Treatment of squamous carcinoma in mice with a vaccine enriched for cells that induce immunity to squamous carcinoma—A new vaccination strategy

Abstract

We report a new vaccination strategy for squamous cell carcinoma (SCC). The vaccine was prepared by transfer of unfractionated DNA-fragments (25 kb) from squamous carcinoma cells (KLN205, DBA/2 origin (H-2 d )) into LM mouse fibroblasts (C3H/ He origin; H-2 k ), a highly immunogenic cell line. To enhance their nonspecific immunogenic properties, the fibroblasts were modified before DNA transfer to secrete IL-2 and to express additional allogeneic MHC class I determinants. As the transferred DNA integrates into the genome of the recipient cells, and is replicated as the cells divide, sufficient DNA to prepare the vaccine could be obtained from as few as 10 7 squamous carcinoma cells (4 mm tumor). Since only a small proportion of the transfected cell-population was expected to have incorporated genes specifying antigens associated with the squamous carcinoma cells (TAA), we devised a novel approach to enrich the vaccine for cells that induce immunity to the SCC.Aliquots of the transfected population were divided into 10 small pools (initial inoculums 5 1 3 10 3 ). We reasoned that if the starting inoculums were sufficiently small, then the distribution of highly immunogenic and weakly immunogenic cells in each pool would not be the same. Cells from individual pools were allowed to increase in number. A portion of the expanded cell populations were maintained frozen/viable for later recovery. The remaining portions were used to immunize na€ ıve DBA/2 mice. Pools containing greater numbers of immunogenic cells were identified by 2 independent assays. Frozen aliquots of cells from the pool that stimulated immunity to the squamous carcinoma to the greatest extent were recovered and subdivided for additional rounds of immune selection. Enhanced immunity to squamous carcinoma mediated by CD81 T cells was induced in tumor-bearing mice treated solely by immunization with the enriched cell-population. ' 2006 Wiley-Liss, Inc.

Development of a DNA-Based Vaccine for Treatment of an Intracerebral Tumor

“…Immunization of tumor-bearing mice with the DNAbased vaccine resulted in the induction of cell mediated immunity directed toward the type of tumor from which the DNA was obtained, and prolongation of survival, consistent with the expression of an array of TAA by the transfected cells. This was the case for mice with melanoma, squamous cell carcinoma and in mice with breast cancer [Chopra et al, 2006;deZoeten et al, 2002;Lichtor et al, 2008]. Multiple undefined genes specifying TAA that characterize the malignant cell population were expressed by cells that took up DNA from the tumor.…”

T-regulatory cells are relatively deficient in squamous carcinomas undergoing regression in mice immunized with a squamous carcinoma vaccine enriched for immunotherapeutic cells

“…18 The A 260 /A 280 ratio of the isolated DNA was 41.8 in each instance. The molecular size of the extracted DNA was approximately 25 kb, as determined by agarose gel electrophoresis.…”

“…18 An analysis by enzyme-linked immunosorbent assay of the culture supernatants of LM-IL-2 cells indicated that 10 6 transduced cells formed 196 pg IL-2/ml/10 6 cells/48 h.…”

“…In prior reports, [17][18][19][20] we described the results of studies in tumor-bearing mice including mice with SCC treated by immunization with a unique vaccine prepared by transfer of sheared genomic tumor-DNA-fragments into LM cells, a mouse fibroblast cell line. As the transferred DNA integrates spontaneously into the genome of the recipient cells, replicates as the cells divide and is expressed, the number of vaccine cells could be conveniently expanded as desired for multiple rounds of immunization.…”

“…DBA/2 mice with established tumors derived from KLN205 cells, a highly malignant squamous carcinoma cell line isolated originally from the Nettesheim lung carcinoma, treated by immunization with vaccines enriched for immunotherapeutic cells survived significantly longer than mice in various control groups, including mice immunized with transfected cells from non-enriched populations. 18 Here, we describe the characteristics of the cell populations infiltrating established squamous carcinomas undergoing regression in mice immunized with vaccines enriched for immunotherapeutic cells. The results in tumor-bearing mice treated successfully by immunization with the enriched vaccines revealed a predominance of CD8 þ T cells and a relative deficiency of T-reg cells in the regressing tumors.…”