
Background BCMA-targeted chimeric antigen receptor (CAR)T-cell therapy promotes transient recovery in patients with multiple myeloma, but relapses occurs commonly. GPRC5D, a surface receptor that is highly expressed in bone marrow samples of multiple myeloma patients, shows a promising potential as a specific CAR-T target. Its expression in MM cells is not dependent on BCMA, which makes it an ideal target for MM treatment, either alone or in combination with BCMA.
Methods In this study, we utilized an in-house phage display synthetic nanobody library to identify two VHHs with high affinity and specificity to GPRC5D.
Results We further demonstrated that GPRC5D CAR T-cells with tandem biepitopic VHHs (OriCAR-017) have superior MM cell lysis in vitro, low toxicity and high anti-tumor effects in vivo, compared to single VHH or bicistronic VHH structures (figures 1–5). A phase 1 trial (Clinical Trials.gov identifier: NCT05016778) with 10 MM patients, including 5 patients who had relapsed after BCMA CAR-T therapy, showed a 100% objective response rate without severe side effects with OriCAR-017 treatment, suggesting the safety and efficacy of OriCAR-017 therapy with best-in-class potential (figure 6).
Conclusions Therefore, OriCAR-017 holds promise for the treatment of MM, either as a first-line therapy or for patients who have relapsed after BCMA CAR T-cell therapy (figure 7).
Selection of VHHs for functional GPRC5D CAR-T cells. (A) Schematic diagram depicting the transmembrane topology and domains of the GPRC5D protein. (B) Schematic diagram of the recombinant lentiviral vectors encoding GPRC5D-targeted VHH CARs generated in this study. (C) Analysis of VHH CARs transduction efficiency. (D) VHH CAR-T cells mediated cytotoxicity was evaluated by a luciferase-based assay of co-cultured MM. 1S cells at E:T ratios of 1:1, 1:3, or 1:9. (E-F) Concentration of cytokines produced by VHH CAR-T cells co-cultured with MM. 1S cells at an E:T ratio of 1:1 was shown.
Evaluation of the antitumor function of OriCAR-017 Cells In Vitro. (A) Schematic diagram of the recombinant lentiviral vectors encoding the GPRC5D-targeted VHH1 and VHH2 CARs generated in this study. (B) Cytotoxicity of OriCAR-017 cells was evaluated by quantitative assessment of LDH in the supernatants of co-cultured CHO or CHO-GPRC5D cells. (C) Cytotoxicity of OriCAR-017 cells was evaluated by luciferase-based assessment of co-cultured MM. 1S cells at the E:T ratio of 1:1, 1:3, or 1:9. (D) Degranulation of OriCAR-017 cells was assessed by the expression of lysosomal protein CD107a. (E-F) Concentrations of IL-2 and IFN-y were measured.
OriCAR-017 cells exhibited superior functional advantage in vitro. (A) Confocal imaging of CAR-T cells. White arrows show MTOC polarization at the IS. (B) Quantification of MTOC to synapse distance, n=10–20.
Evaluation of in vivo antitumor activity and safety of OriCAR-017 cells. (A) In vivo toxicity was evaluated by monitoring the weight of mice during treatment. (B) Antitumor activity of GPRC5D CAR-T in the MM. 1S model. (C) Kaplan-Meier survival curves of mice treated as in (B).
OriCAR-17 demonstrates efficacy in inhibiting orthotopic multiple myeloma tumor models. (A) Schematic of the orthotopic multiple myeloma tumor models used to investigate the CAR-T cells activity in vivo. (B) Total Flux represents the tumor burden of NCI-H929-Luciferase-bearing mice at the indicated time points. (C) Bioluminescence radiance as a surrogate marker for tumor burden. (D) Kaplan-Meier survival curves of NCI-H929-Luciferase-bearing mice treated as in (C).
Safety and efficacy of OriCAR-017 demonstrated in early clinical trial. (A) Clinical responses of all 10 patients with different infusion doses are shown. (B) Percentage of clinical response in all patients after OriCAR-017 T cell infusion. (C) Maximum CRS grade observed in all patients after OriCAR-017 T cell infusion, CRS: cytokine release syndrome. Data cutoff was October 30, 2022.
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