T cell receptor gene therapy: strategies for optimizing transgenic TCR pairing.
ABSTRACT T cell receptor (TCR) gene therapy provides patients with autologous T cells that are genetically engineered with TCRalphabeta chains and constitutes a promising approach for the treatment of tumors and virus infections. Among the current challenges of TCR gene therapy is the optimization of TCRalpha and beta transgene pairing to enhance the functional avidity of therapeutic T cells. Recently, various genetically modified TCRs have been developed that enhance TCR pairing and minimize mispairing, i.e. pairing between transgenic and endogenous TCR chains. Here, we classify such receptors according to their CD3-dependence for surface expression and review their abilities to address functional T cell avidity. In addition, we discuss the anticipated clinical value of these and other strategies to generate high-avidity T cells.
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ABSTRACT: TCR gene therapy represents a feasible and promising treatment for patients with cancer and virus infections. Currently, this treatment rationale is hampered by diluted surface expression of the TCR transgene and generation of potentially self reactive T-cells, both a direct consequence of mis-pairing with endogenous TCR chains. As we reported previously (Gene Ther 16:1369, 2000; J Immunol 180:7736, 2008), TCR mis-pairing can be successfully addressed by a TCR:CD3 fusion protein (i.e., TCR:). Here, we set out to minimize the content of CD3 in TCR:, specific for MAGE-A1/HLA-A1, without compromising TCR pairing and function. Domain-exchange and 3D-modeling strategies defined a set of minimal TCR: variants, which, together with a murinized and cysteine-modified TCR (TCR:mu+cys), were tested for functional TCR expression and TCR pairing. Our data with Jurkat T cells show that the CD3 transmembrane domain is important for cell-surface expression, whereas the CD3 intracellular domain is crucial for T-cell activation. Notably, inability of TCR: to mis-pair was not observed for TCR:mu+cys, which depended exclusively on the transmembrane domain of CD3 and could not be recapitulated by a limited number of structurally defined CD3 transmembrane amino acids. The extracellular CD3 domain was dispensable for TCR:'s ability to prevent TCR mis-pairing, bind pMHC and mediate NFAT activation. In primary human T cells, however, minimal TCR: without CD3's extracellular domain but not TCR: nor TCR:mu+cys revealed compromised cell surface expression and T cell function. Taken together, our study demonstrates that CD3's transmembrane domain dictates TCR:'s inability to TCR mis-pair, but only TCR coupled to complete CD3 and not its minimal variants were functionally expressed in primary T cells.
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ABSTRACT: TCRα- and β-chains cooperatively recognize peptide-MHC complexes. It has been shown that a "chain-centric" TCR hemichain can, by itself, dictate MHC-restricted Ag specificity without requiring major contributions from the paired TCR counterchain. Little is known, however, regarding the relative contributions and roles of chain-centric and its counter, non-chain-centric, hemichains in determining T cell avidity. We comprehensively analyzed a thymically unselected T cell repertoire generated by transducing the α-chain-centric HLA-A*02:01(A2)/MART127-35 TCRα, clone SIG35α, into A2-matched and unmatched postthymic T cells. Regardless of their HLA-A2 positivity, a substantial subset of peripheral T cells transduced with SIG35α gained reactivity for A2/MART127-35. Although the generated A2/MART127-35-specific T cells used various TRBV genes, TRBV27 predominated with >10(2) highly diverse and unique clonotypic CDR3β sequences. T cells individually reconstituted with various A2/MART127-35 TRBV27 TCRβ genes along with SIG35α possessed a wide range (>2 log orders) of avidity. Approximately half possessed avidity higher than T cells expressing clone DMF5, a naturally occurring A2/MART127-35 TCR with one of the highest affinities. Importantly, similar findings were recapitulated with other self-Ags. Our results indicate that, although a chain-centric TCR hemichain determines Ag specificity, the paired counterchain can regulate avidity over a broad range (>2 log orders) without compromising Ag specificity. TCR chain centricity can be exploited to generate a thymically unselected Ag-specific T cell repertoire, which can be used to isolate high-avidity antitumor T cells and their uniquely encoded TCRs rarely found in the periphery because of tolerance. Copyright © 2015 by The American Association of Immunologists, Inc.
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ABSTRACT: Clinical therapy with T cells shows promise for cancer patients, but is currently challenged by incomplete responses and tumor relapse. The exact mechanisms that contribute to tumor relapse remain largely unclear. Here, we treated mouse melanomas with TCR-engineered T cells directed against a human peptide-MHC antigen in immune-competent mice. T cells resulted in significant tumor regression, which was followed by relapse in about 80-90% of mice. Molecular analysis revealed that relapsed tumors harbored non-mutated antigen genes, not silenced by promoter methylation, and functionally expressed surface antigen at levels equal to non-treated tumors. Relapsed tumors resisted a second in vivo T cell treatment, but regained sensitivity to T cell treatment upon re-transplantation in mice. Notably, relapsed tumors demonstrated decreased levels of CD8 T cells and monocytes, which were substantiated by down-regulated expression of chemoattractants and adhesion molecules. These observations were confirmed when using T cells specific for a less immunogenic, endogenous mouse melanoma antigen. We conclude that tumors, when exposed to T cell treatment, can relapse without loss of antigen and develop a milieu that evades recruitment of effector CD8 T cells. Our findings support the concept to target the tumor milieu to aid T cell therapy in limiting tumor relapseMolecular Therapy (2014); doi:10.1038/mt.2014.215.Molecular Therapy 11/2014; 23(2). DOI:10.1038/mt.2014.215 · 6.43 Impact Factor