Kinetics of tumor destruction by chimeric antigen receptor-modified T cells

Abstract

The use of chimeric antigen receptor (CAR)-modified T cells as a therapy for hematologic malignancies and solid tumors is becoming more widespread. However, the infusion of a T-cell product targeting a single tumor-associated antigen may lead to target antigen modulation under this selective pressure, with subsequent tumor immune escape. With the purpose of preventing this phenomenon, we have studied the impact of simultaneously targeting two distinct antigens present on tumor cells: namely mucin 1 and prostate stem cell antigen, both of which are expressed in a variety of solid tumors, including pancreatic and prostate cancer. When used individually, CAR T cells directed against either tumor antigen were able to kill target-expressing cancer cells, but tumor heterogeneity led to immune escape. As a combination therapy, we demonstrate superior antitumor effects using both CARs simultaneously, but this was nevertheless insufficient to achieve a complete response. To understand the mechanism of escape, we studied the kinetics of T-cell killing and found that the magnitude of tumor destruction depended not only on the presence of target antigens but also on the intensity of expression-a feature that could be altered by administering epigenetic modulators that upregulated target expression and enhanced CAR T-cell potency.

Figure 1

T cells can be engineered to recognize and kill pancreatic cancer cells expressing PSCA. (a) Retroviral vector map of the first-generation humanized, codon-optimized CAR-PSCA. (b) Shows the transduction efficiency of CAR-PSCA on primary T cells by detecting the CH2CH3 domain. (c) Shows the phenotype of NT and CAR-PSCA T cells, n = 10. (d) Shows the cytolytic ability of NT and CAR-PSCA T cells in a 6-hour chromium release assay (at a E:T of 10:1) using as a target the PSCA⁺ tumor cell line CAPAN1 (n = 5). CAR, chimeric antigen receptor; LTR, long terminal repeat; scFv, single-chain variable fragment; VH, variable heavy; VL, variable light.

Figure 2

Targeting a heterogeneous tumor with monospecific CAR T cells leads to tumor immune escape. (a) Shows the bioluminescence signal from severe combined immunodeficiency mice engrafted with CAPAN1-eGFP-FFLuc at different time points after treatment with CAR-PSCA or NT T cells (data are plotted as fold change of luminescence intensity compared with day 0). (b) Shows the percentage of residual tumor cells after a 72-hour coculture with a 5:1 E:T ratio, using NT T cells or CAR-PSCA T cells and CAPAN1 tumor cells (n = 4). (c) Shows the antitumor effect of NT and CAR-PSCA T cells on tumor cells that were resistant to an initial T-cell treatment (n = 4). (d) Shows PSCA and MUC1 antigen expression in untreated CAPAN1 tumor cells and in tumor cells following treatment with NT or CAR-PSCA T cells. FFLuc, Firefly luciferase; eGFP, enhanced green fluorescence protein; NT, nontransduced; PSCA, prostate stem cell antigen.

Figure 3

CAR T cells modified to recognize MUC1 kill MUC1⁺ tumor cells. (a) Shows a retroviral vector map of a first-generation MUC1-specific CAR coexpressing a truncated form of CD19 (ΔCD19). (b) Shows the transduction efficiency of CAR-MUC1 on primary T cells—double-positive CH2CH3 and ΔCD19 cells (left panel) and summary data for seven donors (right panel). (c) Shows the T-cell phenotype of NT and CAR-MUC1 T cells (n = 7). (d) Shows the cytolytic activity of NT and CAR-MUC1 T cells in a 6-hour chromium release assay (at a E:T of 10:1) using as targets the MUC1⁺ tumor cell line CAPAN1.

Figure 4

Targeting a heterogeneous tumor with MUC1–specific CAR T cells also leads to tumor immune escape. (a) (left) shows the bioluminescence signal from severe combined immunodeficiency mice engrafted with CAPAN1-eGFP-FFLuc at different time points after treatment with CAR-MUC1 or NT T cells (data are plotted as fold change of luminescence intensity compared with day 0 (right). (b) Shows the percentage of residual CAPAN1 tumor cells after a 72-hour coculture experiment at a 5:1 E:T ratio with NT or CAR-MUC1 T cells (n = 4). (c) Shows the antitumor effect of NT and CAR-MUC1 T cells on tumor cells that were resistant to an initial T-cell treatment (n = 4). (d) Shows PSCA and MUC1 antigen expression on untreated CAPAN1 tumor cells and in tumor cells following treatment with NT or CAR-MUC1 T cells. FFLuc, Firefly luciferase; eGFP, enhanced green fluorescence protein.

Figure 5

Targeting tumors using a dual CAR approach produces superior antitumor activity. (a) Shows the cytolytic effect of NT, CAR-MUC1, CAR-PSCA, or the combination in a 6-hour Cr⁵¹ release assay using CAPAN1 as a target (10:1 E:T ratio; n = 5). *indicates a P value <0.05 using Student's t-test. (b) Shows the number of residual CAPAN1 tumor cells after a 72-hour coculture experiment using the same panel of effectors and targets (E:T of 5:1; n = 5). (c) Shows bioluminescence images of representative mice engrafted with CAPAN-eGFP-FFluc and treated with NT, CAR-MUC1, CAR-PSCA, or the combination, whereas (d) shows summary results for the NT and dual-targeted groups (n = 4) at pre or 28, 42, 56, or 63 days posttreatment. Data are plotted as fold change of luminescence intensity compared with day 0. FFLuc, Firefly luciferase; eGFP, enhanced green fluorescence protein; MUC1, mucin 1; PSCA, prostate stem cell antigen.

Figure 6

Characterizing the tumor immune escape phenomenon using an artificial tumor model. (a) Shows two retroviral vector maps, the first encoding the TAA MUC1, which has a variable number of proline-rich segments that are tandemly repeated (variable number tandem repeat (VNTR)) and coexpressing mOrange. The second encodes the TAA PSCA, which contains six transmembrane portions, and coexpressing GFP. (b) Shows the mOrange and GFP expression on selected transduced 293T cells. (c) Shows residual 293T cells (1:1 mixture of MUC1- and PSCA-expressing cells) 72 hours after treatment with NT or CAR-PSCA T cells at an E:T 10:1 (n = 6). (d) Shows the proportion of residual GFP⁺ and mOrange⁺ tumor cells after CAR-PSCA T-cell treatment. (e) Shows the intensity of GFP, which serves as a surrogate for PSCA antigen expression, (f) shows residual 293T cells (1:1 mixture of MUC1- and PSCA-expressing cells) 72 hours after treatment with NT or CAR-MUC1 T cells at a E:T 10:1 (n = 5). (g) Shows the proportion of residual GFP⁺ and mOrange⁺ tumor cells after treatment with CAR-Muc1, (h) shows the intensity of mOrange as a surrogate for MUC1 antigen expression, (i) shows residual 293T cells (1:1 mixture of MUC1- and PSCA-expressing cells) 72 hours after treatment with NT or or the combination of CAR-MUC1 and CAR-PSCA T cells (n = 5), (j) shows the proportion of residual GFP⁺ and mOrange⁺ tumor cells after treatment with the combination of CAR-MUC1 and CAR-PSCA T cells, and (k) shows the intensity of GFP and mOrange as surrogates for PSCA and MUC1 antigen expression (n = 5). CAR, chimeric antigen receptor; IRES, internal ribosome entry site; LTR, long terminal repeat; MUC1, mucin 1; TM, transmembrane.