Combined Treatment with Short Synthetic Anti-Lymphoma Peptide and CpG ODN; A Therapeutic Vaccine Which cures the A20 Lymphoma in Balb/C Mice.

Background. The short synthetic peptide 302 has been shown to induce rapid membrane disruption of lymphoma cells in vitro and necrosis of local tumors in the A20 lymphoma model in Balb/c mice. In order to stimulate the immune system to generate an anti-tumor response we designed a model where intra-tumor injections of peptide 302 was combined with sc injections of the Toll-like receptor 9 binding synthetic oligonucleotide CpG 1826. Methods. Balb/c mice were inoculated with A20 lymphoma cells sub-cutaneously (s.c.) on the abdomen. When the tumors reached a size of 5–7 mm, 302 peptide was administered directly into the tumors on days 1 and 6. CpG 1826 was injected s.c. on days 1–4 and 6–8. Tumor growth was measured repeatedly during follow-up. A20-specific T-cell responses were detected by culturing peripheral blood lymphocytes from treated animals for 24 hours with A20 lymphoma cells and analyzing for intracellular IFN-γ production by flow cytometry. To dissect the role of T-cell subtypes, the treatment was performed in animals depleted for CD4 or CD8 positive T-cells. In order to show A20 specific immunological memory, cured animals were re-challenged with the A20 lymphoma or the carcinoma cell line CT26. Results. Combined treatment with peptide 302 and CpG 1826 cured 8 out of 10 mice, compared to only 2 out of 10 mice who received peptide 302 alone or CpG 1826 alone. Cured mice were followed for 9 weeks without relapsing. Similar results with the combination of peptide 302 and CpG were observed in a separate experiment. The highest cure rate was achieved when injecting CpG 1826 s.c. in the tumors draining lymph node area as compared to administration of CPG 1826 in a non-draining lymph node region or intra-peritoneal. Animals treated with peptide 302 + CpG 1826 or CpG 1826 alone developed CD8-specific IFN-γ responses against A20 cells. In one separate experiment CD8 knock-out mice did not respond to the treatment, unlike animals depleted for CD4+ cells and normal mice. Only 1 out of 10 cured animals re-challenged with the A20 lymphoma developed a new tumor, a result that was reproduced in a second experiment. Conclusion: Treatment with intra-tumor injections of the anti-lymphoma peptide 302 in combination with CpG 1826 s.c. in the draining lymph node region cured established A20 tumors, induced tumor-specific CD8 positive tumor-reactive T-cells, and induced specific immunological memory. This principle represents a novel therapeutic vaccine approach.