Theresa Hunter

. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Chimeric antigen receptor (CAR) T cell therapies have transformed treatment of B cell malignancies. However, their broader application is limited by complex manufacturing processes and the necessity for lymphodepleting chemotherapy, restricting patient accessibility. We present an in vivo engineering strategy using targeted lipid nanoparticles (tLNPs) for messenger RNA delivery to specific T cell subsets. These tLNPs reprogrammed CD8 + T cells in both healthy donor and autoimmune patient samples, and in vivo dosing resulted in tumor control in humanized mice and B cell depletion in cynomolgus monkeys. In cynomolgus monkeys, the reconstituted B cells after depletion were predominantly naïve, suggesting an immune system reset. By eliminating the requirements for complex ex vivo manufacturing, this tLNP platform holds the potential to make CAR T cell therapies more accessible and applicable across additional clinical indications.

Abstract Methods used to engineer cells for adoptive cell therapies (ACT) utilizing receptors that are constant across many patients (CAR or shared Ag TCRs) typically rely on Lenti-, retro-, or adeno-associated virus to deliver specificity-altering sequences to T cells. However, for personalized therapies such as the generation of neoepitope-specific TCR T cell therapies, use of viral vectors is not feasible due to long manufacturing timelines and prohibitive per-patient costs. PACT Pharma has developed a highly efficient, DNA-mediated (non-viral) proprietary precision genome engineering approach to engineer neoepitope-specific primary human T cells. This method can be widely utilized to generate T cells at research scale, as well as for ex vivo manufacturing. Briefly, genomes of individual primary human CD8 and CD4 T cells are engineered with site-specific nucleases in a single-step transfection process to yield efficient, targeted replacement of the endogenous TCR with the therapeutic neoTCR sequences. In this way, the expression of the endogenous TCR is abolished ensuring natural expression and regulation of the inserted neoTCR. The precision of neoTCR-T cell genome engineering was evaluated by Targeted Locus Amplification (TLA) for off-target integration hot spots or translocations, and by next generation sequencing based off-target cleavage assays and found to lack evidence of unintended outcomes. Engineered neoepitope-specific T cells are highly functional as demonstrated by antigen-specific proliferation, killing and cytokine production. Phenotype and detailed functional characterization of PACTs neoTCR-P1 T cells were performed and are described in a separate abstract. PACT’s precision genome engineering approach enables highly efficient generation of bespoke NeoTCR T cells for personalized adoptive cell therapy for patients with solid tumors. Furthermore, PACT precision genome engineering method is not restricted to the use in T cells and has also been applied successfully to other primary cell types, including natural killer and hematopoietic stem cells. Citation Format: Kyle Jacoby, Robert Moot, William Lu, Diana Nguyen, Barbara Sennino, Andrew Conroy, Bhamini Purandare, Adam J. Litterman, Fabrizia Urbinati, Susan P. Foy, Theresa Hunter, Albert Tai, Michael T. Bethune, Songming Peng, Olivier Dalmas, Alex Franzusoff, Stefanie J. Mandl. Highly efficient, non-viral precision genome engineering for the generation of personalized neoepitope-specific adoptive T cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4783.

Abstract Clinical benefit observed with immuno-oncology trials often depends on the unleashing of a pre-existing intrinsic T cell immune response in each cancer patient. The targets of these intrinsic T cells are commonly ascribed to recognition of patient-specific neoantigens that arose from cancer mutations. PACT Pharma has developed the ability to selectively capture neoantigen-specific CD8+ T cells from peripheral blood of the patient. Leveraging this technology, PACT Pharma is developing personalized, autologous neo-epitope specific TCR-engineered T cell therapies for the eradication of solid tumors. Briefly, using PACT's proprietary TCR isolation technology neoepitope-specific TCRs are cloned and autologous CD8+ and CD4+ T cells from the same patient with cancer are precision genome engineered (using a DNA-mediated (non-viral) method) to express the neoTCR. NeoTCR expressing T cells are then expanded in a manner that preserves a “younger” T cell phenotypes, resulting in a NeoTCR-P1 product in which the majority of the T cells exhibit T memory stem cell and T central memory phenotypes. Upon cognate antigen encounter, NeoTCR-P1 rapidly differentiate into potent effector T cells. Engineered NeoTCR-P1 cells rapidly expand, secrete effector molecules such as perforin and granzyme B, and cytokines such as interferon-gamma (IFN-γ), IL-2 and TNF-alpha (TNF-α). Single cell secretome analysis demonstrates that NeoTCR-P1 cells are highly polyfunctional (secretion of two or more cytokines or effector proteins). These results demonstrate that PACT’s autologous ex vivo engineered NeoTCR-P1 T cells represent a highly personalized adoptive T cell therapy with potential to provide significant clinical benefit to subjects with solid tumors. Citation Format: Barbara Sennino, Andrew Conroy, Bhamini Purandare, Adam Litterman, Kyle Jacoby, Robert Moot, William Lu, Diana Nguyen, Fabrizia Urbinati, Susan Foy, Theresa Hunter, Olivier Dalmas, Michael Bethune, Tim Park, Songming Peng, Alex Franzusoff, Stefanie Mandl. NeoTCR-P1, a novel neoepitope-specific adoptive cell therapy, consists of T cells with ‘younger’ phenotypes that rapidly proliferate and kill target cells upon recognition of cognate antigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1433.