Metabolic engineering against the arginine microenvironment enhances CAR-T cell proliferation and therapeutic activity.

Hematological and solid cancers catabolize the semiessential amino acid arginine to drive cell proliferation. However, the resulting low arginine microenvironment also impairs chimeric antigen receptor T cells (CAR-T) cell proliferation, limiting their efficacy in clinical trials against hematological and solid malignancies. T cells are susceptible to the low arginine microenvironment because of the low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC). We demonstrate that T cells can be reengineered to express functional ASS or OTC enzymes, in concert with different chimeric antigen receptors. Enzyme modifications increase CAR-T cell proliferation, with no loss of CAR cytotoxicity or increased exhaustion. In vivo, enzyme-modified CAR-T cells lead to enhanced clearance of leukemia or solid tumor burden, providing the first metabolic modification to enhance CAR-T cell therapies.

The arginine metabolome in acute lymphoblastic leukemia can be targeted by the pegylated-recombinant arginase I BCT-100.

Arginine is a semi-essential amino acid that plays a key role in cell survival and proliferation in normal and malignant cells. BCT-100, a pegylated (PEG) recombinant human arginase, can deplete arginine and starve malignant cells of the amino acid. Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood, yet for patients with high risk or relapsed disease prognosis remains poor. We show that BCT-100 is cytotoxic to ALL blasts from patients in vitro by necrosis, and is synergistic in combination with dexamethasone. Against ALL xenografts, BCT-100 leads to a reduction in ALL engraftment and a prolongation of survival. ALL blasts express the arginine transporter CAT-1, yet the majority of blasts are arginine auxotrophic due to deficiency in either argininosuccinate synthase (ASS) or ornithine transcarbamylase (OTC). Although endogenous upregulation or retroviral transduced increases in ASS or OTC may promote ALL survival under moderately low arginine conditions, expression of these enzymes cannot prevent BCT-100 cytotoxicity at arginine depleting doses. RNA-sequencing of ALL blasts and supporting stromal cells treated with BCT-100 identifies a number of candidate pathways which are altered in the presence of arginine depletion. Therefore, BCT-100 provides a new clinically relevant therapeutic approach to target arginine metabolism in ALL.

Promiscuous recognition of MR1 drives self-reactive mucosal-associated invariant T cell responses.

Mucosal-associated invariant T (MAIT) cells use canonical semi-invariant T cell receptors (TCR) to recognize microbial riboflavin precursors displayed by the antigen-presenting molecule MR1. The extent of MAIT TCR crossreactivity toward physiological, microbially unrelated antigens remains underexplored. We describe MAIT TCRs endowed with MR1-dependent reactivity to tumor and healthy cells in the absence of microbial metabolites. MAIT cells bearing TCRs crossreactive toward self are rare but commonly found within healthy donors and display T-helper-like functions in vitro. Experiments with MR1-tetramers loaded with distinct ligands revealed significant crossreactivity among MAIT TCRs both ex vivo and upon in vitro expansion. A canonical MAIT TCR was selected on the basis of extremely promiscuous MR1 recognition. Structural and molecular dynamic analyses associated promiscuity to unique TCRß-chain features that were enriched within self-reactive MAIT cells of healthy individuals. Thus, self-reactive recognition of MR1 represents a functionally relevant indication of MAIT TCR crossreactivity, suggesting a potentially broader role of MAIT cells in immune homeostasis and diseases, beyond microbial immunosurveillance.

Isolation of T cell receptors targeting recurrent neoantigens in hematological malignancies.

Mutation-derived neoantigens represent an important class of tumour-specific, tumour rejection antigens, and are attractive targets for TCR gene therapy of cancer. The majority of such mutations are patient-specific and targeting these requires a fully personalized approach. However, some mutations are found recurrently among cancer patients, and represent potential targets for neoantigen-specific TCR gene therapy that is more widely applicable. Therefore, we have investigated if some cancer mutations found recurrently in hematological malignancies encode immunogenic neoantigens presented by common European Caucasoid HLA class I alleles and can form targets for TCR gene therapy. We initially focused on identifying HLA class I neoepitopes derived from calreticulin (CALR) exon 9 mutations, found in ~ 80% of JAK2wt myeloproliferative neoplasms (MPN). Based on MHC class I peptide predictions, a number of peptides derived from mutant CALR (mCALR) were predicted to bind to HLA-A*03:01 and HLA-B*07:02. However, using mass spectrometry and ex vivo pMHC multimer staining of PBMC from MPN patients with CALR exon 9 mutations, we found no evidence that these peptides were naturally processed and presented on the surface of mCALR-expressing target cells. We next developed a protocol utilizing pMHC multimers to isolate CD8+ T cells from healthy human donor PBMC that are specific for mCALR and additional putative neoepitopes found recurrently in hematological malignancies. Using this approach, CD8+ T cells specific for HLA-A*03:01- and HLA-B*07:02-presented mCALR peptides and an HLA-A*11:01-presented mutant FBXW7 (mFBXW7) peptide were successfully isolated. TCRs isolated from mCALR-specific CD8+ T cell populations were not able to recognize target cells engineered to express mCALR. In contrast, mFBXW7-specific CD8+ T cells were able to recognize target cells engineered to express mFBXW7. In conclusion, while we found no evidence for mCALR derived neoepitope presentation in the context of the HLA class I alleles studied, our data suggests that the recurrent pR465H mutation in FBXW7 may encode an HLA-A*11:01 presented neoepitope, and warrants further investigation as a target for T cell based immunotherapy of cancer.