4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors

Abstract

Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic antitumor responses in hematologic malignancies, but tumor regression has rarely occurred using CARs targeting other antigens. It remains unknown whether the impressive effects of CD19 CARs relate to greater susceptibility of hematologic malignancies to CAR therapies, or superior functionality of the CD19 CAR itself. We show that tonic CAR CD3-ζ phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy. Such activation is present to varying degrees in all CARs studied, except the highly effective CD19 CAR. We further determine that CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the antitumor effects of CD19 CARs and for the observations that CD19 CAR T cells incorporating the 4-1BB costimulatory domain are more persistent than those incorporating CD28 in clinical trials.

Figures

Figure 1

GD2.28z CAR T cells have discrepant in vitro and in in vivo activity. (a) CD19 and GD2 antigen expression on the 143B-CD19 osteosarcoma line. Representative of n=5. (b) In vitro51chromium release assay evaluating cytolytic activity of mock-transduced, CD19.28z CAR, and GD2.28z CAR T cells against 143B-CD19. Assay performed 9 days after initial activation. n=3 replicates/point; representative of 4 donors. (c) Growth curves of 143B-CD19 tumors in NSG mice. Mice were inoculated with 106 143B-CD19 periosteally on day 0 followed by adoptive transfer of 107 CAR transduced or mock-transduced T cells on day 14. Mock n=5, CD19.28z CAR n=7, GD2.28z CAR n=7. (d) Left: composition of T cell product adoptively transferred into mice in (c). Middle and right: quantification of T cells within the spleen and tumor 14 days following adoptive T cell transfer into mice from (c). n=8 mice/group. * = p<0.05 by Student’s T-test. Bar graphs represent mean ± SEM. In vivo results are representative of four experiments.

Figure 2

GD2.28z CAR T cells become exhausted during ex vivo expansion. (a) Activation marker expression 4–7 days after initial activation. Representative of 3 donors. (b) Expansion during ex vivo culture. n=3 replicates/point; representative from 3 donors. (c) Quantification of apoptosis of CAR T cells generated from 4 unique donors evaluated 9–10 days following initial activation. (d) Representative exhaustion marker expression 9 days following initial activation. (e) Quantification of exhaustion marker expression pooled from 4 donors, 9–11 days following activation. (f) Exhaustion marker SPICE analysis from (e). (g) ΔΔCT q-RT-PCR expression levels of exhaustion transcription factors relative to mock, 9–11 days following initial activation. n=4 technical replicates; representative of 3 donors. (h) Cytokine production of flow-sorted single-transduced (CD19.28z CAR+ or GD2.28z CAR+) and co-transduced (Co-trans; CD19.28z CAR+ and GD2.28z CAR+) T cells, co-incubated with 143B-CD19 at 9 days after initial activation. n=3 replicates/group; representative of 3 donors. T cells with media: <5 pg/mL IL-2, TNF-α and IFN-γ. (i) Tumor growth curves of NSG mice inoculated with 106 143B-CD19 periosteally on day 0 followed by adoptive transfer of 107 transduced CAR T cells on day 14. n=5 mice/group. (j) Left: composition of T cell product adoptively transferred into mice in (i). Middle and right: quantification of T cells within the spleen and tumor 14 days following adoptive T cell transfer into mice from (i). n=6 mice/group. SPICE analysis: * = p<0.05 by Wilcoxon signed rank test. All other data: * = p<0.05 by Student’s T-test. Bar graphs represent mean ± SEM.

Figure 3

Tonic CAR signaling during ex vivo expansion leads to early exhaustion. (a) Western blot evaluating phosphorylation levels of CAR signaling domains versus total CAR signaling domains, using an anti-phospho-CD3ζ and anti-CD3ζ antibody, respectively. Evaluated day 5 after initial activation. “Basal” phosphorylation evaluated without further stimulation. “After crosslinking” evaluated following incubation with OKT3, anti-CD19 CAR idiotype, or anti-GD2 CAR idiotype antibodies. Representative of 3 donors. (b) Nine amino acid point mutations were introduced to eliminate signaling via the GD2.28z CAR (GD2.mut-28.mut-z CAR). (c) Activation and (d) exhaustion marker expression of GD2.mut-28.mut-z CAR T cells 10 days after initial activation. Representative of 3 donors. (e) Ex vivo expansion and (f-g) exhaustion marker expression of CD19.28z CAR T cells cultured ± anti-idiotype antibody (anti-Id; 0.25 ug/ml) and a crosslinking F(ab’)2 (2.5 ug/ml). n=4 replicates/group; representative of 3 donors. (h) Tumor growth curves of NSG mice inoculated with 106 143B-CD19 periosteally on day 0 followed by adoptive transfer of 107 transduced CAR T cells on day 14. Mice received mock-transduced or CD19.28z CAR cultured with or without anti-idiotype antibody. No anti-idiotype antibody was given to mice. n=8 mice/group. (i) CD19 expression on tumors 10 days following adoptive transfer into mice from (h). (j) Quantification of T cells within the blood 9 days following adoptive T cell transfer into mice from (h). n=3 mice/group. In vivo results are representative of two experiments. * = p<0.05 by Student’s T-test. Bar graphs represent mean ± SEM.

Figure 4

Tonic GD2.28z CAR signaling is antigen independent. (a) In vitro51Cr release assay against the 143B-CD19 osteosarcoma cell line. Both GD2.28z CAR and GD2.mutCDR.28z CAR had comparable transduction efficiencies (85 and 81%, respectively). Assay performed 9 days after initial T cell activation. n=3 replicates/point; representative of 3 donors. (b) Exhaustion marker expression on GD2.mutCDR.28z CAR vs. GD2.28z CAR T cells. Representative of 3 donors. (c) Fluorescence microscopy of T cells transduced with CAR-Cerulean fusion proteins (blue). (d) Quantification of the number of puncta per cell from (c). 30 cells per group; repeated for 3 donors. (e) Exhaustion marker expression levels of a version of the GD2.28z CAR designed to be structurally more like the CD19.28z CAR, based upon removal of the IgG1 hinge and substitution of the GD2 linker with the CD19 linker (GD2.sh.28z CAR). (f) Cytokine release levels upon co-incubation with 143B-CD19 on day 10 of ex vivo culture. # designates values >10,000 pg/mL. n=3 replicates/group; representative of 3 donors. (g) Structure and (h) exhaustion marker expression of a hybrid CAR combining the CDR regions of the CD19.28z CAR and the framework regions of the GD2.28z CAR (CD19-CDR.GD2-FW.28z CAR), 9 days post activation during ex vivo culture. Representative of 3 donors. For cell puncta analysis, * = p<0.0001 by Kolmogorov-Smirnov test. All other data, * = p<0.05 by Student’s T-test. Bar graphs represent mean ± SEM.

Figure 5

4-1BB endodomain ameliorates exhaustion in CAR T cells. (a) Activation marker expression 7 days following initial activation and (b) exhaustion marker expression 9 days after initial activation of GD2 CARs with the CD28 or 4-1BB co-stimulatory domains. (Representative of 4 donors). (c) Quantification of exhaustion marker expression pooled from 4 donors, 9–11 days following initial activation. (d) Exhaustion marker SPICE analysis of (c). (e) Cytokine release upon co-incubation with 143B-CD19 for 24 hours starting on day 10 following initial activation. n=3 replicates/group; representative of 3 donors. (f) Tumor growth curves of NSG mice inoculated with 5 × 105 143B-CD19 periosteally on day 0, then treated with 3 mg cyclophosphamide intraperitoneally on day 2 and with adoptive transfer of 107 transduced CAR T cells on day 4. n=10 mice/group. (g) Quantification of T cells within the blood 8 and 15 days following adoptive transfer into mice from (f). n=8 mice/group. (h) Exhaustion marker expression of CAR T cells from (f) on day 14 following adoptive transfer. (i) Quantification of T cells within the blood of NSG mice inoculated with 106 NALM6-GL followed by adoptive transfer of 5×106 CAR T cells three days later. T cells were quantified on day 8 following adoptive transfer. n=5 mice/group. (j) Exhaustion marker expression of CAR T cells from (i). In vivo results are representative of three experiments. SPICE analysis: * = p<0.05 by Wilcoxon signed rank test. All other data: * = p<0.05 by Student’s T-test. Bar graphs represent mean ± SEM.

Figure 6

Ameliorating effect of 4-1BB signaling is associated with a unique transcriptional profile. (a) Principal component analysis (PCA) of global transcriptional profiles of CAR T cells generated from 3 unique healthy donors, evaluated 9 days following initial activation. Transduction efficiency was >90% for all samples. (b) Heatmap of genes previously reported to impact T cells exhaustion. Exhaustion related transcription factors (TBX21, EOMES, PRDM1, IKZF2), inhibitory receptors (LAG3, HAVCR2, CTLA4, BTLA, CD244), and transcription factors reported to be preferentially expressed in memory vs. exhausted cells (KLF6, JUN, JUNB) were compared. (c) Representative GSEA results from running the unfiltered GD2.BBz CAR vs. GD2.28z CAR T cell rank list against the MSigDB C5 gene ontology sets. (d) Heatmap of genes uniquely dysregulated in exhausted GD2.28z CAR T cells. Genes are those up/downregulated > 2 fold in GD2.28z CAR vs. CD19.28z CAR T cells, up/downregulated > 2 fold in GD2.28z CAR vs. GD2.BBz CAR T cells, and < 2 fold difference in GD2.BBz CAR vs. CD19.28z CAR T cells. Genes associated with response to hypoxia (EGLN3, EGR, PTGIS, ID1), apoptosis (ID1), and metabolism (GLUL, ATP10D, SMPDL3A), or T cell suppressive pathways (CTLA4, CD38, LGMN, CLECL1, ENTPD1, KLRC1/2) were identified.

Methods-only references