Supplementary MaterialsSupplementary 1. MM cells and an extensive evaluation of potential on-target/off-tumor toxicity remain lacking. Through immunohistochemical analyses, we demonstrate that GPRC5D is expressed on malignant bone marrow plasma cells, whereas normal tissue expression is limited to the hair follicle. We developed and evaluated an optimized, human-derived, GPRC5D-targeted CAR T cell therapy. Using a reporter line that provides a specific readout of signaling from the CAR, we identified CAR designs optimized for spacer length (23) and low antigen-independent (tonic) signaling (24C26). Last, we provide preclinical evidence that a GPRC5D-targeted CAR T cell therapy candidate is safe and effective. Despite GPRC5D expression in ALK-IN-6 the hair follicle, we show that anti-cynomolgus and anti-murine cross-reactive GPRC5D CAR T cells do not induce alopecia or cause other clinical signs of damage to the skin in these species. On the basis of these results, we anticipate that GPRC5D shall become a significant clinical target for MM immunotherapy. RESULTS Manifestation of GPRC5D by MM cells In analyzing potential cell surface area focuses on for immunotherapy of MM, we wanted to recognize antigens with near ubiquitous manifestation on MM plasma cells and limited manifestation on essential regular tissue cells. Utilizing the Tumor Cell Range Encyclopedia (CCLE), we examined mRNA manifestation of in silico across 1000 different malignant cell lines, including 30 MM cell lines. Like a control, we examined (Compact disc138), a typical surface area marker of malignant and normal plasma cells. Although can be indicated in MM cell lines extremely, additionally it is indicated in cell lines from nearly all tumor types extremely, with top aerodigestive system tumors getting the highest manifestation ALK-IN-6 (fig. S1A). mRNA was extremely indicated in Rabbit Polyclonal to LRG1 MM cell lines (= 30), but in contrast to mRNA in the esophagus, skin, lung, and liver, among other tissues (fig. S1B), whereas mRNA was not highly expressed in any normal tissues aside from the skin, in which it was variably expressed, in agreement with previous reports (14C16). Furthermore, analysis of RNA expression data on human bone marrow samples showed that primary malignant and normal plasma cells expressed 1000- and 500-fold more mRNA than B cells from peripheral blood, respectively (Fig. 1B and fig. S1C). Open in a separate window Fig. 1. High expression of mRNA in MM cells and variable expression in skin.(A) mRNA expression of in malignant cell lines (= 1036; CCLE, accessed in September 2013, Affymetrix). RMA, robust multiarray average; DLBCL, diffuse large ALK-IN-6 B cell lymphoma; CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; NSC, nonCsmall cell. (B) mRNA expression of in normal tissues according to GTEx RNASeq data (GTEx ENSG00000111291.4). The dashed line represents the expression of in CD138-sorted primary MM cells (BLUEPRINT RNA-seq, = 9). FPKM, fragments per kilobase ALK-IN-6 of transcript per million mapped reads. To evaluate potential correlations between expression and clinical outcomes, we analyzed the Multiple Myeloma Research Foundation (MMRF) CoMMpass trial (NCT0145429), a publicly available longitudinal study with accompanying CD138-sorted RNA-seq expression data from 765 patients (research.themmrf.org/; version IA13). A previous investigation of 48 patients independent of the CoMMpass cohort (20 ) reported that expression above the median correlated with a worse prognosis. Our analysis of the CoMMpass cohort confirms this finding, as expression above the median in this large dataset correlated with shorter progression-free survival (= 0.0031; fig. S2A). expression did not correlate with International Staging System score or any evaluated common cytogenetic abnormality (fig. S2, B and C). Similar to an earlier report (22), we didn’t identify GPRC5D in MM cells using any obtainable or internally developed movement cytometric reagents commercially. These reagents had been incompatible with quantitation of mobile antigen thickness. We used proteins immunohistochemistry (IHC) to judge protein appearance by major malignant plasma cells. The specificity of anti-GPRC5D IHC was validated using K562 cells built expressing GPRC5D and individual MM cell lines endogenously expressing GPRC5D (fig. S3). We also performed multiplex quantitative immunofluorescence (Q-IF) for Compact disc138, BCMA, and GPRC5D on major bone marrow examples; representative pictures are shown in Fig. 2A. Utilizing a cutoff of 50% antigen appearance on Compact disc138+ cells, which includes been found in some studies of BCMA-targeted CAR T cell therapy (“type”:”clinical-trial”,”attrs”:”text message”:”NCT02215967″,”term_id”:”NCT02215967″NCT02215967 and “type”:”clinical-trial”,”attrs”:”text message”:”NCT02658929″,”term_id”:”NCT02658929″NCT02658929), we noticed that 65% (54 of 83) of examples have GPRC5D appearance above this level, 73% (61 of 83) of examples match this threshold for BCMA, and 88% (73 of 83) match this cutoff when appearance of either BCMA or GPRC5D is known as (Fig. 2, ?,BB and ?andC).C). GPRC5D appearance on Compact disc138 cells was indie of BCMA appearance (= 83)..