Volume 119, Issue 4, Pages 1104-1112 (October 2000) Mouse α-fetoprotein–specific DNA-based immunotherapy of hepatocellular carcinoma leads to tumor regression in mice  Christian F. Grimm, Dörte Ortmann, Leonhard Mohr, Sabine Michalak, Tim U. Krohne, Stephan Meckel, Silke Eisele, Jens Encke, Hubert E. Blum, Michael Geissler  Gastroenterology  Volume 119, Issue 4, Pages 1104-1112 (October 2000) DOI: 10.1053/gast.2000.18157 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 DNA expression constructs. G8 myoblast cells or CV-1 cells were transfected/infected with pcD/3-mAFP, pSec-mAFP, or rVV-mAFP, respectively. Subsequently, cell lysates/cell culture supernatant was analyzed for protein expression. (A) Western blot analysis shows a specific gene product of pcD/3-mAFP of ~66 kilodaltons (lane 1). Lane 2, secreted form of mAFP (pSec-mAFP); lane 3, rVV-pSC11; lane 4, rVV-mAFP; lane 5, vaccinia virus expressing a secreted form of mAFP (rVV-SecmAFP). (B) Immunoprecipitation of transfected LMH cells after metabolic labeling for detection of secreted mAFP. Lane 1, mock; lane 2, pSecTagB; lane 3, pSec-mAFP; lane 4, mock; lane 5, pSecTagB; lane 6, pSec-mAFP; lane 7, pSec-mAFP; lane 8, pSecTagB; lane 9, mock; lane 10, pSec-mAFP; lane 11, pSec-mAFP; lane 12, pSec-mAFP. A polyclonal anti-HBsAg serum was used as a negative control antibody (αNc). Gastroenterology 2000 119, 1104-1112DOI: (10.1053/gast.2000.18157) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 mAFP expression in mouse tissue and tumor cells. (A) mAFP expression in the liver of different mouse strains. Hepatic mAFP expression in mice was assessed by Western blotting as described in Materials and Methods in detail. Lane 1, positive control, Hepa1-6 murine HCC cell line; lane 2, BALB/C; lane 3, liver C57BL/6; lane 4, liver C57L/J; lane 5, liver C3H. Because of the strong Western blot signal of Hepa1-6 cells, lane 1 of the blot in A was exposed for just 10 seconds and lanes 2–6 were exposed 1:15 minutes. (B) RT-PCR from thymus and liver of both newborn and 3-week-old C57Bl/6-mice. Total RNA was extracted and RT-PCR was performed with mAFP-specific primer sets as described in Materials and Methods. Lane 1, thymus newborn; lane 2, liver newborn; lane 3, thymus 3 weeks; lane 4, liver 3 weeks; lane 5, kidney 3 weeks; lane 6, Lewis lung carcinoma cells; lane 7, Hepa1-6 cells; lane 8, LMH cells transfected with pcD/3-mAFP; lane 9, H2O. Gastroenterology 2000 119, 1104-1112DOI: (10.1053/gast.2000.18157) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 mAFP-specific antibodies and CTL activity. Anti-mAFP immune responses in sera of immunized animals were detected by metabolic labeling/immunoprecipitation and infection of CV-1 cells with rVV-mAFP. (A) BALB/c mice immunized 3 times (day 1, 14, and 42) intramuscularly with pcD/3-mAFP (lanes 1–3) and pSec-mAFP (lanes 4 and 5). Lane 6 represents serum derived from a mock-DNA immunized animal. (B) C57BL/6 mice immunized 3 times intramuscularly with pcD/3-mAFP (lanes 1–3) and pSec-mAFP (lanes 4–6). Lane 7 represents serum derived from a mock-DNA immunized animal. (C–E) C57BL/6 mice were immunized 3 times using a combined gene gun/intramuscular immunization regimen. In C, animals were immunized with pcD/3-mAFP (lanes 1–4), in D with pcD/3-mAFP + pRJB-GM + pApIL-12p70 (lanes 1–5), and in E with pcD/3-mAFP+ pRJB-GM + pCI-1sIL-18 (lanes 1–5). Lane 6 of each single blot represents serum derived from a mock-DNA immunized mouse, respectively. (F) mAFP-specific CTL responses. Mice were injected 3 times with a total of 125 μg pcD/3-mAFP and the different cytokine expression plasmids. Mock mice were immunized with pcDNA3 (mock) + pApIL-12p70 + pRJB-GM+ pCI-sIL-18 plasmids. Single-spleen cell suspensions were assayed after in vitro stimulation with rVV-mAFP–infected syngeneic spleen cells for 11 days. The effector cells were then tested against MC57G target cells infected with the indicated vaccinia viruses in a 51Cr-release assay at the E:T ratio indicated. Values represent means of triplicate determinations and are derived from responder and unresponder mice. Gastroenterology 2000 119, 1104-1112DOI: (10.1053/gast.2000.18157) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Levels of transaminases in immunized animals. Hepatocellular injury was monitored by measuring serum ALT and AST activities (U/L) in each mouse. Gastroenterology 2000 119, 1104-1112DOI: (10.1053/gast.2000.18157) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 MHC class I expression in tumor cells. 3LL, Hepa1-6, SP2/0 myeloma, and EL-4 lymphoma cells were examined for MHC I by FACS analysis using an anti-mouse H-2Kb/H-2Db specific antibody and a subsequent PE-labeled anti-mouse antibody. Gastroenterology 2000 119, 1104-1112DOI: (10.1053/gast.2000.18157) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Tumor growth in DNA-immunized mice. Hepa1-6 cells (5 × 106) were injected into the right flank of mice. All mice were immunized 14 days after tumor challenge using a combination of gene gun and intramuscular needle injection. At this point, tumors had reached an average volume of 115 mm3 and were comparable between animals in each group. The Lewis lung carcinoma cell line (3LL) was used as an mAFP-negative syngeneic cell line. Tumor incidence and volume were assessed 2 times weekly using calipers. Data are presented as mean volume ± SE. Gastroenterology 2000 119, 1104-1112DOI: (10.1053/gast.2000.18157) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Survival of animals. Tumor appearance and growth to 4000 mm3 were calculated by the Kaplan–Meier method. Gastroenterology 2000 119, 1104-1112DOI: (10.1053/gast.2000.18157) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 CTL activity and identification of antitumoral immune reactivities in vivo. (A) CTL activity against mAFP in tumor-bearing animals. After 5 days of in vitro stimulation, spleen cells were analyzed for cytotoxic activity against mAFP-expressing syngeneic Hepa1-6 cells and mAFP-negative syngeneic Lewis lung carcinoma cells. (B) Therapeutic antitumoral immunity required the participation of both CD4+ and CD8+ T cells. CD4 and CD8 T-cell subpopulations were depleted by intraperitoneal injection of purified hybridoma supernatant. Animals that had rejected the tumor (n = 6) were protected against rechallenge using 5 × 106 Hepa1-6 cells (n = 2). If CD8+ (n = 2) or CD4+ T cells (n = 2) had been depleted in vivo before and during rechallenge, tumor growth was comparable to that of nonimmunized control animals. Gastroenterology 2000 119, 1104-1112DOI: (10.1053/gast.2000.18157) Copyright © 2000 American Gastroenterological Association Terms and Conditions