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Abstract
Background Mesothelin (MSLN) is expressed on a variety of solid tumors, including mesothelioma and ovarian, uterine, gastric, pancreatic, and lung cancers.1 However, efforts to target MSLN using cellular therapies have been hampered by severe on-target, off-tumor toxicities associated with damage to normal tissues expressing MSLN.2 To avoid these toxicities, we have developed a logic-gated engineered cell therapy, Tmod™, which is composed of two chimeric antigen receptors (CARs): an activator that targets a tumor-associated antigen and an inhibitory receptor (blocker) gated by an antigen expressed on normal tissue but lost in tumor cells due to loss of heterozygosity (LOH). A2B694 is an MSLN-specific Tmod construct combining a third-generation MSLN CAR with an LIR-1-based inhibitory receptor specific for human leukocyte antigen A*02 (HLA-A*02).
Methods Lentivirus encoding i) the CAR, ii) the blocker, and iii) an shRNA targeting β2M was used to transduce T cells from HLA-A*02 donors and generate MSLN Tmod cells. In vitro cytotoxicity measurements were performed using fluorescence-based imaging and luciferase readouts. In vivo assessments were performed in NSG mice subcutaneously implanted with “normal” cells (MSLN[+]A*02[+]), or tumor cells (MSLN[+]A*02[-]), in the left and right flanks, respectively. Following engraftment, mice were randomized and treated intravenously with MSLN Tmod cells or controls. Grafts were measured via caliper.
Results MSLN Tmod cells preferentially killed tumor cells (MSLN[+]A*02[-]) over “normal” cells (MSLN[+]A*02[+]) in vitro, unlike clinically active comparator M5 CAR T cells, which indiscriminately killed both target cell types (figure 1A). Soluble MSLN, tested across a 0-2 µg/mL range, did not impact MSLN Tmod function. Additionally, in mixed cell cultures where T cells and tumor and “normal” cells were simultaneously cultured (1:1:1 ratio), MSLN Tmod cells selectively killed tumor targets while sparing “normal” cells. Further, MSLN Tmod cells cycled between activated and blocked states in vitro when repeatedly challenged with tumor or “normal” target cells. Finally, while MSLN CAR T cells killed both “normal” and tumor grafts in vivo, MSLN Tmod cells selectively killed tumor grafts while sparing “normal” grafts (figure 1B, C).
Conclusions A2B694 is an autologous MSLN Tmod cell product that leverages LOH at the HLA locus in cancer cells, providing a mechanism to discriminate between normal and tumor cells. BASECAMP-1 (NCT04981119), an observational study that will identify patients with somatic HLA LOH, is currently recruiting. Eligible patients with metastatic colorectal, pancreatic, or non-small cell lung cancer will be apheresed for a future A2B694 interventional study (EVEREST-2).
References
Hassan R et al. Mesothelin immunotherapy for cancer: Ready for prime time? J Clin Oncol. 2016;34(34):4171-4179.
Tanyi JL et al. Phase I study of autologous T cells bearing fully-humanized chimeric antigen receptors targeting mesothelin in mesothelin-expressing cancers. Presented at: Cellicon Valley ‘21: The Future of Cell and Gene Therapies; May 6-7, 2021; virtual symposium.
