Incorporation of Transmembrane Hydrophobic Mutations in the TCR Enhance Its Surface Expression and T Cell Functional Avidity

Abstract

TCR-gene transfer represents an effective way to redirect the specificity of T lymphocytes for therapeutic purposes. Recent successful clinical trials have underscored the potential of this approach in which efficient expression of the exogenous TCR has been directly linked to the efficacy of T cell activity. It has been also demonstrated that the TCR exhibits a lack of stability associated with the presence of positively charged residues in its transmembrane (TM) region. In this study, we designed an original approach selectively to improve exogenous TCR stability by increasing the hydrophobic nature of the TCRα TM region. Incorporation of hydrophobic residues at evolutionarily permissive positions resulted in an enhanced surface expression of the TCR chains, leading to an improved cellular avidity and anti-tumor TCR activity. Furthermore, this strategy was successfully applied to different TCRs, enabling the targeting of human tumors from different histologies. We also show that the combination of these hydrophobic mutations with another TCR-enhancing approach further improved TCR expression and function. Overall, these findings provide information regarding TCR TM composition that can be applied for the improvement of TCR-gene transfer-based treatments.

Human T Cells Engineered To Express a Programmed Death 1/28 Costimulatory Retargeting Molecule Display Enhanced Antitumor Activity

“…The a-and b-chains from the previously characterized TCRs specific for MART-1 [26][27][28][29][30][31][32][33][34][35] termed F4 (or DMF4) (18,27) and for p53 [264][265][266][267][268][269][270][271][272] (25) were subcloned into the pGEM-4Z/64A vector, as described previously (28). The chimeras PD1/28 and PD1/28BB were created by overlapping PCR, and their amino acid composition is indicated in Fig.…”

“…Freshly isolated PBLs were stimulated for 48 h in the presence of 50 ng/ml OKT3 (eBioscience, San Diego, CA) before transduction. Following stimulation, lymphocytes were transduced with retroviral vectors by transfer to nontreated tissue culture dishes (Nunc, Rochester, NY) that had been precoated with RetroNectin (Takara) and retroviral vectors, as previously described (31).…”

Enhanced antitumor activity mediated by human 4‐1BB‐engineered T cells

“…32 To test the biological activity of 4-1BB-engineered T-cells, we transduced OKT-3-stimulated PBLs with either 4-1BB or NGFR (control gene) and in parallel, we engineered them to express the MART-1-specific TCR F4 that we recently used 28 and that was reported in the first TCR-gene transfer clinical trial. 27 It is important to ensure that the TCR levels expressed on both the NGFR and the 4-1BB populations are similar to negate any possible bias resulting from different T-cell avidities.…”

“…28,29 Briefly, electroporation was performed at 400 V/500 lsec using an ElectroSquare Porator ECM 830 (BTX, San Diego, CA). The amount of in vitrotranscribed mRNA for each chain was 2 lg/10 6 PBMCs.…”

“…The a-and b-chains from the MART-1 [26][27][28][29][30][31][32][33][34][35] -specific TCR termed F4 (or DMF4) 27,28 were subcloned into the pGEM-4Z/64A vector as described previously 28 and in vitro-transcribed mRNA for both TCR chains was introduced into human peripheral blood lymphocytes (PBLs) by electroporation as described previously. 28,29 Briefly, electroporation was performed at 400 V/500 lsec using an ElectroSquare Porator ECM 830 (BTX, San Diego, CA).…”