Selecting highly affine and well-expressed TCRs for gene therapy of melanoma

Abstract

A recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumorreactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well-and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.

Adoptively transferred effector cells derived from naïve rather than central memory CD8 + T cells mediate superior antitumor immunity

“…Two days following initiation of stimulation as described above, cells were transduced with a MSGV1 retroviral vector with a codon optimized sequence encoding the pmel-1 TCR ␣-and ␤-chains linked by an internal ribosome entry site (41). Retrovirus was produced by transient transfection using the PlatE packaging line (Cell Biolabs Inc.) with cotransfection of the pCL-Eco (Imgenex) plasmid.…”

Manipulation of MHC-I/TCR Interaction for Immune Therapy

“…Although the generation of tumorreactive T lymphocytes by transfer of specific TCR genes into autologous or allogenic lymphocytes is feasible, the response rate observed in TCR gene therapy trial is low only 2 out 17 were completely responsed. The selection of wellexpressed and more epitope coverage TCRs with high affinity would improve such treatment (128,129).…”

A Screening Assay to Identify Agents That Enhance T-Cell Recognition of Human Melanomas

“…The chosen TCR reacts specifically to a Melan-A/MART-1 decapeptide ( 26 EAAGIGILTV 35 ) presented by HLA-A2, 38 with appropriate triggering of T-cell activation. 39 We used a lentiviral vector system for transduction of this specific TCR into the J.RT3-T3.5 T cell line, a Jurkat cell derivative that fails to express endogenous surface TCR through a mutation in its beta chain. 47,48 The functional specificity of this transduced Jurkat cell line ( J-TCR-M1) was established by its stimulation (as measured by IL-2 ELISA) only by HLA-A2+ tumor cells presenting the correct Melan-A/MART-1 peptide.…”

“…27 Briefly, a TCR specific for the MART-1 peptide EAAGIGILTV presented in HLA-A2 MHC Class I molecules (1D3 38,39 ) was synthesized by GeneArt (Burlingame, CA) and placed into a third-generation lentiviral transfer vector. 40 The full-length insert (1,820 base pair) containing both chains of the TCR was then subcloned into the lentiviral vector using the restriction sites NheI and SalI.…”