Ines Mende

. 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.

Recently, a new class of human dendritic cell (DC) precursors has been described in the peripheral blood recognized by the mAb M-DC8. These cells represent approximately 1% of PBMC and acquire several characteristics of myeloid DC upon in vitro culture. In this report we show that M-DC8(+) monocytes secrete in response to LPS >10 times the amount of TNF-alpha as M-DC8(-) monocytes, but produce significantly less IL-10. Consistent with a role in inflammatory responses, we found that M-DC8(+) cells localized in the T cell area of inflamed human tonsils and in the subepithelial dome region of Peyer's patches. In patients with active Crohn's disease, abundant M-DC8(+) cells were detectable in inflamed ileal mucosa, which were entirely depleted after systemic steroid treatment. Our results indicate that M-DC8(+) cells are cells of DC phenotype in inflamed mucosa-associated lymphoid tissue that may contribute to the high level of TNF-alpha production in Crohn's disease. We infer that selective elimination of M-DC8(+) cells in inflammatory diseases has therapeutic potential.

Polymeric carriers designed to encapsulate protein antigens have great potential for improving the efficacy of vaccines and immunotherapeutics for diseases such as cancer. We recently developed a carrier system based on polyacrylamide hydrogel microparticles cross-linked with acid-labile moieties. After being phagocytosed by antigen-presenting cells, the protein encapsulated within the carrier is released and processed for subsequent presentation of antigenic epitopes. To understand the impact of particle size on the activation of T-cells following uptake by antigen-presenting cells, particles with mean diameters of 3.5 microm and 35 nm encapsulating a model protein antigen were synthesized by emulsion and microemulsion based polymerization techniques, respectively. In vivo tests demonstrated that both sizes of particles were effective at stimulating the proliferation of T-cells and were capable of generating an antigen-specific cytotoxic T-cell response when coadministered with immunostimulatory DNA. Contrary to previous reports in the literature, our results suggest that there is no significant difference in the magnitude of T-cell activation for the two sizes of particles used in these experiments. This disparity in findings may be related to fundamental differences in material properties of the carriers used in these studies, such as the hydrophilicity of the polyacrylamide particles described here versus the hydrophobic nature of carriers investigated by other groups.