Mesothelin-specific CAR-T cell therapy that incorporates an HLA-gated safety mechanism selectively kills tumor cells

Abstract

Background: Mesothelin (MSLN) is a classic tumor-associated antigen that is expressed in lung cancer and many other solid tumors. However, MSLN is also expressed in normal mesothelium which creates a significant risk of serious inflammation for MSLN-directed therapeutics. We have developed a dual-receptor (Tmod™) system that exploits the difference between tumor and normal tissue in a subset of patients with defined heterozygous gene loss (LOH) in their tumors.

Methods: T cells engineered with the MSLN CAR Tmod construct described here contain (1) a novel MSLN-activated CAR and (2) an HLA-A*02-gated inhibitory receptor (blocker). A*02 binding is intended to override T-cell cytotoxicity, even in the presence of MSLN. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When A*02 is absent from tumors selected for LOH, the MSLN Tmod cells are predicted to mediate potent killing of the MSLN(+)A*02(-) malignant cells.

Results: The sensitivity of the MSLN Tmod cells is comparable with a benchmark MSLN CAR-T that was active but toxic in the clinic. Unlike MSLN CAR-T cells, the Tmod system robustly protects surrogate "normal" cells even in mixed-cell populations in vitro and in a xenograft model. The MSLN CAR can also be paired with other HLA class I blockers, supporting extension of the approach to patients beyond A*02 heterozygotes.

Conclusions: The Tmod mechanism exemplified by the MSLN CAR Tmod construct provides an alternative route to leverage solid-tumor antigens such as MSLN in safer, more effective ways than previously possible.

Keywords: T-lymphocytes; cell engineering; immunologic; immunotherapy; receptors.

Conflict of interest statement

Competing interests: All authors are current or former employees and shareholders of A2 Biotherapeutics, Inc.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Tmod approach to achieve selective cytotoxicity with two targets. (A) The lung (and other vital organs) are surrounded by the MSLN(+) mesothelial lining, creating high risk of on-target, off-tumor toxicity for MSLN-targeted medicines. Selecting patients heterozygous for A*02 whose tumors have lost this allele via LOH gives an opportunity to target MSLN selectively with CAR Tmod cells so they kill tumor cells specifically and spare normal mesothelium. (B) Molecular composition of MSLN-targeted Tmod constructs. The two receptors are co-expressed in a single construct and the encoded fusion protein is cleaved in the cell to generate the activator and blocker. (C) Six display-derived MSLN activators (CAR1-6) and benchmark CARs M5 and SS1 activators were paired with A*02 blocker (solid) or empty vector control (open). Jurkat cells expressing the CAR±blocker (for receptor staining see online supplemental figure 1F) were co-cultured with endogenous MSLN(+) HeLa cells transfected with a titration of A*02:01 mRNA. Response was assessed after 6-hour co-culture. Titrated antigen molecules on the surface of target cells were quantified using the QIFIKIT. IC50 (molecules/cell) values are indicated in the figure. M5 and SS1 CARs are Gen2. (D) Two-dimensional titration of MSLN and A*02 mRNA in MSLN(−) HeLa target cells to establish EC50 and IC50 for the MSLN CAR3 Tmod construct in Jurkat cells. MSLN(−) HeLa target cells were transfected with serially diluted MSLN mRNA and constant A*02 mRNA (left) or serially diluted A*02 mRNA and constant MSLN mRNA (right).

Figure 2

MSLN Tmod functions selectively with human cell lines±MSLN expression. (A) Expression of MSLN in human cell lines assessed by staining with MSLN mAb and flow cytometry. K562 displayed some cross-reactivity to the anti-MSLN antibody, although no functional reactivity to CAR3 or benchmark M5 CAR was observed. (B) Levels of MSLN and A*02 mRNA (CCLE) and protein (QIFIKIT) show correlation. Conversion between protein and mRNA levels was calculated using these standard curves (see online supplemental table 1). (C) EC50 and IC50 of construct in relation to MSLN and HLA-A expression levels in normal (GTEx database) and tumor tissues and cell lines (TCGA, CCLE databases). Conversions between protein and mRNA levels were calculated using the standard curves shown in figure 2B. HeLa and MS751 A*02 transgenic cell line variants shown on the graph (green) better mimic activator and blocker target ratios in normal tissues. Only tissues above the EC50 line express sufficient levels of MSLN to activate CAR3. Tissues above the IC50 line express sufficient levels of A*02 to engage the blocker. Therefore, all normal tissues are expected to be spared by CAR3 Tmod, including the lung. Even those vital tissues in the brain with relatively low HLA-A expression (~100 TPM) are still well above the IC50 for MSLN Tmod blocker function. In addition, MSLN expression in brain is extremely low (~1 TPM), well below the EC50 for activation. Organs were classified as vital or supplementary to vital per ICH Guidance S7A (https://www.fda.gov/media/72033/download). (D) CAR3 selectivity benchmarked against Gen2 M5 CAR on a panel of human cell lines. Activation of CARs in a Jurkat cell functional assay by MSLN(+) or MSLN(−) cell lines showed that both CARs display MSLN-specific activation. (E) MSLN CAR3 Tmod construct selectivity in a Jurkat cell functional assay on a subset of MSLN(+) and MSLN(−) control target cells showed no off-target activity. Note that the blocker slightly reduces background or tonic activation compared with CAR alone. This has been observed generally by us and by others (Hwang et al, 2021). Two-way ANOVA with a multiple comparisons test was used to calculate significance between CAR3 and CAR3 Tmod. Data shown for D–E depict the average of technical replicates. All experiments were run independently at least three times.

Figure 3

MSLN CAR3 Tmod construct is potent and selective in primary T cell assays. (A) MSLN CARs and CAR3 Tmod cytotoxicity in primary T cells with various MSLN(+)A*02(−)RFP(+) tumor or MSLN(+)A*02(+)GFP(+) “normal” target cells. A*02:MSLN (B:A) target antigen ratios for each cell line are noted. M5 CAR is Gen2. E:T=1:1. Data are representative of multiple (3+) independent repeats with at least 4 different donors. (B) Secreted IFN-γ and (C) T cell activation assessed by forward-scatter shift was measured 48 hours after co-culture of transduced T cells with tumor or “normal” target cells (same as in figure 3A). Two-way ANOVA with a multiple comparisons test was used to calculate significance (n=3 technical replicates). (D) sMSLN does not affect CAR-T activity. Acute cytotoxicity of tumor or “normal” target cells by M5 benchmark CAR or CAR3 was not affected by the presence of 500 ng/mL sMSLN (Acro Bio). E:T=0.6:1. (E) Staining of transiently transfected CAR(+) Jurkat cells with labeled sMSLN monomer (dashed) or tetramer (solid) analyzed by flow cytometry shows that the sMSLN is structurally intact and able to bind.

Figure 4

MSLN CAR3 Tmod construct is selective, serially-cytotoxic, and reversible in primary T cell assays. (A) MSLN CAR3 Tmod cells selectively kill RFP(+) tumor cells and spare GFP(+) “normal” cells in mixed tumor/”normal” cell cultures. Due to the adherent nature of the HeLa cell line, dead/dying target cells tend to remain as clusters on the surface. White arrows point to examples of dead/dying RFP(+) tumor cells. (B) Representative cytotoxicity in mixed “normal” and tumor co-cultures with “normal”:tumor=9:1 (see online supplemental figure 2C for other ratios). E:T=0.6:1. (C) MSLN CAR3 Tmod construct mediates selective, serially-cytotoxic, and reversible cytotoxicity. After 48 hours of co-culture with tumor or “normal” HeLa target cells, T cells were co-cultured for a second round with either tumor or “normal” target cells; E:T=1.2:1. RACA, repeat antigen-challenge assay; Rev., reversibility assay. Data shown for C depicts the average of technical triplicates and is representative of at least 2 independent experimental repeats. (D) MSLN CAR3 Tmod killing of tumor target cells and blocking of “normal” target cells is not affected by the presence of PD-L1 induced by overnight treatment of HeLa target cells with 50 ng/mL IFN-γ (see online supplemental figure 2E). Blockade with anti-PD-1 antibody similarly has no effect on CAR3 Tmod.

Figure 5

Tmod construct mediates selective killing of tumor cells in a xenograft model. (A) Schematic diagram of the dual-flank tumor and “normal” MS751 xenograft model. (B) Bioluminescence imaging of “normal” and tumor cells. Bioluminescence values are to the right of the color scale in flux units of photons/sec/cm2/sr. Day 0=pre–T cell injection; days 8 and 15=post–T cell injection. (C) Graft sizes assessed by caliper measurement. Left: Tmod cells (blue arrow) kill tumor cells equivalent to CAR-Ts (red arrow). Right: CAR-Ts kill, while Tmod cells spare “normal” cells. Two-way ANOVA with a multiple comparisons test was used to calculate significance between CAR3 and CAR3 Tmod at each timepoint. n=10 animals per group.

Figure 6

MSLN Tmod system can be extended to autologous T cells. (A) MSLN SS1 CAR Tmod construct kills MSLN(+) A*02(−) tumor HeLa target cells but no longer blocks in the presence of autologous A*02 as a result of cis-binding. B2M knockout (KO) by CRISPR restores blocker availability as demonstrated by binding to A*02 tetramer. (B) B2M KO by CRISPR in A*02(+) T cell donors restores blocking on MSLN(+)A*02(+) “normal” HeLa target cells similar to A*02(−) T cell donors. E:T=1.2:1 (C) Representative co-culture images at 48 hours for figure 6B. (D) Similar to SS1 Tmod, CAR3 Tmod has reduced binding to A*02 tetramer in A*02(+) T cells. B2M knockdown (KD) with shRNA restores blocker availability. (E) B2M shRNA also restores blocking of cytotoxicity on MSLN(+)A*02(+) “normal” HeLa cells. (F) Interestingly, CAR3 paired with a humanized A*02 blocker retains the ability to block killing of “normal” cells in A*02(+) donor T cells, even in the absence of B2M KO or KD.

Figure 7

MSLN Tmod system can be extended to other blockers. (A) Jurkat cells expressing MSLN CAR3 and A*03, B*07 or A*11 blocker constructs were blocked in the presence of increasing blocker antigen on endogenous MSLN(+) HeLa target cells. (B) MSLN CAR3 A*11 Tmod cytotoxicity in primary T cells with MSLN(+)A*11(−)GFP(+) tumor or MSLN(+)A*11(+)GFP(+) “normal” HeLa target cells shows selective blocking in the presence of blocker antigen. Note that both tumor and “normal” target cells used here expressed GFP but are recolored for consistency. A*11:MSLN (B:A ≈8) target antigen ratio is noted. E:T=0.8:1. (C) Representative co-culture images at 48 hours for figure 7B.