Gene Therapy With a High-Affinity Single-Chain p53-Specific TCR Mediates Potent Anti-Tumor Response Without Inducing Gvhd In Vivo

The adoptive transfer of tumor-reactive cells is a promising approach in the treatment of human malignancies, but the challenge of isolating T cells with high-avidity for tumor antigens in each patient has limited its widespread application. Using HLA-A2.1 transgenic mice, we have demonstrated the feasibility of T-cell receptor (TCR) gene transfer into T cells to circumvent self-tolerance to the widely expressed human p53(264-272) tumor-associated antigen and developed approaches to generate high-affinity CD8-independent TCR. However, a safety concern of TCR gene transfer is the risk of pairing between introduced and the naturally expressed endogenous TCR chains, resulting in the generation of self-reactive T cells (off-target autoimmunity). We first genetically modified p53TCR constructs to minimize mispairing and improve correct pairing of the introduced TCR. We and others have shown that, cysteine modifications are able to increase the expression of the introduced TCR but fail to prevent mispairing formation in mouse and human T cells. To further enhance preferential TCR pairing, cell surface expression and TCR function, we introduced additional cysteine residues into the TCR α and β chain constant domains along with codon-optimization of the TCR sequences and cloning of the TCR constructs into one single 2A-based retroviral vector. To overcome TCR mispairing formation, we designed a single chain (sc) TCR by connecting the variable TCRa domain to the TCRb chain via a short peptide linker co-expressed with a truncated constant TCR a domain. Beside off-target toxicity, adoptive transfer of high-avidity T cells may potentially cause severe on-target toxicity for normal cells expressing low level of antigens. In this respect, pre-clinical in vivo studies are still missing.

Here, we evaluated the safety issues raised by the risk of p53TCR gene transfer-associated on/off-target toxicities in relevant mouse models of adoptive transfer.

In vitro studies showed that, scTCR-modified CD4⁺ and CD8⁺ T cells displayed similar high-avidity compared to the full-length TCR, as determined by peptide titration in cytotoxicity assays and were able to mediate specific lysis of p53 mutant A2.1⁺ tumor cells. Though, genetic modifications preserved the antigen specificity of these TCRs, the full-length version of the TCR could not prevent mispairing-mediated lethal off-target autoimmunity in vivo. In sharp contrast, T cells engrafted with the modified scTCR did not induce graft-versus-host disease (GVHD) following adoptive transfer. We next assessed the potential of scTCR-modified T cells to cause on-target autoimmunity in vivo, using mice which express the human wild type p53 and A2.1K^b (Hupki-A2.1K^b). We found that lymphodepleting preconditioning regimens plus vaccination-induced expansion of transferred TCR-specific T cells did not result in a depletion of hematopoietic cells, as mice recovered normal white blood cell counts, including lymphocytes and monocytes and survived without any sign of toxicity. Importantly, our study demonstrated that high-avidity scTCR-engineered human T cells were able to eradicate established tumors and persist for more than 6 months after infusion without inducing xeno-GVHD in NodScid IL-2R gamma chain-null mice.

Taken together, our study provided evidence that an optimized high-affinity scTCR-specific for the broadly expressed tumor-associated antigen p53(264-272) can eradicate p53⁺A2.1⁺ tumor cells in vivo without inducing off-target or self-directed toxicities in humanized mouse models of adoptive T-cell transfer. These data strongly support the improved safety and therapeutic efficacy of high-affinity scp53TCR for TCR-based immunotherapy of p53-associated malignancies.