No abstract

We have shown that the pulsing of dendritic cells ( DCs ) with human papillomavirus type 16 ( HPV -16 ) antigen proteins by lipofection stimulates class I -restricted cytotoxic T lymphocyte ( CTL ) response against primary cervical cancer cells. Also, we have shown that adeno -associated virus ( AAV ) was able to effectively deliver a cytokine gene into DCs. It has been our hypothesis that the delivery of antigen genes into DCs, resulting in endogenous and continuous antigen protein expression, may result in an improvement in T -cell priming by DCs. Here, DCs are pulsed ( infected ) with an AAV vector containing the HPV -16 E6 gene. After infection, transduced E6 gene mRNA expression and vector chromosomal integration could be identified in infected DCs. Furthermore, priming rosettes formed at early times when the AAV / E6 vector was used. Most importantly, AAV / E6 vector pulsing of DCs induced, after only 7 days of priming, a strong CTL response against primary cervical cancer cell lines, compared to bacterial E6 protein lipofection. Killing was significantly blocked by the addition of anti -MHC class I antibodies. Fluorescence -activated cell sorter ( FACS ) analysis of resulting primed cell populations revealed higher levels of CD8 + T cells by AAV -based pulsing, with little evidence of CD56 ( NK ). FACS analysis of the DC populations revealed that AAV / E6 vector -pulsed DCs had higher levels of CD80 and lower levels of CD86 than protein -pulsed DCs. These data suggest that rAAV may be appropriate for antigen pulsing of DCs for immunotherapy protocols. Finally, our protocol represents an advance in regards to the time needed for generating a CTL response compared to other techniques. Cancer Gene Therapy ( 2001 ) 8, 948 -957

Adeno‐associated virus (AAV) is able to efficiently deliver a cytokine gene into dendritic cells (DC). Improvements in T cell priming by DC might be effected by the delivery of antigen genes into DC, resulting in continuous protein expression, as most proteins have short half‐lives. In this study, a recombinant AAV vector containing the human papillomavirus (HPV)‐16 E7 gene was used to pulse/infect DC and compared to the pulsing of DC by the lipofection of bacterially produced E7 protein. Pulsing of DC with AAV/antigen (Ag) gene was found to be superior to pulsing with protein in six different assay systems: (1) the level of antigen transfer into DC as determined by intracellular staining; (2) the level of MHC class I‐restricted killing in cytotoxic T lymphocyte (CTL) assays;(3) the level of IFN‐γ expression; (4) the level of DC‐T cell priming clusters generated; (5) the level of CD80 and CD83 expression on DC; and (6) in the resulting CD8:CD4 ratio. Finally, AAV/Ag gene pulsing resulted in strong CTL activity after only 7 days of priming. These data suggest that AAV vectors may offer advantages over the commonly used protein‐pulsing technique and that AAV vectors may be useful for the stimulation of CTL activity and adoptive immunotherapy protocols.

No abstract

Recent studies in tumor immunology indicate that malignant cells frequently express normal testicular-specific proteins. Because these proteins show restricted normal tissue distribution, they are usually highly immunogenic and may be potential targets for immunotherapy. In the present study, we have used a pair of sequence-specific primers in reverse transcription-polymerase chain reaction (

BACKGROUNDThe authors previously identified sperm protein 17 (Sp17) as being expressed in patients with multiple myeloma. The restricted expression of Sp17 in normal tissue makes it an ideal target for tumor vaccine. In the current study, the authors extended their research to include ovarian carcinoma.METHODSA pair of sequence specific primers was used in reverse transcriptase‐polymerase chain reaction to determine the gene expression of Sp17. A recombinant Sp17 protein was used with monocyte‐derived dendritic cells and autologous peripheral blood mononuclear cells to generate Sp17 specific cytotoxic T‐lymphocytes (CTLs). The successful generation of Sp17 specific CTLs was confirmed using standard 51chromium‐release assays.RESULTSSp17 was found to be expressed in the primary tumor cells from 70% of the patients with ovarian carcinoma. Human leukocyte antigen (HLA) class I‐ restricted Sp17 specific CTLs were generated successfully from the peripheral blood of three patients with ovarian carcinoma at the time of disease presentation. These CTLs were able to lyse autologous Epstein‐Barr virus‐transformed lymphoblastoid cells in a Sp17‐dependent manner. Target lysis was HLA class I‐dependent and could be blocked by antibodies against monomorphic HLA class I but not HLA class II molecules. The CTLs also lysed Sp17‐positive autologous tumor cells, suggesting that Sp17 is processed and presented in association with the HLA class I molecules in Sp17‐ positive tumor cells in a concentration and configuration that could be recognized by recombinant protein‐primed CTLs. Tumor cell killing by the CTLs appeared to be mediated through the perforin pathway. Flow cytometric analysis of the CTLs indicated that they predominantly were CD8 in phenotype and produced interferon‐γ and scant amounts of interleukin‐4.CONCLUSIONSThe results of the current study suggest the potential of Sp17 as a target for immunotherapy in patients with ovarian carcinoma. Cancer 2002;94:2447–53. © 2002 American Cancer Society.DOI 10.1002/cncr.10506

BackgroundTo report about initial technical and clinical experience in preoperative radiation treatment of rectal cancer with volumetric modulated arcs with the RapidArc® (RA) technology.MethodsTwenty-five consecutive patients (pts) were treated with RA. All showed locally advanced rectal adenocarcinoma with stage T2-T4, N0-1. Dose prescription was 44 Gy in 22 fractions (or 45 Gy in 25 fractions). Delivery was performed with single arc with a 6 MV photon beam. Twenty patients were treated preoperatively, five did not receive surgery. Twenty-three patients received concomitant chemotherapy with oral capecitabine. A comparison with a cohort of twenty patients with similar characteristics treated with conformal therapy (3DC) is presented as well.ResultsFrom a dosimetric point of view, RA improved conformality of doses (CI95% = 1.1 vs. 1.4 for RA and 3DC), presented similar target coverage with lower maximum doses, significant sparing of femurs and significant reduction of integral and mean dose to healthy tissue. From the clinical point of view, surgical reports resulted in a down-staging in 41% of cases. Acute toxicity was limited to Grade 1-2 diarrhoea in 40% and Grade 3 in 8% of RA pts, 45% and 5% of 3DC pts, compatible with known effects of concomitant chemotherapy. RA treatments were performed with an average of 2.0 vs. 3.4 min of 3DC.ConclusionRA proved to be a safe, qualitatively advantageous treatment modality for rectal cancer, showing some improved results in dosimetric aspects.

Sperm protein 17 (Sp17) is a highly immunogenic cancer-testis antigen expressed by tumour cells from up to 30% of patients with multiple myeloma (MM). We recently successfully generated Sp17-specific human leucocyte antigen (HLA)-A1 and B27-restricted cytotoxic T lymphocytes (CTLs) from the peripheral blood of a healthy donor. Because CTLs were able to kill HLA-matched fresh myeloma cells, it may be possible to generate and administer myeloma-specific donor T cells to MM patients following allogeneic stem cell transplantation to enhance graft-versus-myeloma (GVM) without inducing graft-versus-host disease (GVHD). To determine how widely applicable this approach is, we have determined the ability to generate Sp17-specific CTLs from four consecutive healthy donors with other HLA class I phenotypes. We found that Sp17-specific HLA class I-restricted CTLs could be easily generated from all four donors. Sp17-specific CTLs were primarily CD8 in phenotype and produced interferon-g and very little interleukin-4. These T cells killed target cells primarily via the perforin-mediated route. These results therefore suggest that myeloma-specific donor T-cell infusion that targets Sp17 to selectively enhance GVM could be applicable to patients with Sp17MM.