Phenotype, Function, and Safety of a p53 TCR Bicistronic GMP-Suitable Retroviral Construct.

Abstract

Malignant transformation of normal cells is frequently correlated with the involvement of so called tumor-associated antigens (TAA). Such proteins, that are often overexpressed in tumor cells, can be recognized by cytotoxic CD8+ T cells (CTL) if presented as peptides on MHC (Major-Histocompatibility-Complex)-class I molecules. Due to self-tolerance mechanisms, the peripheral T cell repertoire is devoid of efficient TAA-specific, tumor-reactive CTL with high affinity, limiting the successful development of antigen-specific immunotherapeutic strategies based on such tumor-reactive T cells. The aim of this project is the preclinical development of an adoptive immunotherapy against p53-positive tumors. The p53 tumor-suppressor protein is an antigen, which is markedly upregulated in multiple tumors. Such overexpression of p53 correlates with the presence of mutated variants of p53, which inactivate p53’s normal function as tumor-suppressor. Tumors with wildtype (wt) p53 also exhibit abnormalities in p53 expression, metabolism, and function, which arise from alterations of an impressive repertoire of p53-interacting gene products. Hence, perturbation of p53 regulatory pathways and metabolism occurs in most, if not all, types of human malignancies and makes class I MHC bound peptides naturally processed from the wt p53 protein almost universal targets for tumor-specific CD8+ CTL. The generation of CTL, bearing a high-affinity p53-specific T-cell receptor (TCR) reactive against such peptide/MHC-class I complexes presented on tumors, would display a promising strategy for cancer immunotherapy. Through adoptive transfer of autologous PBMCs, retrovirally transduced with genes encoding such TCR, self-tolerance against p53 in tumor patients could be circumvented leading to selective destruction of p53-expressing tumors without harming healthy tissue. Previous studies have described strategies to circumvent self-tolerance against p53 using HLA-A2.1 transgenic mice. Immunization of such mice with peptides derived from human p53 has generated efficient high-affinity p53A2.1-specific CTL clones. Moreover the TCR from one of these clones that specifically recognizes a wt p53 peptide (aa 264–272) could be cloned for the transduction into human T-cells. We could show that the introduction of this p53(264–272)A2.1-specific-TCR into human PBMCs via retroviral transduction produced effective and high affine T cells of high affinity capable of recognizing and lysing p53+ tumor cells while sparing normal p53-expressing cells. Apart from CD8+ T cells, p53(264–272)A2.1-specific-TCR-transduced CD4+ helper T cells (Th) too were able to recognize peptide-loaded p53(264–272)A2-complexes as well as p53/HLA-A2-positive tumor cells. Moreover both T cell subsets were cooperative and interacted synergistically with dendritic cell intermediates and tumor targets. The adoptive transfer of such gene-modified T cells could display a highly effective therapy against p53-positive tumors. To demonstrate the therapeutic potential of p53(264–272)/HLA-A2-specific TCR transgenic cells for a possible clinical translation, we have initiated preclinical studies for the adoptive transfer of such cells. Here, we demonstrate the function and safety in vitro and in vivo of a retroviral GMP-suitable vector construct coding for the p53(264–272)/HLA-A2-specific TCR designed for clinical application.