Francisco Garcia-Pons

H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies.

H‐2 class I‐negative, HLA‐A2.1‐transgenic HHD mice were used for a comparative evaluation of the immunogenicity of HLA‐A2.1‐restricted human tumor‐associated cytotoxic T lymphocyte (CTL) epitopes. A hierarchy was established among these peptides injected into mice in incomplete Freunds adjuvant which correlates globally with their capacity to bind and stabilize HLA‐A2.1 molecules. Co‐injection of a helper peptide enhanced most CTL responses. In contrast, classical HLA class I‐transgenic mice which still express their own class I molecules did not, in most cases, develop HLA‐A2.1‐restricted CTL responses under the same experimental conditions. Different monoepitope immunization strategies of acceptable clinical usage were compared in HHD mice. Recombinant Ty‐virus‐like particles, or DNA encoding epitopes fused to the hepatitis B virus middle envelope protein gave the best results. Using this latter approach and a melanoma‐based polyepitope construct, CTL responses against five distinct epitopes could be elicited simultaneously in a single animal. Thus, HHD mice provide a versatile animal model for preclinical evaluation of peptide‐based cancer immunotherapy.

Identification of a human telomerase reverse transcriptase peptide of low affinity for HLA A2.1 that induces cytotoxic T lymphocytes and mediates lysis of tumor cells

Telomerase reverse transcriptase (TRT) is a tumor-associated antigen expressed in the vast majority of human tumors and is presently one of the most promising target candidates for a therapeutic cancer vaccine. TRT is also expressed at low level in selected tissues and should be considered a self antigen. In the present study we sought to develop cytotoxic T lymphocytes (CTL) responses directed against human (h)TRT peptides with low relative affinity for which the available repertoire is to be preferentially spared from tolerance. This was accomplished by using analogue peptides of hTRT whose relative affinity for the MHC was increased by a targeted (→Tyr) substitution in position one. By immunizing HLA A2.1 transgenic mice with these analogue peptides, we identified one such low relative affinity peptide (p572) that is endogenously processed and presented by HLA A2.1 in tumor cells, and is recognized by specific CTL. We used the highly immunogenic analogue peptide to successfully induce TRT-specific CTL in cancer patients and normal donors. CTL against p572-lysed human and mouse tumor cells but not activated autologous B cells. This peptide represents, therefore, an important candidate component of a cancer vaccine based on a TRT substrate and validates the strategy of targeting peptides with low affinity for the MHC for cancer immunotherapy.

Role of T cell help and endoplasmic reticulum targeting in protective CTL response against influenza virus.

We report on the induction of primary and long‐term memory cytotoxic T lymphocyte (CTL) responses against the nucleoprotein of the influenza virus A/PR8/34 in mice immunized with plasmid DNA targeted to B lymphocytes in the spleen. We found that the magnitude of the CTL response and the size of the pool of memory CTL was greater when the CTL response was induced in presence of T cell help. Interestingly, immunization with a signal sequence‐competent transgene was markedly superior to immunization with a transgene lacking the endoplasmic reticulum (ER) targeting sequence, in inducing CTL. We also found a correlation between in vivo protection from lethal virus challenge and (1) the availability of T cell help and (2) ER targeting. Immunization of dendritic cell‐deficient mice suggests that B lymphocytes function as antigen‐presenting cells in this model of immunization. Collectively, the results suggest that somatic transgene immunization is a conceptually new approach to induce effective anti‐viral CTL responses and to assess the parameters critical for long‐lasting and protective CTL responses in vivo.

Peptides derived from the onconeural HuD protein can elicit cytotoxic responses in HHD mouse and human

Anti-Hu syndrome is a paraneoplastic neurologic disease seemingly associated with an efficient antitumoral immune response against HuD protein expressed by both small cell lung cancer (SCLC) and neurons. Since anti-Hu antibodies are not pathogenic, and oligoclonal CD8(+) T cells infiltrate neoplastic and nervous tissues, we examined MHC class I-restricted immunogenicity of human HuD. Among 14 HuD-derived peptides potentially immunogenic in HLA-A*0201 restriction, 10 had actual in vitro binding capacity to the HLA molecule, 8 elicited specific cytotoxic T lymphocytes (CTLs) in a humanized murine model after peptidic vaccination, 2 also elicited specific CTLs in healthy humans, and 1 was naturally processed and presented to the immune system.