Wt P53 Peptides Research Articles

T cell receptors employ diverse strategies to target a p53 cancer neoantigen

Adoptive cell therapy with tumor-specific T cells can mediate durable cancer regression. The prime target of tumor-specific T cells are neoantigens arising from mutations in self-proteins during malignant transformation. To understand T cell recognition of cancer neoantigens at the atomic level, we studied oligoclonal T cell receptors (TCRs) that recognize a neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by the major histocompatibility complex class I molecule HLA-A2. We previously reported the structures of three p53R175H-specific TCRs (38-10, 12-6, and 1a2) bound to p53R175H and HLA-A2. The structures showed that these TCRs discriminate between WT and mutant p53 by forming extensive interactions with the R175H mutation. Here, we report the structure of a fourth p53R175H-specific TCR (6-11) in complex with p53R175H and HLA-A2. In contrast to 38-10, 12-6, and 1a2, TCR 6-11 makes no direct contacts with the R175H mutation, yet is still able to distinguish mutant from WT p53. Structure-based in silico mutagenesis revealed that the 60-fold loss in 6-11 binding affinity for WT p53 compared to p53R175H is mainly due to the higher energetic cost of desolvating R175 in the WT p53 peptide during complex formation than H175 in the mutant. This indirect strategy for preferential neoantigen recognition by 6-11 is fundamentally different from the direct strategies employed by other TCRs and highlights the multiplicity of solutions to recognizing p53R175H with sufficient selectivity to mediate T cell killing of tumor but not normal cells.

CD8+ T cell recognition of polymorphic wild-type sequence p53(65-73) peptides in squamous cell carcinoma of the head and neck.

The TP53 tumor suppressor gene contains a well-studied polymorphism that encodes either proline (P) or arginine (R) at codon 72, and over half of the world's population is homozygous for R at this codon. The wild-type sequence (wt) p53 peptide, p53(65-73), has been identified as a CD8+ T cell-defined tumor antigen for use in broadly applicable cancer vaccines. However, depending on the TP53 codon 72 polymorphism of the recipient, the induced responses to the peptides incorporating R (p53(72R)) or P (p53(72P)) can be "self" or "non-self." Thus, we sought to determine which wt p53(65-73) peptide should be used in wt p53-based cancer vaccines. Despite similar predicted HLA-A2-binding affinities, the p53(72P) peptide was more efficient than the p53(72R) peptide in HLA-A2 stabilization assays. In vitro stimulation (IVS) of CD8+ T cells obtained from healthy HLA-A2(+) donors with these two peptides led to the generation of CD8+ T cell effectors in one-third of the samples tested, at a frequency similar to the responsiveness to other wt p53 peptides. Interestingly, regardless of their p53 codon 72 genotype, CD8+ T cells stimulated with either p53(72P) or p53(72R) peptide were cross-reactive against T2 cells pulsed with either peptide, as well as HLA-A2(+) head and neck cancer (HNC) cell lines presenting p53(72P) and/or p53(72R) peptides for T cell recognition. Therefore, the cross-reactivity of CD8+ T cells for the polymorphic wt p53(65-73) peptides, irrespective of their p53 codon 72 polymorphism, suggests that employing either peptide in wt p53-based vaccines can result in efficient targeting of this epitope.

Abstract 2414: Comparable effect of p53 peptide vaccine in adjuvant or pulsed on dendritic cells in ovarian cancer patients: A gynecologic oncology group study

Abstract Purpose: Peptide antigens have been administered by different approaches in cancer vaccine therapy, including direct injection or pulsed onto dendritic cells; however, the optimal delivery method is still debatable. In this study, we describe the immune response elicited by the wild-type (wt) p53 vaccine using two approaches. Experimental Design: Twenty-one HLA-A2.1 patients with stage III, IV, or recurrent ovarian cancer overexpressing the p53 protein with no evidence of disease were treated in two cohorts. Arm A received subcutaneous wt p53:264-272 peptide admixed with Montanide and GM-CSF. Arm B received wt p53:264-272 peptide-pulsed dendritic cells intravenously. Interleukin-2 (IL-2) was administered in both cohorts in alternative cycles. The immunologic response was assessed by ELISPOT and tetramer assays. Results: Nine of 13 patients (69%) in arm A and 5 of 6 patients (83%) in arm B had an immunologic response. The vaccine caused no serious systemic side effects. IL-2 administration resulted in grade 3 and 4 toxicities in both arms and directly induced expansion of T regulatory cells. The median overall survival was 40.8 and 29.6 months for arm A and B, respectively; the median progression-free survival was 4.2 and. 8.7 months, respectively. Conclusion: We demonstrated that using either vaccination approach could generate comparable specific immune responses against the wt p53 peptide with minimal toxicity. Accordingly, our findings support the utilization of the less demanding subcutaneous method. Furthermore, as IL-2 may add toxicity and induce T regulatory cells, which can suppress the immune response, therefore, it may not be needed in future trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2414.

CD4+ T cell responses to HLA-DP5-restricted wild-type sequence p53 peptides in patients with head and neck cancer

Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines. Evidence has been accumulating which indicates that CD4+ Th cells have an important role in generating and maintaining antitumor immune responses. To elucidate the nature of CD4+ Th responses to wt p53 epitopes in patients with squamous cell carcinoma of the head and neck (SCCHN), peripheral blood mononuclear cells (PBMCs) from HLA-DP5+ patients were stimulated with HLA-DP5-restricted wt p53 peptides, p53(108-122) or p53(153-166), and tested for the release of IFN-gamma and IL-5 in ELISPOT assays. Immunohistochemistry for p53 accumulation in tumors, and ELISA for serum antibodies to p53 were also performed. Eleven (57.9%) of 19 HLA-DP5+ patients but none of 5 healthy donors had detectable Th1 and/or Th2 responses to wt p53 peptides by ELISPOT assay. Among these 11 responding patients, 9 (81.8%) and all 11 (100%) patients had a tumor burden and p53 accumulation, respectively. On the other hand, two responding patients were in post-operative condition. Interestingly, among nine patients with a tumor burden, four patients with early disease showed either Th1-polarized or mixed Th1/Th2 responses, while five patients with advanced disease showed either Th2-polarized or mixed Th1/Th2 responses. Our results suggest that wt p53(108-122) and p53(153-166) peptides stimulate both Th1- and Th2-type CD4+ T cell responses in patients with SCCHN, and anti-p53 Th responses may persist even after surgical resection of the tumor; however, the presence of a tumor and its progression may affect the nature of immune responses to wt p53 peptides.

Toward the development of multi-epitope p53 cancer vaccines: An in vitro assessment of CD8 + T cell responses to HLA class I-restricted wild-type sequence p53 peptides

Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines. Six HLA-A2 or HLA-A24-restricted wt p53 peptides were evaluated for their ex vivo immunogenicity and their potential for use in cancer vaccines. Peripheral blood mononuclear cells (PBMC) obtained from HLA-A⁎0201 + and/or HLA-A⁎2402 + normal donors and subjects with squamous cell carcinoma of the head and neck (SCCHN) were analyzed for p53 peptide-specific reactivity in ELISPOT IFN-γ assays. CD8 + T cells in 7/10 normal donors (HD) and 11/23 subjects with SCCHN responded to at least one of the wt p53 peptides. CD8 + T cell precursors responsive to wt p53 epitopes were detected in the circulation of most subjects with early disease, and an elevated blood Tc 1/Tc 2 ratio distinguished wt p53 peptide responders from non-responders. The identification of multiple wt p53 peptides able to induce cytolytic T lymphocytes in most subjects with cancer promotes the development of multi-epitope p53 vaccines.

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

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.

The Wild-Type Sequence (wt) p5325–35 Peptide Induces HLA-DR7 and HLA-DR11-Restricted CD4+ Th Cells Capable of Enhancing the Ex Vivo Expansion and Function of Anti-wt p53264–272 Peptide CD8+ T Cells

Tumor peptide-based vaccines are more effective when they include tumor-specific Th cell-defined as well as CTL-defined peptides. Presently, two overlapping wild-type sequences (wt) p53 helper peptides, p53(108-122) and p53(110-124), have been identified as HLA-DR1- and/or HLA-DR4-restricted epitopes. These HLA-DR alleles are expressed by approximately 35% of subjects with cancer. To identify Th cell-defined wt p53 peptides suitable for use on the remaining subject population, a dendritic cell (DC)-based coculture system was developed. CD4+ T cells isolated from PBMC obtained from HLA-DR4- normal donors were stimulated ex vivo with autologous DC transfected with wt p53 or mutant p53 cDNA. Reactivity of T cells was tested in ELISPOT IFN-gamma assays against DC pulsed individually with a panel of algorithm-predicted, multiple HLA-DR-binding wt p53 peptides. The wt p53(25-35) peptide was identified as capable of inducing and being recognized by CD4+ T cells in association, at a minimum, with HLA-DR7 and -DR11 molecules, each of which is expressed by approximately 15% of the population. In addition, the presence of anti-p53(25-35) CD4+ Th cells was shown to enhance the in vitro generation/expansion of HLA-A2-restricted, anti-wt p53(264-272) CD8+ T cells, which from one donor were initially "nonresponsive" to the wt p53(264-272) peptide. The wt p53(25-35) peptide has attributes of a naturally presented Th cell-defined peptide, which could be incorporated into antitumor vaccines applicable to a broader population of subjects for whom a wt p53 helper peptide is presently unavailable, as well as used for monitoring anti-p53 Th cell activity in cancer subjects receiving p53-based immunotherapy.

Crystallographic Analysis of an 8-mer p53 Peptide Analogue Complexed with MDM2

The most potent inhibitor of the p53-MDM2 interaction reported to date is an 8-mer p53 peptide analogue (Novartis peptide), which contains 6-chlorotryptophane (Cl-Trp) and phosphonomethylphenylalanine (Pmp) as key residues for the enhanced activity. We report here a crystal structure of the co-complex between MDM2 and the Novartis peptide solved at 1.8 A resolution. The structural basis for the role of the two aromatic residues are delineated by comparing the present structure with crystal structures of the MDM2 co-complex bound to other inhibitors including the wt-p53 peptide itself.

Impact of p53-based immunization on primary chemically-induced tumors

In mice as well as humans, cytotoxic T lymphocytes (CTL) specific for wild-type-sequence (wt) p53 peptides have been shown to react against a wide range of tumors, but not normal cells. As such, they are attractive candidates for developing broadly applicable cancer vaccines. Of particular interest is the potential of using p53-based vaccines in high-risk individuals to prevent cancer. Methylcholanthrene, an immunosuppressive polycyclic hydrocarbon carcinogen implicated as a causative agent in human cancers, has long been used to induce murine tumors with a high incidence of genetic alterations and sensitivity to wt p53-specific CTL. To analyze the potential of p53-based vaccines on primary tumors, we evaluated the efficacy of DNA and dendritic cell vaccines targeting wt p53 peptides given to methylcholanthrene-treated mice in the protection or therapy settings. The results indicate that the efficacy of these vaccines relative to reducing tumor incidence were severely compromised by vaccine-induced tumor escape. As compared to tumors induced in non-immunized mice, a higher incidence of epitope-loss tumors was detected in tumors from the immunized mice. The increase in tumor escape arose as a consequence of either increased frequencies of mutations within/flanking p53 epitope-coding regions or downregulation of expression of the major histocompatibility complex Class I molecules that present these epitopes for T cell recognition These findings are consistent with current views of immunoselection occurring in patients receiving tumor peptide-based immunotherapy, and impact on the design and implementation of p53-based vaccines, in particular, those aimed at treating individuals at high risk for developing cancer.