2020) Preclinical evaluation of an affinity-enhanced MAGE-A4-specific T-cell receptor for adoptive T-cell therapy, OncoImmunology, 9:1, 1682381, ABSTRACT A substantial obstacle to the success of adoptive T cell-based cancer immunotherapy is the sub-optimal affinity of T-cell receptors (TCRs) for most tumor antigens. Genetically engineered TCRs that have enhanced affinity for specific tumor peptide-MHC complexes may overcome this barrier. However, this enhancement risks increasing weak TCR cross-reactivity to other antigens expressed by normal tissues, potentially leading to clinical toxicities. To reduce the risk of such adverse clinical outcomes, we have developed an extensive preclinical testing strategy, involving potency testing using 2D and 3D human cell cultures and primary tumor material, and safety testing using human primary cell and cell-line crossreactivity screening and molecular analysis to predict peptides recognized by the affinity-enhanced TCR. Here, we describe this strategy using a developmental T-cell therapy, ADP-A2M4, which recognizes the HLA-A2-restricted MAGE-A4 peptide GVYDGREHTV. ADP-A2M4 demonstrated potent anti-tumor activity in the absence of major off-target cross-reactivity against a range of human primary cells and cell lines. Identification and characterization of peptides recognized by the affinity-enhanced TCR also revealed no cross-reactivity. These studies demonstrated that this TCR is highly potent and without major safety concerns, and as a result, this TCR is now being investigated in two clinical trials (NCT03132922, NCT04044768). ARTICLE HISTORY

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-β, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-β. Truncating the intracellular signaling domain from TGF-β receptor (TGFβR) II produces a dominant-negative receptor (dnTGFβRII) that dimerizes with endogenous TGFβRI to form a receptor that can bind TGF-β but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02–restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157–165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254–262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-β inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFβRII (e.g., GSK3845097). TGF-β isoforms and a panel of TGF-β–associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-β–positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non–small cell lung cancer setting. Coexpression of dnTGFβRII may therefore improve the efficacy of TCR-transduced T cells.

Major histocompatibility complex class-1-related protein (MR1), unlike human leukocyte antigen (HLA) class-1, has until recently been reported to be monomorphic. Tumor cell-specific MR1 restricted T cell receptors (TCRs) have been described, offering potential therapeutic application for cancer treatment. We show that human T cells expressing a TCR derived from an MR1-restricted T cell clone, termed MC.7.G5 (7G5.TCRT), retain MR1-directed cytotoxicity. However, activity is not pan-cancer, as initially reported with the clone MC.7.G5. Recognition is restricted by an allelic variant of MR1 (MR1*04) which is present at approximately 1% of the population at the heterozygote level. The 7G5 TCR is not cancer specific, as 7G5.TCRT and 7G5.TCRT-like TCRs react to both cancer and healthy cells expressing MR1*04 alleles. These data demonstrate that healthy individuals can harbor T cells reactive to an MR1 variant displaying self-ligands expressed in cancer and benign tissues. Targeting MR1 in cancer will require identification of cancer-specific presented ligands, and careful confirmation of cancer specificity of TCRs. MR1*04 may behave as an alloantigen warranting further study.

cells in peripheral blood mononuclear cells (PBMCs) treated with gastro-sphere conditioned medium in comparison with non-gastro-sphere conditioned medium. It was also demonstrated that T cell expansion was not significantly decreased in gastro-sphere conditioned medium treatment compared with the parental (non-gastrospheres) and control group. Conclusions: We concluded that gastro-sphere-soluble factors increase the percentage of Treg and Th17 cells more than its parental soluble factors. This suggested the possible existence of differentiation factors such as key cytokines among tumor cell secretions, which contribute to T cell differentiation. While it has been shown that gastro-sphere secretions can be possibly more immunomodulator than its parental cell secretions and lead to limited T cell expansion. Legal entity responsible for the study: