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TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans., Increasing TCR cell surface expression can potentiate T cell responses to low-concentrations of antigen. Here the authors identify aminoacids in human TCR variable domains that impact its surface expression, and demonstrate how editing these residues can improve T cell activation and effector function without altering antigen specificity.

INTRODUCTION We have previously described AUTO1, a CD19 CAR with a fast off-rate CD19 binding domain, designed to reduce CAR-T immune toxicity and improve engraftment. Its clinical activity has been tested in r/r paediatric and adult B-ALL. Cumulatively, this data confirms the intended function of the receptor, with low levels of CRS/ICANS and long-term engraftment of CAR T-cells observed in both patient groups. Recently, CAR-T therapy has been explored in indolent lymphomas such as follicular (FL) and mantle cell lymphoma (MCL), but a high incidence of toxicity including Grade 3-4 ICANS has been reported. We have initiated testing of AUTO1 in the setting of indolent and high-grade B-NHL and CLL (NCT02935257). METHODS Manufacturing: CAR T-cell products were generated using a semi-automated closed process from non-mobilised leukapheresate. Study design: Subjects ≥ 16y underwent lymphodepletion with fludarabine (30mg/m 2 x3) and cyclophosphamide (60mg/kg x1) prior to AUTO1 infusion, with the exception of the DLBCL cohort who additionally received a single dose of pembrolizumab (200mg) on day -1 to potentiate CAR-T expansion. AUTO1 dose varies based on the indication. Split dosing of 230 x10^6 CD19 CAR T-cells at day 0 and day 9 is employed in the CLL cohort. A single dose of 200 x10^6 CD19 CAR T-cells is delivered to patients with B-NHL. Study endpoints include feasibility of manufacture, grade 3-5 toxicity and remission rates at 1 and 3 months. RESULTS As of 17th May 2021, we recruited 13 patients: 7 with FL, 4 with MCL, 1 DLBCL and 1 CLL. Apheresis and product manufacture was successful in all 13 patients and 9 patients were infused: 7 with FL and 2 with MCL. Three patients (1 DLBCL, 1 CLL and 1 MCL) were pending infusion at time of data cut-off and 1 patient (MCL) died due to Covid-19 prior to infusion. Patients treated with AUTO1 had a median age of 56 years (range 39-68y), had received a median of 3 prior lines of treatment (range 2-5) and all patients had stage IV disease at screening. Grade 1 CRS was reported in 4/9 and Grade 2 CRS in 1/9. 1/9 developed MAS which resolved with anakinra/dexamethasone. No ICANS was observed on study. Excellent CAR engraftment was observed and 9/9 patients were in CMR by 18FDG PET-CT post-treatment. At a median of 6.1 months (range 4.0-8.1m), 8/9 patients were disease free at last follow-up. One patient died in CMR at month 5.6 of COVID-19. CONCLUSION AUTO1 has a tolerable safety profile in adult patients with r/r B-NHL despite high disease burden. Early data shows 100% complete remission rates and excellent CAR engraftment/expansion. Additional MCL, CLL and DLBCL patients, updated data and longer follow up will be presented. Disclosures Roddie: Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau. Hartley: Astra Zeneca: Ended employment in the past 24 months; ADC Therapeutics: Consultancy, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Farzaneh: Autolus: Consultancy, Current equity holder in publicly-traded company. Lowdell: Autolus: Consultancy, Current equity holder in publicly-traded company. Linch: Autolus: Consultancy, Current equity holder in publicly-traded company. Pule: Autolus: Current Employment, Current equity holder in publicly-traded company. Peggs: Autolus: Consultancy, Current equity holder in publicly-traded company.