Human Dendritic Cells Transfected with RNA Encoding Prostate-Specific Antigen Stimulate Prostate-Specific CTL Responses In Vitro

Abstract

Although immunological tolerance to self Ags represents an important mechanism to prevent normal tissue injury, there is growing evidence that tolerance to tumor Ags, which often represent normal peripherally expressed proteins, is not absolute and can be effectively reverted. Prostate-specific Ag (PSA) is a self Ag expressed by both normal and malignant prostatic epithelium, and therefore offers a unique opportunity to examine the ability of self Ags to serve as specific CTL targets. In this study, we investigated the efficacy of autologous dendritic cells (DC) transfected with mRNA encoding PSA to stimulate CTL against PSA Ags in vitro. Ag in form of RNA carries the advantage to encode multiple epitopes for many HLA alleles, thus permitting induction of CTL responses among many cancer patients independent of their HLA repertoire. In this study, we show that PSA mRNA-transfected DC were capable of stimulating primary CTL responses against PSA Ags in vitro. The PSA-specific CTL did not cross-react with kallikrein Ags, a protein, which shares significant homology with PSA, suggesting that harmful autoimmune toxicity may not represent a significant problem with this approach. PSA RNA-transfected DC generated from male or female healthy volunteers or from cancer patients were equally effective in stimulating PSA-specific CTL in vitro, implying that neither natural tolerance to PSA Ags nor tumor-mediated T cell anergy may represent major barriers for CTL generation against the self Ag PSA. This study provides a preclinical rationale for using PSA RNA-transfected DC in active or adoptive immunization protocols.

Epstein‐Barr virus (EBV) latent membrane protein‐1‐specific cytotoxic T lymphocytes targeting EBV‐carrying natural killer cell malignancies

“…We here applied an efficient approach to load APC with N-terminally truncated LMP1 using in vitro transcribed mRNA introduced via electroporation. Such mRNA-loaded DC have been proven to be able to stimulate the immune system in vitro and in vivo [21,22,[38][39][40][41][42]. In addition, this provides a very safe tool for human clinical studies, as mRNA is not immunogenic, has a relatively short half-life, and lacks the potential for integration into the host genome.…”

“…There is accumulating evidence that APC transduced with in vitro transcribed mRNA encoding certain Ag are potent inducers of CTL specific to tumor-associated Ag [21][22][23][24], even overcoming immunological tolerance to self Ag [25]. The advantages seem to derive from (1) complete deletion of antigenicity of vector backbone sequences; (2) highly reproducible yields with in vitro transcription; (3) high efficiency of transduction using electroporation.…”

Immunotherapy with allotumour mRNA-transfected dendritic cells in androgen-resistant prostate cancer patients

“…Cancer vaccines may represent an alternative form of therapy for such patients. It has been shown that autologous dendritic cells (DCs) pulsed with peptides specific for PSA (Heiser et al, 2000), prostate-specific membrane antigen (PSMA) (Tjoa et al, 1996) or telomerase reverse transcriptase (TERT) (Vonderheide et al, 1999) are capable of stimulating potent CTL responses in vitro and data from clinical trials are now available (Heiser et al, 2002;Kaufman et al, 2004). Since prostate cancer cells are genetically unstable and as such represent 'shifting targets' (Shaffer and Scher, 2003), single-agent vaccines may result in selection of genetic variants that escape the immune attack.…”

Efficient antitumor immunity in a murine colorectal cancer model induced by CEA RNA‐electroporated B cells

“…For in vitro transcription, pcDNA3-EGFP and pcDNA3-CEA plasmids were linearized with Sma I, purified by phenol/chloroform extraction and ethanol precipitation and used as DNA templates [39]. In vitro transcription was performed using T7 RNA polymerase (mMESSAGE mMACHINE kit; Ambion, Austin, TX) according to the manufacturer's instructions.…”