An immunoproteomic approach to characterize the CAR interactome and signalosome

Abstract

Adoptive transfer of T cells that express a chimeric antigen receptor (CAR) is an approved immunotherapy that may be curative for some hematological cancers. To better understand the therapeutic mechanism of action, we systematically analyzed CAR signaling in human primary T cells by mass spectrometry. When we compared the interactomes and the signaling pathways activated by distinct CAR-T cells that shared the same antigen-binding domain but differed in their intracellular domains and their in vivo antitumor efficacy, we found that only second-generation CARs induced the expression of a constitutively phosphorylated form of CD3ζ that resembled the endogenous species. This phenomenon was independent of the choice of costimulatory domains, or the hinge/transmembrane region. Rather, it was dependent on the size of the intracellular domains. Moreover, the second-generation design was also associated with stronger phosphorylation of downstream secondary messengers, as evidenced by global phosphoproteome analysis. These results suggest that second-generation CARs can activate additional sources of CD3ζ signaling, and this may contribute to more intense signaling and superior antitumor efficacy that they display compared to third-generation CARs. Moreover, our results provide a deeper understanding of how CARs interact physically and/or functionally with endogenous T cell molecules, which will inform the development of novel optimized immune receptors.

Fig. 1. CARs alter gene expression.

(A) Schematic of the transmembrane and co-stimulatory domains contained in the PSCA-specific second-generation 28t28Z and third-generation 8t28BBZ CARs studied in these experiments. (B and C) Affymetrix microarray analyses were performed in GFP-transduced human lymphocytes and PSCA-28t28Z or PSCA-8t28BBZ CD8⁺ CAR-T cells before (Pre) or after (Post) adoptive transfer into NSG mice. Principal component analysis (B) and Hierarchical clustering of differentially-expressed genes (C) are from the analysis of at least 3 biological replicates. (D) Flow cytometry analysis of CD69 and CD25 abundance on pre-infusion (day 12) CD8⁺ T cells that expressed GFP or CARs, as indicated. Histograms are representative of 4 independent experiments. (E) Number of differentially-expressed genes in PSCA-8t28BBZ (blue) or PSCA-28t28Z (red) CAR-T cells. Gene-set enrichment analysis (bar charts) was performed on the genes differentially expressed by each population or both populations compared to GFP controls. Data are from the analysis of at least 3 biological replicates. (F) Flow cytometry analysis of the in vitro proliferation of T cells transduced with GFP-control or the indicated CARs compare to non-transduced cells in the same culture. Histograms are representative of 4 independent experiments.

Fig. 2. Proteomic-based CAR interactome analysis.

(A) Schematic of the immunoprecipitation tandem liquid chromatography and mass spectrometry experiment on PSCA-28t28Z and PSCA-8t28BBZ CAR-T cells and GFP controls. (B and C) Proteomics data analysis of the proteins enriched in lysates of CAR-T cells immunoprecipitated for CAR. Ingenuity canonical pathway analysis (B) or top interaction partners (C) with PSCA-28t28Z (left) and PSCA-8t28BBZ (right) are from the analysis of 3 biological replicates. Arrow indicates CD3ζ (CD247).

Fig. 3. CARs interact with CD3ζp21.

(A) Flow cytometry analysis of CAR abundance on PSCA-28t28Z (red), PSCA-8t28BBZ (blue), or GFP (gray)-transduced T cells. Histograms are representative of 3 independent experiments. (B and C) Western blot analysis of CD3ζ interaction in lysates of T cells transduced with PSCA-28t28Z, PSCA-8t28BBZ, or GFP and immunoprecipitated for the CAR scFv. Blots (C) are representative of 8 independent experiments. (D) Schematic CAR designs with different transmembrane, hinge and co-stimulatory domains. (E and F) Western blot analysis of CD3ζ association in lysates of T cells transduced with the indicated CAR and immunoprecipitated for CAR scFv. Blots (E) are representative of 10 independent experiments. Quantified band intensity values (F) compared to the endogenous CD3ζ p16 (red) or CD3ζ-CAR p55/p75 (blue) are data with means ± SD from all experiments. **P<0.01; ***P<0.001; ****P<0.0001 by Friedman ANOVA with Dunn’s multiple comparison test.

Fig. 4. Second-generation CARs constitutively phosphorylate CAR and CD3ζp21/23.

(A to B) Western blot analysis of pCD3ζ within the indicated CAR (A) and with the associated CD3ζ p21/p23 isoform (B) in lysates of T cells transduced with the indicated CAR and immunoprecipitated for CAR scFv. Blots (A and B) are representative of 3 independent experiments. Quantified band intensity values (C) are data with means ± SD from all experiments. (D) Tandem MS/MS analysis of pTyr¹⁴² (top) and pTyr¹²³ (bottom) of immunoprecipitated CD3ζ species within 21–25kDa, from lysates of PSCA-8tBBZ CAR-T cells. Spectra are representative of the analysis of 3 biological replicates. *P<0.05; **P<0.01; ns, not significant by Paired one-way ANOVA with Fisher’s post-test

Fig. 5. The CAR endodomain size determines its ability to interact with CD3ζp21.

(A) Flow cytometry analysis of CAR abundance on PSCA-8t28Z (green) and PSCA-8t(+24)28Z (light blue) and GFP (gray)-transduced T cells. Histograms (left) are representative of 5 independent experiments. The frequency of CAR⁺ cells was quantified for each donor (lines). (B and C) Western blot analysis of CD3ζ association with the indicated CAR in lysates of CAR T cells immunoprecipitated for CAR scFv. Blots (B) are representative of 7 independent experiments. Quantified band intensity values (C) compared to the endogenous CD3ζ p16 (red) or CD3ζ-CAR p55/p75 (blue) are data with means ± SD from all experiments. (D) ELISA analysis of IFNγ production by CAR-T cells normalized to the amount produced by GFP-T cells after overnight incubation with HPAC, H2110 cells or media alone (No tumor). Data and means ± SEM are from 3 independent experiments with lines connecting each independent donor analyzed. **P<0.01, ***P<0.001, and ns; not significant by Paired t-test (A and D), Friedman ANOVA with Dunn’s multiple comparison test (C) or two-way ANOVA (D).

Fig. 6. CAR signaling upon antigen binding.

(A) Experimental design for the comparison of PSCA-28t28Z and PSCA-8t28BBZ signalosome. Heavy isotope-labeled HPAC tumor cells were co-cultured with unlabeled GFP-transduced or CAR-T cells for 1 hour, then lysed and trypsin digested. Tandem mass spectrometry analysis was performed on pY peptides concentrated by immunoprecipitation and pS/pT peptides from the flow-through. (B and C) Proteomics data analysis of the 40/751 phospho-proteins that were differentially abundant in CAR-T cells when compared to background controls. Ingenuity pathway analysis (B) is representative of the analysis of 3 biological replicates. Abundance data on selected phosphopeptides relevant to TCR signaling in PSCA-28t28Z (red), PSCA-8t28BBZ (blue), or control CAR-T cells (black) and means ± SEM are from all samples. *P<0.05, **P<0.01, and ***P<0.001 by t-test.