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Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy
Published on: December 17, 2019
GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells
Eric L Smith1,2, Kim Harrington3, Mette Staehr1
1Cellular Therapeutics Center, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
Abstract:
Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138+ MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell-derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.
Insights
Chimeric antigen receptor (CAR) T cell therapy targeting G protein-coupled receptor, class C group 5 member D (GPRC5D) shows promise for multiple myeloma (MM). GPRC5D CAR T cells effectively eradicated MM, including in BCMA-resistant cases, offering a new treatment avenue.
Area of Science:
- Oncology
- Immunotherapy
- Molecular Biology
Background:
- Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) shows promise for multiple myeloma (MM), but relapses occur due to BCMA-negative cells.
- G protein-coupled receptor, class C group 5 member D (GPRC5D) is a potential new target, with prior mRNA detection in MM but elusive protein expression confirmation.
- Identifying novel targets is crucial for overcoming therapeutic resistance in multiple myeloma.
Purpose of the Study:
- To confirm GPRC5D protein expression on multiple myeloma cells.
- To develop and evaluate GPRC5D-specific CAR T cells for multiple myeloma treatment.
- To assess the efficacy of GPRC5D-targeted CAR T cells, particularly in BCMA-refractory models.
Main Methods:
- Quantitative immunofluorescence was used to confirm GPRC5D protein expression on primary multiple myeloma cells.
- A phage display library was panned to identify GPRC5D-specific single-chain variable fragments (scFvs).
- Multiple CAR constructs were generated and screened using a Nur77 reporter system, with in vivo validation in a murine MM xenograft model.
Main Results:
- GPRC5D protein was confirmed on CD138+ multiple myeloma cells, independent of BCMA expression.
- GPRC5D-targeted CAR T cells (GPRC5D(109)) demonstrated specificity, cytotoxicity against MM cell lines and primary samples, and cytokine release.
- GPRC5D(109) CAR T cells eradicated MM in vivo, including in a BCMA antigen escape model, and showed comparable activity to anti-BCMA CAR T cells without significant toxicity in tested species.
Conclusions:
- GPRC5D is a viable protein target for CAR T cell therapy in multiple myeloma.
- GPRC5D(109) CAR T cell therapy is effective against multiple myeloma, including cases resistant to BCMA-targeted therapy.
- This approach offers a potential new treatment strategy for advanced multiple myeloma, irrespective of prior BCMA-targeted treatment.
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