Comparing CAR and TCR engineered T cell performance as a function of tumor cell exposure

Abstract

Chimeric antigen receptor (CAR) T cell therapies have resulted in profound clinical responses in the treatment of CD19-positive hematological malignancies, but a significant proportion of patients do not respond or relapse eventually. As an alternative to CAR T cells, T cells can be engineered to express a tumor-targeting T cell receptor (TCR). Due to HLA restriction of TCRs, CARs have emerged as a preferred treatment moiety when targeting surface antigens, despite the fact that functional differences between engineered TCR (eTCR) T and CAR T cells remain ill-defined. Here, we compared the activity of CAR T cells versus engineered TCR T cells in targeting the B cell malignancy-associated antigen CD20 as a function of antigen exposure. We found CAR T cells to be more potent effector cells, producing higher levels of cytokines and killing more efficiently than eTCR T cells in a short time frame. However, we revealed that the increase of antigen exposure significantly impaired CAR T cell expansion, a phenotype defined by high expression of coinhibitory molecules and effector differentiation. In contrast, eTCR T cells expanded better than CAR T cells under high antigenic pressure, with lower expression of coinhibitory molecules and maintenance of an early differentiation phenotype, and comparable clearance of tumor cells.

Keywords: CAR; T cell receptor; TCR; activation-induced cell death; antigen exposure; chimeric antigen receptor; comparison; exhaustion; solid tumors; tumor load.

Figure 1.

CAR T cells have stronger short-term effector functions than eTCR T cells. Retrovirally transduced and MACS purified CD8+ CAR T and eTCR T cells were assessed for effector functions in targeting CD20-expressing malignancies. (a) Overview of used constructs. CAR constructs and the 1E9 TCR construct were cloned into retroviral expression systems. CD20-targeting CARs were designed on either ofatumumab (Ofa)- or rituximab (RTX)-derived scFvs combined with CD28 stalk, transmembrane, and signaling domains. FMC63-28z CAR was used as a CD19-targeting control. 1E9 TCR is constant domain murinized and cysteine modified. (b) Specificity of CD20-targeting constructs was assessed by IFNγ ELISA after overnight coculture. 5000 T cells were incubated for 16 h with target cells at indicated E:T ratios. Data points show averaged duplicate values from three different experiments using individual donors. (c)5000 T cells were coincubated overnight with long-term expanded ALL cells that cells that naturally differ in their CD20 expression. E:T ratios are as indicated. Data depict averaged duplicate values from four experiments using T cells derived from independent donors. (d) Comparison of cytokine secretion as a function of target cell exposure at an E:T ratio of 1:9 as depicted in (c). (e) Representative killing efficacy of CAR- and TCR-transduced T cells in 6 h ⁵¹Cr release assay. 2500 target cells were labeled for 1 h with Na₂⁵¹CrO₄ and incubated with T cells at indicated E:T ratios. Statistics in (b), (c), and (d) show Fisher’s least significant difference test with comparisons as indicated. Comparisons were paired for donors.

Figure 2.

Both CAR T cells and TCR T cells proliferate when encountering target cells, but CD20 CAR T cell expansion correlates negatively with target antigen expression. To assess antigen specific proliferation of T cells as a function of target antigen expression, T cells were labeled with CFSE and incubated with irradiated target cells for 4 days at an E:T ratio of 1:2 in the presence of IL-2. CFSE dilution was assessed by flow cytometry.(a) Representative histograms of CFSE dilution after 4 days of coculture with indicated target cells. (b) Antigen-specific expansion as T cell counts after 4 days, normalized to T cells counts without target cells. Data pooled from different experiments using 3 individual donors, performed in duplicates. Error bars show SD. Statistics depict Fisher’s LSD test, comparing 1E9 eTCR T cell or Ofa-28z CAR T cell counts when encountering CD20-negative ALL GD and CD20-high ALL CM target cells.

Figure 3.

CAR T cells are more susceptible to activation-induced cell death than eTCR T cells. CAR T and TCR T cells were incubated with varying ratios of CD20-high ALL CM to assess activation-induced cell death (AICD). (a) Exemplary FACS plot after staining for the presence of cleaved caspase-3/7. (b) AICD levels after overnight culture as normalized frequencies of T cells staining positive for activated Caspase-3/7 and (c) number of viable T cells normalized to nonstimulating conditions after overnight coculture. Pooled data from four experiments using different donors, performed in duplicates. Statistics depict pairwise comparisons of 1E9 TCR and Ofa-28z T cells per E:T ratio using repeated measure ANOVA (matched for donors) and Fisher’s LSD test. Error bars depict SD.

Figure 4.

eTCR T cells express lower levels of coinhibitory molecules after activation than CAR T cells. T cells were incubated with ALL CM (CD20 high) at indicated E:T ratios for 72 h hours and assessed for expression of coinhibitory molecules PD-1 and LAG3. (a) Representative FACS plots of PD-1 and LAG3 expression 72 h after coculture. Gates were set based on FMO controls. (b/c) gMFI of PD1 (b) and LAG3 (c) on T cells 72 h after coculture. Pooled data from four experiments using different donors; pairwise comparisons were performed using repeated measures ANOVA (paired for donors) and Fisher’s LSD test.

Figure 5.

High antigen exposure drives effector memory differentiation of CAR T cells, while eTCR T cells maintain a central memory-like phenotype. Antigen-induced T cell differentiation was assessed by flow cytometry. A) Representative FACS plots of CD45RA and CD62L expression of T cells after coculture with CD20-high ALL CM at indicated E:T ratios for 72 h hours. (b) and (c) T cells were MACS sorted on CD62L+ cells and subsequently incubated with indicated target cells at an E:T ratio of 1:3 for 72 h. (b) shows technical duplicate values and mean of cell counts of CD62L+ T cells from one representative experiment. (c) compares CD62L+ T cell counts after encounter of ALL RL or ALL CM as described in B for 5 different experiments and donors. Statistics depict the two-sided ratio paired T test.

Figure 6.

eTCR T cells outperform CAR T cells under high antigenic pressure. T cells were incubated with nonirradiated target cells at different E:T ratios for 7 days in the presence of 100IU/ml IL-2. (a) T cell counts after 7 days of coculture. Summary data of 5 different experiments using different donors. (b) Antigen-specific proliferation (normalized cell count to TCM) derived from (a). (c) Antigen-specific proliferation of 1E9 TCR T cells and Ofa-28z CAR T cells after encounter of nonirradiated ALL RL, ALL BV, or ALL CM. (d) Remaining viable ALL cells after 7 days of coculture from the same experiments shown in (c). Error bars in (a) and (b) depict SEM. Statistics in (a)-(d) compare 1E9 TCR and Ofa-28z using repeated measures ANOVA matched for donors and Fisher’s LSD test.