Also, in the latter group, both the CD4+ and the CD8+ PD-1+ T cells remained at similar levels by day 45, but were reduced between 30% and 50% by day 72, respectively ( Figure 5C ). since 70% of the mice controlled the tumor growth and survived, whereas the remaining 30% developed tumors and died by day 72. In contrast, 100% of the mice in the control groups died by day 30. The anti-DEC-205:16E5 conjugate was found to induce 16E5-specific memory T cells, with a Th1/Th17 profile. Both CD4+ and CD8+ T cells contributed to the observed protection. Finally, treating mice that had developed tumors with an anti-PD-1 mAb, delayed the tumor growth for more than 20 days. These results show that targeting 16E5 to DEC-205, alone or combined with an immune checkpoint blockade, could be a promising protocol for the treatment of the early stages of HPV-associated cancer. to the DEC-205 receptor by conjugation with a specific anti-DEC-205 mAb to stimulate antigen presentation by DCs. Moreover, potent protective responses against different infectious brokers and cancer have been achieved when used together with a maturation stimulus (33C43). Thus, targeting tumor antigens to DCs through DEC-205 is usually a promising alternative for the treatment of malignant tumors. The aim of this work was to evaluate whether targeting the 16E5 oncoprotein to DEC-205, present in DCs, could induce an effective protective immune response against a 16E5-expressing tumor cell line in a therapeutic model. We found that small amounts of 16E5, chemically conjugated to a rat anti-DEC-205 mAb and inoculated s.c. in mice with Poly I:C as adjuvant, induced a powerful specific protective response against the 16E5-expressing BMK-16/myc tumor cells. The procedure cured 70% of the experimental mice. This protection was found to be dependent on memory CD4+ and CD8+ T cells with a Th1/Th17 type phenotype. In addition, the administration of an anti-PD-1 mAb in mice with a retarded tumor growth (30%) caused an even greater delay of the process. Material and Methods Mice Specific-pathogen-free, 6- to 8-week-old female BABL/c mice were provided by the animal house at the National Institute of Public Health (Cuernavaca, Morelos, Mexico). For experimental procedures, mice were housed in the same facility following the guidelines of the institutional Ethics Committee and the Mexican National Regulation on animal care and experimentation, under a standard light/dark cycle (12 h/12?h) and provided with food and water gene (BMK-16/c-myc). MA-104 cells from Rhesus monkey kidney were purchased from ATCC (CRL-2378.1). Under conditions, the cells were grown in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 g/mL streptomycin and 2 mM L-glutamine, and they were incubated at 37C in humidified air containing 5% CO2. All cell culture reagents were from Invitrogen. Monoclonal Antibodies Production The rat hybridomas producing the IgG2a mAb against mouse DEC-205 (NLDC-145) and the rat isotype control (IgG2a) (III-10) were donated by Dr. Ralph Steinman (Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York). The mouse hybridoma against Histidine tag (6His) (clone 2R-2A6) was generated at Dr. Gutierrez-Xicotencatls laboratory and characterized as IgG1 isotype (unpublished results). For the production of the mAbs, the hybridomas were expanded in CD Hybridom serum-free medium supplemented with 0.2% FBS and 2 mM L-glutamine and purified as previously described (41). Briefly, the mAbs rich supernatants were precipitated with ammonium sulfate (50% w/v) for 1?h at room temperature, followed by centrifugation at 11,000 g for 15?min. The pellets containing the mAbs were re-suspended in one-tenth of the original volume with PBS/0.01% Tween-20 and dialyzed against PBS at 4C for 16?h. Finally, the mAbs were purified by affinity chromatography with a sepharose-protein Lodenafil G column (Hiptrap, General Electric), according to the suppliers protocol. Production and WDFY2 Purification of the HPV16 E5 Protein The HPV16 gene was cloned and the protein was produced under conditions using the Rapid Translation System (RTS proteo Master, Roche) equipment, as previously described (45). Briefly, the 6His-tagged 16E5 recombinant protein Lodenafil (His-16E5) was produced in the RTS at 22C for 16?h with continuous stirring, and it was purified by affinity chromatography on a Ni-NTA column (Qiagen), following the providers protocol. Fractions from Lodenafil the chromatography were analyzed by immune Western blot to verify the presence and identity.
