By virtue of its tissue-specific expression, carcinoembryonic antigen (CEA) is an important self, tumor-associated antigen, which is expressed by different human adenocarcinomas and also serves as a target for active-specific immunotherapy. Similar to humans, CEA expression in mice transgenic for the human CEA gene (CEA.Tg) occurs predominantly along the gastrointestinal tract. CEA.Tg mice were crossed with mice bearing a mutation in the Apc gene (MIN mice), and the CEA.Tg/MIN progeny developed multiple intestinal neoplasms, which overexpress CEA to levels that are reminiscent of those reported for tubulovillous intestinal adenomas from patients. CEA.Tg/MIN mice were vaccinated with an aggressive diversified prime/boost vaccine regimen: (a) a primary vaccine consisting of recombinant vaccinia virus-expressing CEA and a triad of costimulatory molecules (TRICOM): B7.1, ICAM-1, and LFA-3 (rV-CEA-TRICOM); and (b) a booster vaccine using CEA-TRICOM in a recombinant avipox (fowlpox) virus (rF-CEA-TRICOM). Granulocyte/macrophage colony-stimulating factor was administered as a biological adjuvant with all vaccinations, either as a recombinant protein (with rV-CEA-TRICOM) or as a recombinant avipox virus (with rF-CEA-TRICOM). That vaccine regimen generated strong CEA-specific host immune responses in CEA.Tg/MIN mice, which resulted in (a) a delayed onset of adult anemia and weight loss, (b) a significant reduction in the number of intestinal tumors, and (c) improved overall survival. No evidence of autoimmunity directed against normal tissues expressing CEA was observed in mice in which the CEA-based vaccine significantly reduced intestinal tumor load. The CEA.Tg/MIN mice present a clinically relevant model in which different CEA-based vaccine strategies can be tested on the spontaneous onset of intestinal tumorigenesis.

CEA3 , a Mr180,000–200,000 oncofetal antigen, is a member of the Ig superfamily and expressed on a high percentage of adenocarcinomas, particularly those of the colon, pancreas, breast, lung, rectum, and stomach (1, 2). Because of its limited normal tissue expression and its overexpression on carcinomas, CEA is also considered a self, tumor-associated antigen and a target for passive (3) and active immunotherapy (4, 5, 6, 7, 8, 9, 10). Recent clinical data have established that different vaccine strategies can generate human B and T cells that recognize CEA, providing additional evidence that CEA is a target for eliciting immune responses against a variety of cancer types (4, 5, 6, 7, 8, 9, 10).

Preclinical murine models expressing the complete human CEA gene as a transgene (11, 12) have been generated and CEA is expressed predominately along the GI tract, as in humans (13). In addition to CEA expression in normal tissues, CEA.Tg mice used in this study have high serum levels of CEA that presents additional peripheral tolerance to the host immune system (14). Yet, tolerance to CEA has been overcome by vaccinating CEA.Tg mice with recombinant orthopox or avipox-CEA viruses, murine fibroblasts expressing CEA, and an oral CEA-based DNA vaccine, as shown by the generation of anti-CEA Ig antibodies, Ig class switching, TH1 type CEA-specific CD4+ responses, and CD8-dependent cytotoxicity (14, 15, 16, 17, 18). Besides generating CEA-specific host immunity, CEA-based vaccines have elicited antitumor immunity against CEA-expressing tumors (14, 15, 16, 17, 18). The use of transplantable CEA-expressing tumor cells in those studies presents some important limitations, including the retroviral insertion of CEA without associated regulatory elements into cells that normally do not express the gene and the growth of those tumors cells at ectopic sites (s.c., lung, liver) not authentic to colorectal cancer. From an immunological perspective, the rapid growth rate of the transplanted tumors not only fails to mimic the growth characteristics of carcinomas but also requires a shortened interval between cancer vaccine administrations, which probably does not permit optimal host immunity.

In this study, CEA.Tg mice were bred with ApcMIN (MIN) mice that are heterozygous for a mutant allele of the mouse homologue of the human APC gene (19). MIN is a fully penetrant, autosomal dominant, nonsense mutation (codon 850) believed to initiate tumorigenesis in utero and results in multiple spontaneous intestinal neoplasms in adult mice (19, 20). Mice (CEA.Tg/MIN) carrying both the MIN and human CEA genes develop numerous intestinal neoplasms with strong CEA expression in all tumor cells, as well as CEA expression in normal GI tissues (21, 22). An immunotherapeutic protocol, consisting of recombinant poxviruses expressing CEA and TRICOM (B7.1, ICAM-1, and LFA-3) (23), combined with GM-CSF, induced strong anti-CEA host immune responses that significantly suppressed intestinal tumor load and improved long-term survival of CEA.Tg/MIN mice. CEA.Tg/MIN mice can serve as an important preclinical model for the evaluation of cancer vaccines alone or in combination with other anticancer therapeutic modalities.

Mice.

Mice expressing the gene for human CEA [CEA.Tg, Line 2682, C57BL/6 (H-2b), heterozygous] were obtained from John Thompson (University of Freiburg, Freiburg, Germany). MIN (C57BL/6J-ApcMIN/+) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Both colonies were maintained with continuous backcrossing with C57BL/6 mice. Mice with spontaneously arising CEA-expressing intestinal tumors were derived by breeding female CEA.Tg with male MIN mice. All mice entered onto this study were the offspring of the CEA.Tg x MIN cross and maintained in microisolator cages and fed Purina Rodent Certified Chow 5002 ad libitum. Animal care was in compliance with recommendations of the Guide for Care and use of Laboratory Animals, National Research Council.

Genotyping.

Fecal and blood samples were taken from the CEA.Tg x MIN F1 offspring at weaning. Mice carrying the CEA transgene were identified by the presence of fecal CEA protein detected using a solid-phase, double-determinant, anti-CEA ELISA kit (AMDL, Inc., Tustin, CA; Ref. 15), and allele-specific PCR analysis of DNA isolated from the blood (24) identified the Apc mutation. Approximately 25% of the offspring were positive for CEA expression as well as for the presence of the MIN mutation, and those mice were designated CEA.Tg/MIN. Mice expressing MIN, but not CEA, were designated MIN and those expressing CEA, but not MIN mutation, were designated CEA.Tg. Mice that were negative for both CEA and MIN were designated C57BL/6. Genotypes of all mice were rechecked at the completion of the study.

Vaccines, Adjuvants, and Injection Schema.

Details for the construction and production of the recombinant vaccinia (23) and avipox (fowlpox) viruses (23, 25) have been published. Vaccines are referred to as either CEA-based or non-CEA-based vaccines. The CEA-based vaccines were comprised of (a) a recombinant vaccinia virus or a (b) recombinant avipox (fowlpox) virus containing genes encoding human CEA and three murine costimulatory molecules B7.1, ICAM-1 and LFA-3 and designated either rV-CEA-TRICOM or rF-CEA-TRICOM. The non-CEA-based vaccines are the same recombinant vaccinia and avipox viruses expressing the TRICOM but not the human CEA gene (designated either rV-TRICOM or rF-TRICOM). A description of the construction of the recombinant avipox (fowlpox) virus expressing murine GM-CSF (rF-GM-CSF) has been reported (15). The parental virus for the generation of recombinant fowlpox-GM-CSF was plaque purified from a tissue culture-adapted vaccine strain of fowlpox virus. rF-GM-CSF was constructed via homologous recombination in vivo between the parental fowlpox DNA and a plasmid vector that contains the murine GM-CSF gene.

All vaccines were given s.c. in 100 μl of HBSS at the base of the tail. Recombinant vaccinia-based vaccines, rV-CEA-TRICOM or rV-TRICOM, were the primary vaccines and administered at a dose of 108 pfu in combination with 20 μg of rGM-CSF (PeproTech, Inc., Rock Hill, NJ). Recombinant GM-CSF protein was then injected for an additional 3 consecutive days at the vaccination site. Booster vaccinations were also administered s.c. in 100 μl containing 108 pfu of either rF-CEA-TRICOM or rF-TRICOM combined with 107 pfu of rF-GM-CSF. A group of mice received injections of the vehicle (HBSS) alone and were designated vehicle control.

Intestinal Extract Preparation and Solid-Phase RIAs.

Three to five mice from each group listed in Table 1 were sacrificed at 150–160 days of age. The entire intestine was removed and its contents flushed with cold DPBS. For the CEA.Tg and C57BL/6 mice, 3–5-cm sections were isolated from the proximal/distal jejunum and ileum. For the CEA.Tg/MIN and MIN mice, neoplastic lesions (n = 20–30) were microisolated using a dissecting microscope from those same intestinal areas. Extracts were prepared and the protein concentrations determined as described previously (26). Forty μg of extract protein were dried to each well of a 96-well plate, and the binding of an anti-CEA monoclonal antibody 125I-COL-1 (27) and an irrelevant antibody, BL3, was performed as a solid-phase RIA (28). Both antibodies were labeled with Na125I using the Iodogen method (Pierce Chemical, Rockford, IL), which yielded specific activities of 15–30 μCi/μg and 65–75% of the input 125I bound to the IgG. Immunoreactivity of 125I-COL-1 was assessed using wells of 96-well plates sensitized with 50 ng of CEA. The percentage of input radiolabeled antibody bound was >90%. Each antibody was added to triplicate wells of 96-well plates and allowed to incubate overnight at 4°C. The wells were cut and counted using a LKB Riagamma (model 1274) gamma counter. In each assay, cpm from wells to which no antibody or 125I-BL3 (400–600 cpm) was subtracted from the cpm from the wells that received 125I-COL-1. CEA levels were also measured in the same protein extracts using the anti-CEA ELISA kit (AMDL, Inc.) according to the manufacturer’s instructions. Internal low and high standards for CEA were included in all assays.

Serum CEA and Anti-CEA Antibody Responses.

CEA antibodies were measured by ELISA as described previously (14). Microtiter plates were sensitized overnight at 4°C with 100 ng/well CEA (International Enzymes, Fallbrook, CA) or ovalbumin (Sigma Chemicals, St. Louis, MO). Wells were blocked with DPBS containing 5% BSA, followed by a 1-h incubation of diluted mouse serum (1:10–1:31,250). Antibodies bound to the wells were detected with horseradish peroxidase-conjugated goat antimouse IgG (Kirkegaard & Perry Labs., Inc., Gaithersburg, MD) using an ELISA microplate autoreader at A490 nm. Triplicates of positive and negative controls and serum samples were run for all assays.

Proliferation Assay.

Complete details of the T-cell proliferation assay have been described previously (15). Splenocytes were enriched for T cells that were incubated in flat-bottomed, 96-well plates at a cell density of 1.0–1.5 × 105 cells/well with 5 × 105 irradiated (2000 rad) syngeneic CEA.Tg mouse splenocytes containing 50–3.125 μg/ml CEA or ovalbumin. Proliferation was measured after 5 days of incubation at 37°C by adding [3H]thymidine (1 μCi/well; Amersham Corp., Chicago, IL) to the wells 18 h before harvesting. Cells were harvested and counted by liquid scintillation spectroscopy as described previously (15).

Cytokine Production Assays.

Splenic T cells from CEA.Tg/MIN mice were isolated and grown in flat-bottomed, 96-well plates at a cell density of 2 × 104FS cells/well, 5 × 105 irradiated (2000 rad) syngeneic CEA.Tg mouse splenocytes/well and 50 μg CEA/ml. Supernatant from designated wells for each treatment group was harvested after 48 h, and IFN-γ levels were measured using the appropriate ELISA assay (Endogen, Inc., Cambridge, MA).

Cytotoxicity Assay.

Splenocytes from 2–3 mice/group were pooled and single cell suspensions generated that were added to complete medium containing RPMI 1640 supplemented with 15 mm HEPES (pH 7.4), 2 mml-glutamine, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, 10 mg/ml gentamicin, 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT), and 50 μm 2-mercaptoethanol. Twenty to 25 million splenocytes were added in 10 ml to T-25 flasks along with 10 μg/ml of a CEA 8-mer peptide, CEA526–533 (EAQNTTYL; Ref. 29). T-cell cultures were restimulated 6 days later in 24-well plates. T cells (200,000) were added along with 5 × 106 irradiated syngeneic splenocytes, 1 μg of CEA peptide/ml and 10 units/ml recombinant human interleukin 2 (Proleukin; Chiron Corp., Emeryville, CA). Cytolytic activity was assessed 6 days later using EL-4, a murine lymphoma cell line to which exogenous CEA peptide or a control peptide, vesicular stomatitis virus N52–59, RGYVYQGL, were added (1 μg/ml). Peptides were purchased (>95% pure) from Multiple Peptide Systems, Inc. (San Diego, CA), diluted in DMSO to a stock concentration of 10 mg/ml and stored at −80°C; subsequent dilutions were done in HBSS.

Tumor Scoring, Histopathology, and IHC Staining.

Mice were killed by CO2 inhalation and the entire GI tract removed. The small and large intestines were isolated by cutting at the point just distal to the gastric/duodenal border (pyloric sphincter) and the rectum at the anus. The small intestine was cut into four 40-mm long sections, which corresponded to the duodenum, proximal and distal jejunum, and the ileum and placed on dampened filter paper with DPBS. The entire large intestine (cecum and colon; ∼60 mm) was also excised and similarly prepared. Using microdissecting scissors, each segment was opened longitudinally, and the mucosal surface was rinsed free of content with DPBS. Using a dissecting microscope (×10 magnification), each segment was scored (blinded to the scorer) for the presence of tumors. The smallest gross tumor scored was ∼1 mm, and tumors were divided according to size <2 mm, 2–5 mm, and >5 mm. The sum of the number of intestinal and colonic tumors was the measure of total GI tumor burden.

For histopathological evaluation, intestine and colon tissues were fixed in 10% neutral buffered formalin, embedded in paraffin blocks, and processed by routine histological methods for H&E staining. The largest and smallest tumors from each gut segment were examined microscopically. Proliferative epithelial lesions had microscopic morphology typical of that described for MIN mice (20, 30). Lesions were classified as intestinal intraepithelial neoplasms (dysplasia, carcinoma in situ) if they did not involve the full thickness of the mucosa and did not compress adjacent tissue. Adenomas involved the full thickness of the mucosa and compressed adjacent tissue and adenocarcinomas invaded the muscularis mucosae. The majority of gross lesions were either intraepithelial neoplasms or adenomas. For IHC staining, sections were dried overnight in a 45°C oven before staining. Staining for CEA was performed on 4-μm tissue sections and air-dried on Superfrost Plus slides. The Vector M.O.M. Kit (Vector Laboratories, Burlingame, CA), a modification of the avidin-biotin complex method for localizing mouse monoclonal antibodies on mouse tissue was used in conjugation with a murine anti-CEA monoclonal antibody, COL-1 (dilution 1:1000; Ref. 27). COL-1 was incubated on the slides for 30 min at room temperature. An isotype-matched monoclonal antibody with irrelevant antigen specificity was used as a negative control. Positive and negative controls for CEA expression were CEA-expressing MC-38 cells, designated MC-38-CEA-2, and the CEA-negative parental MC-38 tumor cell line (29).

Determination of Anti-DNA and ANA Serum Levels.

Both anti-DNA and ANA serum titers were measured by Anilytics, Inc. (Gaithersburg, MD). Anti-DNA titers were measured using sodium sulfate precipitates of undiluted serum samples in a RIA. Titers > 2.5 IU/ml were considered positive. An ELISA test was used to measure ANA using two pools of nuclear antigens: pool A consisted of single-stranded DNA, SSA, SSB, and Jo-1; pool B consisted of double-stranded DNA, ribonucleoprotein, histones, samarium, and scl-70. Results were scored as to the overall reactivity: 0, negative; 1–4, weakly, moderately, strongly, very strongly reactive.

Long-Term Survival Studies.

CEA.Tg/MIN mice were divided into three treatment groups: CEA-based vaccine (n = 14); non-CEA-based vaccine (n = 10); and vehicle control (n = 12). Also included were MIN mice (n = 5) that were given the CEA-based vaccine. Mice were genotyped immediately after weaning and given the appropriate vaccine or vehicle control by 30 days of age, and booster vaccines were given monthly for the duration of the study. Any mouse whose weight fell for 4 consecutive weeks and whose hematocrit level was ≤25 was sacrificed, age recorded, and the GI tract examined for tumor burden.

Statistical Analysis.

Statistical significance for differences in body weights, hematocrit, and number of intestinal tumors was based on Student’s two-tailed t test. Significance differences in overall survival were evaluated using the Kaplan-Meier test. All Ps reported are two-sided and have not been adjusted for the multiplicity of evaluation performed on the data. P < 0.05 was considered significant.

CEA.Tg/MIN Experimental Model.

Initial studies were designed to determine the approximate levels of CEA expression in normal versus neoplastic intestinal tissue. Normal intestinal tissue was isolated from CEA.Tg and C57BL/6 mice, whereas neoplastic intestinal tissue (predominately adenomas) was taken from CEA.Tg/MIN and MIN mice as outlined in the “Materials and Methods.” CEA levels in protein extracts, as measured using an anti-CEA monoclonal antibody (COL-1) in a direct-binding, solid-phase RIA and CEA EIA, were ∼3-fold higher in the neoplastic tissue when compared with levels in extracts from normal intestines (Table 1). Higher CEA expression levels in the intestinal neoplastic tissues were also observed with IHC staining. Both CEA.Tg/MIN and CEA.Tg mice had measurable serum CEA levels, and the presence of intestinal tumors in the CEA.Tg/MIN mice was not associated with increased serum CEA levels. Comparison of both the number and location of intestinal and colonic tumors found in MIN mice were approximately the same whether those mice were either CEA.Tg or CEA negative (Fig. 1).

Vaccine Effects on Body Weight and Hematocrit Levels of CEA.Tg/MIN and MIN Mice.

CEA.Tg/MIN or MIN (CEA-negative) mice received the CEA-based, the non-CEA-based vaccine, or the vehicle control. As explained in the “Materials and Methods,” the CEA-based vaccine was comprised of rV-CEA-TRICOM combined with rGM-CSF followed by monthly boosts with rF-CEA-TRICOM combined with rF-GM-CSF, whereas the non-CEA-based vaccine was rV-TRICOM combined with rGM-CSF followed by monthly boosts with rF-TRICOM combined with rF-GM-CSF. Vehicle control mice received injections of HBSS. CEA.Tg/MIN mice, like MIN mice (19, 20), developed adult-onset anemia accompanied by severe, progressive weight loss, overt changes that are linked with the formation of intestinal tumors. Individual mouse body weights from five groups of mice were measured weekly, and hematocrit levels were determined every 4–6 weeks. As shown in Table 2, the average total weight gain for CEA.Tg/MIN mice that received either the non-CEA-based vaccine or the vehicle control was similar to that of MIN mice given the CEA-based vaccine or vehicle control. Mice in all four control groups achieved their maximum weight before 120 days of age, usually gaining 5–6 g. In addition, by 120 days of age, the mice in those four groups were either anemic or borderline anemic as indicated by their hematocrit values (≤36; Table 2). In contrast, CEA.Tg/MIN mice that received the CEA-based vaccine gained weight and by 150 days of age, their overall average weight gain was 10.1 ± 1.6 g; hematocrit levels remained in the normal range, suggesting that vaccine administration helped maintain normal health status of those mice (Table 2).

Comparative Anti-CEA Immunity Generated in CEA.Tg/MIN and Other Mice.

It was of interest to examine whether the weight gain and normal hematocrits of the CEA.Tg/MIN mice might be associated with the presence of CEA-specific host immunity. After receiving the primary vaccination followed by 3 monthly booster vaccinations of either the CEA-based or non-CEA-based vaccine, groups of CEA.Tg/MIN mice were evaluated for the presence of anti-CEA humoral and cellular immune responses. CEA.Tg/MIN mice vaccinated with the CEA-based vaccine had developed strong anti-CEA serum Ig levels (Fig. 2,A) and CEA-specific CD4 T-cell proliferative responses (Fig. 2,B). Furthermore, CD8 T-cell responses after vaccination using the CEA-based vaccines were also present as indicated by CEA-peptide-specific IFN-γ production (Fig. 2,C) and lysis of peptide-pulsed targets (Fig. 2 D). No measurable CEA-specific immune responses were detected in CEA.Tg/MIN that were vaccinated with the non-CEA-based vaccine or those that received vehicle control. CEA-specific humoral, CD4 and CD8 cellular responses were also found in MIN that were vaccinated with the CEA-based vaccine (data not shown).

Vaccine Effects on Tumor Formation and Overall Survival.

Total number of GI tumors in CEA.Tg/MIN mice that were vaccinated with the CEA-based vaccine, non-CEA-based vaccine, or the vehicle control were determined at ∼150 days of age (Fig. 3). The average number of tumors found in either CEA.Tg/MIN mice treated with the vehicle control or the non-CEA-based vaccine was 36.7 ± 6.2 and 46.8 ± 6.2, respectively. There was a significant reduction in the average number of tumors (22.4 ± 6.8) found in CEA.Tg/MIN mice that received the CEA-based vaccine when compared with either control group (Fig. 3).

In a separate long-term survival study (Fig. 4), CEA.Tg/MIN mice were vaccinated with the CEA-based vaccine, non-CEA-based vaccine, or the vehicle control, whereas a group of MIN mice (CEA-negative littermates) received the CEA-based vaccine. All mice in the three control groups, CEA.Tg/MIN mice given either the non-CEA-based vaccine or vehicle control, and the MIN mice vaccinated with the CEA-based vaccine, were sacrificed by 25–27 weeks because of progressive weight loss and anemia. For comparison, at 27 weeks of age, 80% of the CEA.Tg/MIN mice that received the CEA-based vaccine were alive with stable body weights and normal hematocrits. At 40 weeks, 50% of those mice remained alive; however, by week 49, all mice were anemic with progressive weight loss that required sacrifice. Macroscopic examination revealed numerous neoplastic lesions.

Histopathology, Hematology, and IHC.

Spleen, pancreas, lung, liver, and kidneys from individual CEA.Tg/MIN mice that received either the CEA-based vaccine, non-CEA-based vaccine, or the vehicle control were examined for gross and microscopic lesions (Table 3). In all three groups, spleens were enlarged and most other organs were pale when taken from mice that had multiple intestinal proliferative lesions. Histopathological analyses revealed that the enlarged spleens were attributable to increased extramedullary hematopoiesis, and the paleness of other organs seemed associated with severe anemia because no other pathology was found (Table 3). Sporadic hydronephrosis was found in all three groups of mice. Serum samples from individual mice were analyzed for the presence of anti-DNA and ANA titers, and no changes were observed in any of the three groups of CEA.Tg/MIN mice (Table 3).

Intestinal tissues from CEA.Tg/MIN mice treated with the CEA-based vaccine were analyzed histopathologically (H&E staining) and by IHC for CEA expression by using anti-CEA monoclonal antibody COL-1 (Fig. 5). H&E staining revealed typical intestinal adenomas (Fig. 5, A and C) from CEA.Tg/MIN mice in which the CEA-based vaccines did not reduce tumor load. Fig. 5,E illustrates basophilic cells with hyperchromatic, piled-up nuclei and the presence of mitotic figures within an adenoma. Overexpression of CEA as evidenced by relative COL-1 staining intensity was found in adenomas (Figs. 5, B and D) when compared with adjacent normal epithelial. Fig. 5,F is a higher magnification of CEA expression in an intestinal adenoma from a CEA.Tg/MIN mouse in which the CEA-based vaccine did not suppress adenoma formation. Histopathological analyses of the intestines from CEA.Tg/MIN mice in which the CEA-based vaccine suppressed adenoma formation revealed normal intestinal architecture (Fig. 5,G) and CEA expression in intestinal crypts (Fig. 5 H).

MIN mice carry a germ-line mutation of the murine Apc gene, which results in the formation of multiple intestinal adenomas (19, 20). In humans, a homologous germ-line mutation in the tumor suppressor gene, adenomatous polyposis coli (APC), predisposes individuals to an inherited form of colon cancer, familial adenomatous polyposis, characterized by the early development of multiple colorectal adenomas, some of which can subsequently form carcinomas (31). Somatic mutations of the APC gene are found in the early stages of ∼85–90% of sporadic colorectal cancers (31, 32). In this study, MIN mice bred with mice carrying the human CEA gene result in offspring that spontaneously develop multiple intestinal tumors that overexpress CEA. Previous reports from this laboratory have shown the ability to generate (a) host immune responses to CEA, a self, tumor antigen, and (b) antitumor immunity against transplantable CEA-expressing tumors in the CEA.Tg mice vaccinated with different vaccine regimens (14, 15). Introduction of the MIN genotype presents an experimental murine model that (a) develops spontaneous intestinal neoplasms expressing CEA in an authentic tissue site and (b) provides a 4–5 month life span during which cancer vaccines can be administered at intervals that better mimic those being used in clinical studies.

Besides its restricted expression in normal tissues, overexpression of CEA in neoplastic tissues is regarded as an important property that allows CEA to be a target for immunotherapy (1, 2). CEA expression levels have been reported to be ∼2–6-fold higher in tubulovillous adenomas of patients when compared in protein extracts from histologically normal mucosa from healthy donors (33). In agreement with previous studies (21, 22), CEA expression was ∼3-fold higher in the intestinal tumors of CEA.Tg/MIN mice when compared with normal intestinal samples from CEA.Tg mice. Not only does this overexpression of CEA offer an opportunity to be exploited by the host immune system, the similarities between humans and the CEA.Tg/MIN mice provides additional evidence for the use of this experimental model in preclinical cancer vaccine studies.

Recombinant orthopox vectors, both vaccinia and avipox, have been preferred candidates for cancer vaccines because numerous genes can be inserted and the vector-driven immunogen elicits strong immune responses against weak immunogens such as CEA (34, 35). Diversified prime/boost vaccine regimen in this study used two vectors: (a) a replication competent, recombinant vaccinia-CEA-TRICOM virus that elicits a strong immune response to CEA but whose repeated use is limited because of antivaccinia host immunity; and (b) a nonreplicating, recombinant avipox-CEA-TRICOM virus that can be administered multiple times as a boost. The diversified prime/boost vaccine protocol has been shown to be superior to administering multiple injections of the same recombinant vaccine (36). Insertion of TRICOM into both the recombinant vaccinia and avipox vaccines results in a more vigorous anti-CEA immune response as well as antitumor immunity (23, 25). GM-CSF was included as a biological adjuvant because of its ability to increase antigen-presenting cell infiltration at the vaccination site (37) and regional lymph nodes (15), resulting in a more vigorous T-cell response to CEA (15). Moreover, recent evidence demonstrates that GM-CSF elicits optimal adjuvant effects when delivered as a recombinant avipox virus.4

CEA.Tg/MIN mice present a formidable challenge for the immune system to generate not only anti-CEA host immunity but also to affect intestinal tumor formation. From the MIN standpoint, tumorigenesis is believed to initiate in utero(38), followed by aberrant crypt foci formation (ACFMin, 38) at 2 weeks of age, and the large tumor burden as a result of numerous intestinal tumors. CEA expression in normal tissues and its presence in circulating serum also presents a considerable degree of peripheral tolerance for the immune system to overcome. Therefore, an aggressive vaccination schema, primary vaccination by ages 30–35 days with monthly booster vaccinations, was adopted. The first indications that the CEA-based vaccine was eliciting favorable results was the health status of the CEA.Tg/MIN mice. CEA.Tg/MIN mice, like MIN mice, develop adult-onset anemia (hematocrit values < 35) commensurate with progressive weight loss. Anemia and progressive weight loss typically appeared before 120 days of age in the CEA.Tg/MIN that were treated with the vehicle control or vaccinated with the non-CEA-based vaccine. Overall weight gain in those mice as well as MIN mice rarely exceeded 5–6 g, whereas CEA.Tg/MIN mice vaccinated with the CEA-based vaccine maintained normal hematocrit levels and often gained >10 grams of body weight. When analyzed at necropsy, those CEA.Tg/MIN mice that received the CEA-based vaccine had a significant reduction in the number of intestinal tumors when compared with CEA.Tg/MIN mice that received the non-CEA-based vaccine (P < 0.01) or vehicle control (P < 0.05). Of the 13 CEA.Tg/MIN mice that received the CEA-based vaccine, 5 had dramatic responses (0–3 tumors), 4 had partial responses (4–25 tumors), and the remaining 4 had no response. The administration of the vaccine devoid of the CEA transgene did not suppress tumor formation, thus providing a compelling argument that the generation of anti-CEA host immunity was, indeed, involved in tumor formation. Additional study is needed to elucidate the exact mechanism as well as those events that result in incomplete suppression of tumor formation and/or escape from immune regulation. The absence of any antitumor effects in some mice could be attributable to the inability of the vaccine to induce sufficient antitumor immunity and/or the development of tumor escape mechanisms. Tumor escape can occur because of tumor-related events (i.e., loss/reduction of expression of major histocompatibility antigens, transforming growth factor β production, Fas-FasL interactions) and/or changes within the immune system (i.e., down-regulation of T cell ζ-chain, generation of T suppressor cells). The present findings argue that CEA loss variants do not explain why some CEA.Tg/MIN mice were unresponsive to the CEA-based vaccine (i.e., no reduction in tumor load) because IHC staining revealed strong CEA expression in those tumors (Fig. 5, B, D, and F). Nonetheless, the CEA.Tg/MIN mice present an excellent model to investigate those other phenomena of immune evasion.

Vaccines that target tissue-specific, self-antigens in mouse models are capable of activating autoreactive T cells that elicit autoimmune pathology (39, 40, 41, 42). Other animal studies have provided ample evidence that vaccination protocols can generate substantial antitumor immunity with little, or no, autoimmunity (43, 44). One of the more intriguing issues of this and previous studies (14, 15, 25) has been the ability of the orthopox-based vaccines to induce significant antitumor host immunity with little, if any, autoimmunity directed against normal CEA-expressing tissues in CEA.Tg mice. Histopathology and IHC staining of normal intestinal tissues revealed no significant changes in intestinal architecture or CEA expression levels in CEA.Tg/MIN mice in which the CEA-based vaccine dramatically reduced the number of intestinal tumors (Fig. 5, G and H). The most common pathological finding was enlarged spleens associated with extramedullary hematopoiesis, which, probably, was a compensation for anemia. Neither anti-DNA nor ANA serum antibodies were elevated in any mice receiving the CEA-based vaccine. Those results were underscored by the long-term survival of CEA.Tg/MIN mice that received the CEA-based vaccine. At 40 weeks of age, ∼50% of those mice were still alive. By week 49, however, all were dead, not because of any autoimmune-associated pathology, but rather, because those mice developed anemia, progressive weight loss, and intestinal tumors, the same disease progression that occurred by weeks 25–27 in the different control groups of mice. The absence of autoimmunity in those mice in which the CEA-based vaccine mediated significant antitumor immunity remains to be determined. Some possible explanations include (a) differential susceptibility of tumor and normal tissues to the immune effector arms and (b) braking of the autoreactive T-cell activity by tolerizing antigen-presenting cells, the presence of regulatory T cells or terminating vaccination (45). Investigating those possibilities will be crucial for the ongoing development of overexpressed self, tumor antigens as immunotherapy targets.

Fig. 1.

Number of tumors along the GI tract of MIN mice that were either CEA.Tg (▪) or CEA negative (). Data are the mean ± SE, 5 mice/group.

Fig. 1.

Number of tumors along the GI tract of MIN mice that were either CEA.Tg (▪) or CEA negative (). Data are the mean ± SE, 5 mice/group.

Close modal
Fig. 2.

Serum anti-CEA Ig reactivity (A), CEA-specific CD4 proliferative response (B), CD8-dependent IFN-γ production (E:T cell ratio, 25:1; C), and lytic activity (D) of T cells isolated from CEA.Tg/MIN mice. CEA.Tg/MIN mice received four injections of either the CEA-based vaccine (•), non-CEA-based vaccine (▴) or vehicle control (▪). Splenocytes isolated from 3–4 mice/group were combined and the immune assays were carried out as described in “Materials and Methods.” Data are the mean ± SE of triplicate determinations from a representative experiment that was repeated with similar results.

Fig. 2.

Serum anti-CEA Ig reactivity (A), CEA-specific CD4 proliferative response (B), CD8-dependent IFN-γ production (E:T cell ratio, 25:1; C), and lytic activity (D) of T cells isolated from CEA.Tg/MIN mice. CEA.Tg/MIN mice received four injections of either the CEA-based vaccine (•), non-CEA-based vaccine (▴) or vehicle control (▪). Splenocytes isolated from 3–4 mice/group were combined and the immune assays were carried out as described in “Materials and Methods.” Data are the mean ± SE of triplicate determinations from a representative experiment that was repeated with similar results.

Close modal
Fig. 3.

Total number of intestinal tumors in CEA.Tg/MIN mice that received the CEA-based vaccine (A), non-CEA-based vaccine (B), or vehicle control (C). Each • represents a single mouse, and the ------ represent the mean number of tumors for each group.

Fig. 3.

Total number of intestinal tumors in CEA.Tg/MIN mice that received the CEA-based vaccine (A), non-CEA-based vaccine (B), or vehicle control (C). Each • represents a single mouse, and the ------ represent the mean number of tumors for each group.

Close modal
Fig. 4.

Long-term survival of vaccinated CEA.Tg/MIN and MIN mice. CEA.Tg/MIN mice were vaccinated with the CEA-based vaccine (n = 12, ▴), non-CEA-based vaccine (n = 10, ▪), or vehicle control (n = 12, •). MIN mice (n = 5, ▵) also received the CEA-based vaccines. Vaccines were administered as outlined in the “Materials and Methods.” P < 0.001 versus CEA.Tg/MIN mice given the non-CEA-based vaccine or vehicle control or MIN mice that received the CEA-based vaccine.

Fig. 4.

Long-term survival of vaccinated CEA.Tg/MIN and MIN mice. CEA.Tg/MIN mice were vaccinated with the CEA-based vaccine (n = 12, ▴), non-CEA-based vaccine (n = 10, ▪), or vehicle control (n = 12, •). MIN mice (n = 5, ▵) also received the CEA-based vaccines. Vaccines were administered as outlined in the “Materials and Methods.” P < 0.001 versus CEA.Tg/MIN mice given the non-CEA-based vaccine or vehicle control or MIN mice that received the CEA-based vaccine.

Close modal
Fig. 5.

H&E (A, C, E, and G) and COL-1 immunohistochemical staining (B, D, F, and H) of intestinal tissues from CEA.Tg/MIN mice that received the CEA-based vaccine. A and C are examples of adenomas in the small intestine (jejunum) from CEA.Tg/MIN mice in which the CEA-based vaccine did not reduce adenoma formation. A and C, adenoma (arrows) with compression of adjacent villi (v; ×10X. B, CEA expression in the adenoma illustrated in A (×10). D, strong CEA expression in adenoma illustrated in C (×10). E, cells in the adenoma illustrated in C are basophilic with hyperchromatic, piled-up nuclei with scattered mitotic figures (arrows; ×20). F, strong CEA expression in tumor cells (×20). G, normal gut architecture in a CEA.Tg/MIN mice in which the CEA-based vaccine prevented tumor formation (×25). H, crypts in the normal intestine shown in G express CEA (arrow; ×40).

Fig. 5.

H&E (A, C, E, and G) and COL-1 immunohistochemical staining (B, D, F, and H) of intestinal tissues from CEA.Tg/MIN mice that received the CEA-based vaccine. A and C are examples of adenomas in the small intestine (jejunum) from CEA.Tg/MIN mice in which the CEA-based vaccine did not reduce adenoma formation. A and C, adenoma (arrows) with compression of adjacent villi (v; ×10X. B, CEA expression in the adenoma illustrated in A (×10). D, strong CEA expression in adenoma illustrated in C (×10). E, cells in the adenoma illustrated in C are basophilic with hyperchromatic, piled-up nuclei with scattered mitotic figures (arrows; ×20). F, strong CEA expression in tumor cells (×20). G, normal gut architecture in a CEA.Tg/MIN mice in which the CEA-based vaccine prevented tumor formation (×25). H, crypts in the normal intestine shown in G express CEA (arrow; ×40).

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This project has been funded, in part, with federal funds from National Cancer Institute, NIH, Grant N01-C0-12400.

3

The abbreviations used are: CEA, carcinoembryonic antigen; GI, gastrointestinal; Ig, immunoglobulin; CEA.Tg, CEA transgenic; MIN, multiple intestinal neoplasia; TRICOM, triad of costimulatory molecules; GM-CSF; granulocyte/macrophage colony-stimulating factor; ANA, antinuclear antibody; pfu, plaque-forming units; IHC, immunohistochemistry; DPBS, Dulbecco’s phosphate-buffered saline.

4

E. Reali, J. Schlom, and J. W. Greiner. Development of a recombinant avian poxvirus expressing granulocyte-macrophage colony-stimulating factor as a biological adjuvant for poxvirus and protein/peptide-based vaccines, manuscript in preparation.

Table 1

CEA expression levels in normal/neoplastic intestinal tissues and sera

GenotypeGI tissueCEA expression
RIAa (cpm 125I-COL-1 bound/40 μg protein)EIAb (μg CEA/mg protein)IHCcSerum CEAd (ng/ml)
CEA.Tg/MIN Neoplastic 44,400 ± 2,150 3.27–4.45 ++ 42.2 ± 7.6 
CEA.Tg Normal 12,160 ± 990 1.35–1.80 60.8 ± 14.9 
MIN Neoplastic NEG NEG NEG NEG 
C57BL/6 Normal NEG NEG NEG NEG 
GenotypeGI tissueCEA expression
RIAa (cpm 125I-COL-1 bound/40 μg protein)EIAb (μg CEA/mg protein)IHCcSerum CEAd (ng/ml)
CEA.Tg/MIN Neoplastic 44,400 ± 2,150 3.27–4.45 ++ 42.2 ± 7.6 
CEA.Tg Normal 12,160 ± 990 1.35–1.80 60.8 ± 14.9 
MIN Neoplastic NEG NEG NEG NEG 
C57BL/6 Normal NEG NEG NEG NEG 
a

Mice from each group (3–5/group) were sacrificed at 150 days of age, and normal and neoplastic intestinal tissues were removed and protein extracts prepared as outlined in “Materials and Methods.” Forty μg of extract protein were dried to each well of a 96-well plate, and the binding of 125I-COL-1 and an irrelevant, isotype-matched antibody, BL3, was performed as a solid-phase RIA. Data represent the mean ± SE of the total 125I-COL-1 (−125I-BL3) for each group of mice. NEG, <500 cpm 125I-COL-1 bound.

b

CEA levels were measured in the same protein extracts (diluted to 1.0 mg protein/ml) using the anti-CEA ELISA kit (AMDL, Inc.). Data represent the range of CEA levels for 3–5 individual mice/group. NEG, <5 ng CEA/mg protein.

c

IHC staining intensity of COL-1 in intestinal tumors of CEA.Tg/MIN and normal intestines of CEA.Tg mice. For each slide, 3–5 different fields were scored independently by a board-certified pathologist. Scoring was based on either the absence of staining, NEG, or relative staining intensities: +, weak, pale brown; and ++, strong, dark brown immunoprecipitate.

d

Sera CEA levels were measured as described in the “Materials and Methods.” Data represent the mean ± SE of 5–8 mice/group. NEG, <5 ng CEA/ml serum.

Table 2

Effect of CEA-based vaccine on weight gain and hematocrit levels

GenotypeVaccineNo. of miceΔ Body weight (g)aHematocrit (%)
90 days120 days150 days
CEA.Tg/MIN CEA-based 12 10.1 ± 1.6b 46.9 ± 3.1 44.2 ± 4.8b 41.0 ± 5.5b 
 Non-CEA-based 10 6.0 ± 1.1 39.7 ± 5.9 33.5 ± 3.7 29.8 ± 3.5 
 None 12 5.8 ± 0.9 42.9 ± 2.8 32.9 ± 3.9 30.8 ± 2.2 
MIN CEA-based 13 5.5 ± 0.7 43.3 ± 2.1 35.5 ± 3.3 28.5 ± 2.2 
 None 12 5.1 ± 0.4 44.1 ± 2.7 34.8 ± 4.0 31.0 ± 3.1 
GenotypeVaccineNo. of miceΔ Body weight (g)aHematocrit (%)
90 days120 days150 days
CEA.Tg/MIN CEA-based 12 10.1 ± 1.6b 46.9 ± 3.1 44.2 ± 4.8b 41.0 ± 5.5b 
 Non-CEA-based 10 6.0 ± 1.1 39.7 ± 5.9 33.5 ± 3.7 29.8 ± 3.5 
 None 12 5.8 ± 0.9 42.9 ± 2.8 32.9 ± 3.9 30.8 ± 2.2 
MIN CEA-based 13 5.5 ± 0.7 43.3 ± 2.1 35.5 ± 3.3 28.5 ± 2.2 
 None 12 5.1 ± 0.4 44.1 ± 2.7 34.8 ± 4.0 31.0 ± 3.1 
a

Maximum amount of gram body weight gained. Mean ± SE.

b

P < 0.05 (versus CEA.Tg/MIN mice given either the non-CEA-based vaccine or the vehicle control, and MIN mice that received either the CEA-based vaccine or the vehicle control).

Table 3

Histopathology and serum anti-DNA and ANA titers of CEA.Tg/MIN micea

VaccineMouse no.Vaccine (n)HistopathologybSerum analyses
SPLULKAnti-DNAcANAd
CEA-based EHe 2.1 
 HN 2.0 
 3.1 
 EH HN 0.0 
 EH 3.2 
Non-CEA-based EH HN 1.1 
 EH 2.0 
 EH 0.9 
 EH 1.6 
Untreated none EH HN 4.4 
 none EH 2.5 
 none EH 1.9 
 none EH 0.5 
 none EH 1.6 
VaccineMouse no.Vaccine (n)HistopathologybSerum analyses
SPLULKAnti-DNAcANAd
CEA-based EHe 2.1 
 HN 2.0 
 3.1 
 EH HN 0.0 
 EH 3.2 
Non-CEA-based EH HN 1.1 
 EH 2.0 
 EH 0.9 
 EH 1.6 
Untreated none EH HN 4.4 
 none EH 2.5 
 none EH 1.9 
 none EH 0.5 
 none EH 1.6 
a

Mice were sacrificed between 150–175 days of age.

b

Tissues examined for histopathology: S, spleen; P, pancreas; LU, lungs; L, liver; K, kidneys.

c

Anti-DNA titers were measured using sodium sulfate precipitates of undiluted serum samples in a RIA. Titers > 2.5 IU/ml were considered positive.

d

An ELISA test was used to measure antinuclear antibody using two pools of nuclear antigens: pool A consisted of single-stranded DNA, SSA, SSB, and Jo-1; pool B consisted of double-stranded DNA, ribonucleoprotein, histones, samarium, and scl-70. Results were scored as to the overall reactivity: 0, negative; 1–4, weakly, moderately, strong, very strongly reactive.

e

Histopathological findings: N, normal; EH, extramedullary hematopoiesis; HN, hydronephrosis.

We thank Donald Hill, Garland Davis, and Keith Rogers for their excellent technical assistance. We also thank Debra Weingarten for editorial assistance in the preparation of this manuscript.

1
Gold P., Freedman S. O. Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques.
J. Exp. Med.
,
121
:
439
-462,  
1965
.
2
Thompson J. A., Grunert F., Zimmermann W. Carcinoembryonic antigen gene family: molecular biology and clinical perspectives.
J. Lab. Clin. Anal.
,
5
:
344
-366,  
1991
.
3
Goldenberg D. M., Kim E. E., Deland F. H., Bennett S., Primus F. J. Radioimmunodetection of cancer with radioactive antibodies to carcinoembryonic antigen.
Cancer Res.
,
40
:
2984
-2992,  
1980
.
4
McAneny D., Ryan C. A., Beazley R. M., Kaufman H. L. Results of a Phase I trial of a recombinant vaccinia virus that expresses carcinoembryonic antigen in patients with advanced colorectal cancer.
Ann. Surg. Oncol.
,
3
:
495
-500,  
1996
.
5
Samanci A., Yi Q., Fagerberg J., Strigard K., Smith G., Ruden U., Wahren B., Mellstedt H. Pharmacological administration of granulocyte/macrophage-colony-stimulating factor is of significant importance for the induction of a strong humoral and cellular response in patients immunized with recombinant carcinoembryonic antigen.
Cancer Immunol. Immunother.
,
47
:
131
-147,  
1998
.
6
Conry R. M., Khazaeli M. B., Saleh M. N., Allen K. O., Barlow D. L., Moore S. E., Craig D., Arani R. B., Schlom J., LoBuglio A. F. Phase I trial of a recombinant vaccinia virus encoding carcinoembryonic antigen in metastatic adenocarcinoma: comparison of intradermal versus subcutaneous administration.
Clin. Cancer Res.
,
5
:
2330
-2337,  
1999
.
7
Marshall J. L., Hawkins M. J., Tsang K. Y., Richmond E., Pedicano J. E., Zhu M-Z., Schlom J. Phase I study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen.
J. Clin. Oncol.
,
17
:
332
-337,  
1999
.
8
von Mehren M., Arlen P., Tsang K. Y., Rogatko A., Meropol N., Cooper H. S., Davey M., McLaughlin S., Schlom J., Weiner L. Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas.
Clin. Cancer Res.
,
6
:
2219
-2228,  
2000
.
9
Fong L. H., Hou Y., Rivas A., Benicke C., Yuen A., Fisher G. A., Davis M. M., Engleman E. G. Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy.
Proc. Natl. Acad. Sci. USA
,
98
:
8809
-8814,  
2001
.
10
Marshall J. L., Hoyer R. J., Toomey M. A., Faraguna K., Chang P., Richmond E., Pedicano J. E., Gehan E., Peck R. A., Arlen P., Tsang K. Y., Schlom J. Phase I study in cancer patients of a diversified prime and boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus to elicit anti-carcinoembryonic antigen immune responses.
J. Clin. Oncol.
,
18
:
3964
-3973,  
2000
.
11
Eades-Perner A-M., van der Putten H., Hirth A., Thompson J., Neumaier M., von Kleist S., Zimmermann W. Mice transgenic for the human carcinoembryonic antigen gene maintain its spatiotemporal expression pattern.
Cancer Res.
,
54
:
4169
-4176,  
1994
.
12
Clarke P., Mann J., Simpson J. F., Rickard-Dickson K., Primus F. J. Mice transgenic for human carcinoembryonic antigen as a model for immunotherapy.
Cancer Res.
,
58
:
1469
-1477,  
1998
.
13
Eades-Perner A-M., Zimmermann W. Carcinoembryonic antigen-transgenic mice: a model for tumor immunotherapy.
Tumor Biol.
,
16
:
56
-61,  
1995
.
14
Kass E., Schlom J., Thompson J., Guadagni F., Graziano P., Greiner J. Induction of protective host immunity to carcinoembryonic antigen (CEA), a self antigen in CEA transgenic mice, by immunizing with a recombinant vaccinia-CEA virus.
Cancer Res.
,
59
:
676
-683,  
1999
.
15
Kass E., Panicali D. L., Mazzara G., Schlom J., Greiner J. W. Granulocyte/macrophage-colony stimulating factor produced by recombinant avian poxviruses enriches the regional lymph nodes with antigen-presenting cells and acts as an immunoadjuvant.
Cancer Res.
,
61
:
206
-214,  
2001
.
16
Mizobata S., Tompkins K., Simpson J. F., Shyr Y., Primus F. P. Induction of cytotoxic T cells and their antitumor activity in mice transgenic for carcinoembryonic antigen.
Cancer Immunol. Immunother.
,
49
:
285
-295,  
2000
.
17
Niethammer A. G., Primus F. J., Xiang R., Dolman C. S., Ruehlmann J. M., Ba Y., Gillies S. D., Reisfeld R. A. An oral DNA vaccine against human carcinoembryonic antigen (CEA) prevents growth and dissemination of Lewis lung carcinoma in CEA transgenic mice.
Vaccine
,
20
:
421
-429,  
2001
.
18
Xiang R., Primus F. J., Ruehlmann J. M., Niethammer A. G., Silletti S., Lode H. N., Dolman C. S., Gillies S. D., Reisfeld R. A. A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T-cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice.
J. Immunol.
,
167
:
4560
-4565,  
2001
.
19
Moser A. R., Pitot H. C., Dove W. F. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse.
Science (Wash. DC)
,
247
:
322
-324,  
1990
.
20
Su L-K., Kinzler K. W., Vogelstein B., Preisinger A. C., Moser A. R., Luongo C. V., Gould K. A., Dove W. F. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene.
Science (Wash. DC)
,
256
:
668
-670,  
1992
.
21
Thompson J. A., Eades-Perner A-M., Ditter M., Muller W. J., Zimmermann W. Expression of transgenic carcinoembryonic antigen (CEA) in tumor-prone mice: an animal model for CEA-directed tumor immunotherapy.
Int. J. Cancer
,
72
:
197
-202,  
1997
.
22
Wilkinson R. W., Ross E. L., Poulsom R., Iiyas M., Straub J., Snary D., Bodmer W. F., Mather S. J. Antibody targeting studies in a transgenic murine model of spontaneous colorectal tumors.
Proc. Natl. Acad. Sci., USA
,
98
:
10256
-10260,  
2001
.
23
Hodge J. W., Sabzevari H., Yafal A. G., Gritz L., Lorenz M. G. O., Schlom J. A triad of costimulatory molecules synergize to amplify T-cell activation.
Cancer Res.
,
59
:
5800
-5807,  
1999
.
24
Paulsen J. E., Steffensen I-L., Andreassen A., Vikse R., Alexander J. Neonatal exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine via breast milk or directly induces intestinal tumors in multiple intestinal neoplasia mice.
Carcinogenesis (Lond.)
,
20
:
1277
-1282,  
1999
.
25
Grosenbach D. W., Barrientos J. C., Schlom J., Hodge J. W. Synergy of vaccine strategies to amplify antigen-specific immune responses and antitumor effects.
Cancer Res.
,
61
:
4497
-4505,  
2001
.
26
Colcher D., Bird R., Roselli M., Hardman K. D., Johnson S., Pope S., Dodd S. W., Pantoliano M. W., Milenic D. E., Schlom J. In vivo tumor targeting of a recombinant single-chain antigen binding protein.
J. Natl. Cancer Inst. (Bethesda)
,
82
:
1191
-1197,  
1990
.
27
Muraro R., Wunderlich D., Thor A., Lundy J., Noguchi P., Cunningham R., Schlom J. Definition by monoclonal antibodies of a repertoire of epitopes on carcinoembryonic antigen differentially expressed in human colon carcinoma versus normal adult tissues.
Cancer Res.
,
45
:
5769
-5780,  
1985
.
28
Guadagni F., Witt P. L., Robbins P. F., Schlom J., Greiner J. W. Regulation of carcinoembryonic antigen expression in different human colorectal tumor cells by interferon-γ.
Cancer Res.
,
50
:
6248
-6255,  
1990
.
29
Schmitz J., Schlom J., Reali E., Hodge J. W., Patel A., Davis G., Greiner J. W. Identification of an interferon-γ-inducible carcinoembryonic antigen (CEA) CD8+ T-cell epitope which mediates tumor killing in CEA transgenic mice.
Cancer Res.
,
62
:
5058
-5064,  
2002
.
30
Paulsen J. E., Steffensen I-L., Loberg E. M., Husoy T., Namork E., Alexander J. Qualitative and quantitative relationship between dysplastic aberrant crypt foci and tumorigenesis in the Min/+ mouse colon.
Cancer Res.
,
61
:
5010
-5015,  
2001
.
31
Kinzler K. W., Vogelstein B. Lessons from hereditary colorectal cancer.
Cell
,
87
:
159
-170,  
1996
.
32
Miyoshi Y., Nagase H., Ando H., Horii A., Ichii S., Nakatsuru S., Aoki T., Miki Y., Mori T., Nakamura Y. Somatic mutations of the APC gene in colorectal tumors: mutation cluster region in the APC gene.
Hum. Mol. Genet.
,
1
:
229
-233,  
1992
.
33
Guadagni F., Roselli M., Cosimelli M., Spila A., Cavaliere F., Arcuri R., D’Alessandro R., Fracasso P. L., Casale V., Vecchione A., Casciani C. U., Greiner J. W., Schlom J. Quantitative analysis of CEA expression in colorectal adenocarcinoma and serum: lack of correlation.
Int. J. Cancer
,
72
:
949
-954,  
1997
.
34
Smith G. L., Moss B. Infectious poxvirus vectors have capacity for at least 25,000 base pairs of foreign DNA.
Gene (Amst.)
,
25
:
21
-28,  
1983
.
35
Stephens E. B., Compans R. W., Earle P., Moss B. Surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors.
EMBO J.
,
5
:
237
-245,  
1986
.
36
Hodge J. W., McLaughlin J. P., Kantor J. A., Schlom J. Diversified prime and boost protocols using recombinant vaccinia virus and recombinant non-replicating avian pox virus to enhance T-cell immunity and antitumor responses.
Vaccine
,
15
:
759
-768,  
1997
.
37
Kielian T., Nagai E., Ikubo A., Rasmussen C. A., Suzuki T. Granulocyte/macrophage-colony-stimulating factor released by adenovirally transduced CT26 cells leads to the local expression of macrophage inflammatory protein 1α and accumulation of dendritic cells at vaccination sites in vivo.
Cancer Immunol. Immunother.
,
48
:
123
-131,  
1999
.
38
Moser A. R., Dove W. F., Roth K. A., Gordon J. I. The Min (Multiple Intestinal Neoplasia) mutation: its effects on gut epithelial cell differentiation and interaction with a modifier system.
J. Cell Biol.
,
116
:
1517
-1526,  
1992
.
39
Fong L., Ruegg C. L., Brockstedt D., Engleman E. G., Laus R. Induction of tissue-specific autoimmune prostatitis with prostatic acid phosphatase immunization.
J. Immunol.
,
159
:
3113
-3117,  
1997
.
40
Dittel B. N., Visintin I., Merchant R. M., Janeway C. A., Jr. Presentation of the self antigen myelin basic protein by dendritic cells leads to experimental autoimmune encephalomyelitis.
J. Immunol.
,
163
:
32
-39,  
1999
.
41
Overwijk W. W., Lee D. S., Surman D. R., Irvine K. R., Touloukian C. E., Chan C-C., Carroll M. W., Moss B., Rosenberg S. A., Restifo N. P. Vaccination with a recombinant vaccinia virus encoding a “self” antigen induces autoimmune vitiligo and tumor cell destruction in mice: requirement for CD4+ T lymphocytes.
Proc. Natl. Acad. Sci. USA
,
96
:
2982
-2987,  
1999
.
42
Ludewig B., Ochsenbein A. F., Odermatt B., Paulin D., Hengartner H., Zinkernagel R. M. Immunotherapy with dendritic cells directed against tumor antigens shared with normal host cells results in severe autoimmune disease.
J. Exp. Med.
,
191
:
795
-803,  
2000
.
43
Dranoff G., Jaffee E., Lazenby A., Golumbek P., Levitsky H., Brose K., Jackson V., Hamada H., Pardoll D., Mulligan R. C. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity.
Proc. Natl. Acad. Sci. USA
,
90
:
3539
-3543,  
1993
.
44
van Elsas A., Sutmuller R. P. M., Hurwitz A. A., Ziskin J., Villasenor J., Medema J-P., Overwij W. W., Restifo N. P., Melief C. J. M., Offringa R., Allison J. P. Elucidating the autoimmune and antitumor effector mechanisms of a treatment based on cytotoxic T lymphocyte antigen-4 blockade in combination with a B16 melanoma vaccine: comparison of prophylaxis and therapy.
J. Exp. Med.
,
194
:
481
-489,  
2001
.
45
Gilboa E. The risk of autoimmunity associated with tumor immunotherapy.
Nat. Immune.
,
2
:
789
-792,  
2001
.