Znt. J . Cancer: 48, 900-907 (1991) 0 1991 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancer

A RECOMBINANT VACCINIA VIRUS EXPRESSING HUMAN CARCINOEMBRYONIC ANTIGEN (CEA) Howard KAUFMAN', Jeffrey SCHLOM~ and Judy KANTOR Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Carcinoembryonicantigen (CEA) is a 180-kDa glycoprotein expressed on most gastrointestinal carcinomas. A 2.4-kb cDNA clone, containing the complete coding sequence, was isolated from a human colon tumor cell library and inserted into a vaccinia virus genome. This newly developed construct was characterized by Southern blotting, D N A hybridization studies, and polymerase chain reaction analysis. The CEA gene was stably integrated into the vaccinia virus thymidine kinase gene. The recombinant was efficiently replicated upon serial passages in cell cultures and in animals. The recombinant virus expresses on the surface of infected cells a protein product recognized by a monoclonal antibody (COL- I) directed against CEA. Immunization of mice with the vaccinia construct elicited a humoral immune response against CEA. Pilot studies also showed that administration of the recombinant CEA vaccinia construct was able to greatly reduce the growth in mice of a syngeneic murine colon adenocarcinoma which had been transduced with the human CEA gene. The use of this new recombinant CEA vaccinia construct may thus provide an approach in the specific active immunotherapy of human GI cancer and other CEA expressing carcinoma types.

Carcinoembryonic antigen (CEA), originally described by Gold and Freedman (1965), has become one of the most widely studied oncofetal tumor-associated antigens. Although CEA is not specific for colon cancer, it has clinical utility in the surveillance of the post-operative patient following primary tumor resection. The development of monoclonal antibodies (MAbs) directed against CEA has led to improvements in the diagnostic imaging of primary colon tumors and the immunolocalization of metastatic disease (Sikorska et al., 1988; Goldenberg et al., 1987; Mach et al., 1981). Several more recent approaches, including anti-idiotypic antibodies and radiolabelled and drugconjugated MAbs, have prompted interest in targeting CEA for immunotherapy. CEA is generally considered to be weakly immunogenic in humans; that is, little evidence exists for humoral or cellmediated immunity to CEA in normal or cancer patients. The co-presentation of CEA with a strong immunogen thus represents a logical approach to inducing an anti-CEA response for tumor immunotherapy. Recent advances in recombinant vaccinia virus technology provide a powerful method for such antigenic co-presentation. Vaccinia virus is highly irnmunogenic and stimulates both humoral and cell-mediated immune responses; this cell-mediated immunity may be especially important in tumor rejection (Hellstrom and Hellstrom, 1969). Vaccinia virus is also capable of presenting tumor antigens along with cellular major histocompatibililty complex antigens (Moss and Flexner, 1987). Thus, immunization with a recombinant vaccinia virus provides a strong stimulus to the immune system. Such constructs protect against a variety of infectious diseases (Moss et al., 1984; Wachsman et al., 1988; Spriggs et aE., 1988; Fisher-Hoch et al., 1989). Protection against tumor challenge has also been demonstrated in animal models using recombinant vaccinia viruses (Lathe et al., 1987; Bernards et al., 1987; Estin et al., 1988). The introduction of foreign genetic material into vaccinia virus has become possible following recent advances in molecular biology and genetic engineering. Vaccinia virus recombinants are generally stable and have been reported to properly

replicate and transcribe foreign genes inserted under the regulation of a vaccinia promoter (Coupar et al., 1988). Foreign gene products are expressed, and post-translational modification occurs normally (Stephens et al., 1986). The successful insertion and expression of as many as 25,000 base pairs of DNA into vaccinia virus has been reported (Smith and Moss, 1983). Although most of the recombinant constructs have utilized prokaryotic genes, the construction of recombinant vaccinia viruses expressing eukaryotic genes is possible when cDNA clones are available. CEA is a highly glycosylated protein with a molecular weight of 180 kDa. CEA belongs to a larger family of genes located on chromosome 19 which are related by structure and evolution to the immunoglobulin supergene family (Thompson and Zirnmerman, 1988). The function of CEA is unknown at this time. However, it has been reported to function as a homophilic molecule in in vitro systems (Benchimol et al., 1989; Oikawa et al., 1989). The CEA gene has been isolated and the sequence of a cDNA clone reported (Oikawa et al., 1987). The cDNA clone contains a coding region of 2,065 nucleotides as well as a portion of both the 3'- and 5'-untranslated regions; this sequence of cDNA was incorporated into a vaccinia virus genome. The molecular characterization of this recombinant vaccinia virus-CEA construct is now reported, and the potential use of this viral construct as a vaccine for the active immunotherapy of human colon cancer shall be discussed. MATERIAL AND METHODS

Cells TK- 143B, a human sarcoma cell line with a deficient thymidine kinase (TK) gene, was used for selection and characterization of the recombinant vaccinia virus. The TK- 143B cells were obtained from the ATCC (CRL 8303). CV-1 (ATCC; CCL 70), a monkey embryonic kidney cell line, was used for the initial transfections and subsequent propagation of wild-type and recombinant vaccinia virus high-titer lysates. Virus Vaccinia virus of the WR strain (V-WR) was provided by Dr. B. Moss (NIAID, Bethesda, MD). This strain of vaccinia virus is a specially adapted neurotropic strain for use in rodents. The virus was stored at - 70°C. Titers were obtained by standard viral titer assays (Mackett et al., 1985). The V-WR virus contains a non-essential TK gene in the Hind111 J fragment of the viral genome.

*To whom correspondence and requests for reprints should be addressed. at the Laboratorv of Tumor Immunoloev and Bioloev. National Cancer Institute, Nationai Institutes of Health, BTilding 10, 6 O m 8B-07, Bethesda, MD 20892. 'Present address: Department of Surgery, Boston University School of Medicine, Boston, MA 02118. Received: January 18, 1991 and in revised form March 28, 1991

RECOMBINANT CEA VACCINIA VIRUS

Construction of recombinant vaccinia virus The construction of recombinant vaccinia viruses has been described in detail (Mackett et al., 1985). Briefly, a human cDNA clone, from a human colon carcinoma cell cDNA Iibrary constructed in our laboratory, was used to isolate a 2.4kb Sma I fragment. This fragment, containing the entire coding sequence for CEA, 95 nucleotides of the 5' untranslated region and 264 nucleotides of the 3' untranslated region, was inserted into the Sma I restriction site of the PSC-11 plasmid. This plasmid, also obtained from Dr. Moss, was developed as a method of inserting and selecting foreign genes into a live vaccinia virus genome (Mackett et al., 1984). This plasmid also contains viral TK gene segments which direct the insertion of the chimeric plasmid construct into the non-essential TK gene of the parental wild-type vaccinia virus (Fig. I), as well as the E . coli LacZ gene under the control of the vaccinia P11 promoter. LacZ codes for beta-galactosidase, which provides a method of selection for appropriate recombinant viral particles. Transfectionprocedure Recombination was performed by infecting a 60-mm tissue culture dish of nearly confluent CV-1 cells with 0.20 plaqueA Sma I

5

Sma I '

1

2106 bp

95bP

+

264bp

'

f 3'

c

CEA

Pi

t PI I

I

P2

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FIGURE1 - Diagram of the PSC 11-CEA plasmid construct. A Sma I restriction site for insertion of foreign genetic segments is in juxtaposition to the vaccinia 7.5P promoter aligning the viral promoter with the start site of the cloned gene. The E . coli LacZ gene coding for P-galactosidase is under the regulation of the vaccinia virus P11 promoter. The LacZ gene and the Sma I cloning site are both contained within segments of the right (TK-R) and left (TK-L) vaccinia TK gene sequences. These viral sequences direct the insertion of the recombinant plasmid into the wild-type vaccinia TK gene. Vaccinia TK is a non-essential viral gene, and homologous recombination with the PSC 11 cloning plasmid results in a TK-deficient virus. (a) The insert gene segment is a cDNA clone of CEA containing 95 bp of 5'-untranslated region, 264 bp of 3'-untranslated region, and 2,106 bp of coding sequences. P, and P, are primers used for PCR DNA amplification. The cDNA was blunt-end ligated into the PSC 11 Sma I cloning site. (b) The resulting chimeric construct, designated PSC 11-CEA, was analyzed by restriction endonuclease mapping with BamHl .

90 1

forming unitdcell (pfukell) of wild-type vaccinia virus (VWR). While the infection was in progress, a solution was prepared consisting of 1 ml transfection buffer (0.14 M NaCl, 5 ~ lKCI, l ~ 1 mM Na,HPO,, 0.1% dextrose, and 20 mM HEPES; PH adjusted to 7.0-7.1), 5 pg of the chimeric PSC 11-CEA plasmid DNA, and 1 pg of vaccinia virus DNA as carrier. The solution was vortexed for 1 min, flicked gently after the addition of 50 pl of 2.5 M CaCl,, and kept at room temperature for 20 min. After the 2-hr infection period at 37"C, the viral inoculum was aspirated off, and the monolayer was washed twice with phosphate-buffered saline (PBS). The DNA precipitate was added to the dish in a dropwise fashion and left at room temperature for 30 min. Five milliliters of fresh culture medium supplemented with 5 % FCS were added, and the dish was incubated at 37°C for 3 hr. After the 3-hr incubation, the medium was aspirated from the dish and replaced with 5 ml of fresh culture medium supplemented with 5% FCS, then the dish was incubated at 37°C for another 48 hr. The cells were scraped into the medium and collected by centrifugation. The virus was released by 3 cycles of freeze-thawing followed by sonication for 1 min in a 450-watt water bath sonicator. Recombinant viral clones were selected by growth on TK - 143B cells in the presence of 5-bromo-deoxyuridine (BUdR) and 5-bromo-4-chloro-3-indoyl-beta-D-galactosidase (X-Gal). Appropriate blue recombinant viruses were purified by 5 rounds of plaque purification and grown into a high-titer lysate using standard techniques (Mackett et al., 1982). Southern blotting The recombinant vaccinia genome was analyzed by viral DNA extraction, restriction endonuclease digestion with HindIII, and Southern blotting. The digestion of wild-type vaccinia DNA with HindIII revealed a typical restriction pattern (McCarron et al., 1978) which included a 5.1-kb HindIII J fragment containing a segment of the viral TK gene. The recombinant virus lacked this HindIII J fragment due to the insertion of the chimeric plasmid construct into the viral TK gene. Vaccinia viral DNA extraction A nearly confluent monolayer of TK- 143B cells was infected with 30 pfuicell of either V-WR or recombinant vaccinia virus (rV-CEA). The infection was allowed to proceed until a maximal cytopathic effect was observed, usually after about 24 hr. The cells were scraped into the medium and pelleted by centrifugation, and the cell pellet was resuspended in 50 p1of PBS. A mixture of 300 p1 of low-salt buffer (20 m Tris-HCI, PH 8.0, 10 mM EDTA, and 0.75% SDS) and 20 p1 of proteinase K (10 mg/ml) was added to the cell sample and vortexed for 1 min. The mixture was shaken at 37°C overnight. The DNA was then extracted twice with phenol/chloroform and twice with chloroform, and precipitated in 0.3 N sodium acetate and ethanol. HindIII digestion and Southern blotting The extracted viral DNA was digested with HindIII, subjected to electrophoresis on a 0.6% agarose gel at 80 volts, transferred to a nylon membrane, and hybridized with a radiolabelled vaccinia virus DNA probe. The blot was analyzed for the presence or absence of the HindIII J fragment and hybridized with other radiolabelled probes to determine the size of the recombinant J fragment. DNA hybridization and polymerase chain reaction The presence of the CEA gene in the recombinant vaccinia genome was determined by DNA hybridization studies and analysis by the polymerase chain reaction (PCR). Following selection and purification of recombinant vaccinia viruses, a

902

KAUFMAN ET A L .

nearly confluent layer of TK- 143B cells growing in a 60-mm tissue culture dish were infected with 10 pfdcell rV-CEA or V-WR. The cell monolayer was incubated at 37°C for 24 hr and then used for DNA hybridization or PCR analysis. The dishes used for DNA hybridization were treated by applying a nylon membrane directly over the cell monolayer for 10 min. The nylon membrane was then denatured, neutralized, and soaked in 2X SSC for several minutes. The DNA was crosslinked to the nylon membrane by exposure to UV radiation for 2 min using a DNA transfer lamp (Fotodyne, New Berlin, WI). The membrane was then hybridized with a radiolabelled CEA probe. The dishes used for PCR analysis were treated with an agarose overlay following viral infection, which fixed the location of viral plaques on the dish. Isolated plaques were picked with a sterile “tooth-pick,’’ placed in 1 ml PBS without calcium or magnesium, boiled for 10 min, cooled on ice, and subjected to standard PCR analysis using a Perkin-Elmer-Cetus DNA thermal cycler (Norwalk, CT). The PCR primers used in this study were designed to amplify a 2.1-kb fragment. These primers initiate outside the CEA coding region, at nucleotide -40 (PI) and at nucleotide 2230 (PJ (Fig. la). A plaque infected with wild-type vaccinia virus was used as a negative control in the PCR reaction. Following 30 cycles of amplification, samples were collected and electrophoresed on a 1% agarose gel, transferred to a nylon membrane, and hybridized with the radiolabelled CEA probe as described above. Protein expression Protein expression and localization were determined by immunofluorescent staining with a MAb COL- 1 directed against CEA. A nearly confluent monolayer of TK- 143B cells was infected with 30 pfuicell of V-WR or rV-CEA and incubated at 37°C for 5 hr. The viral inoculum was aspirated, the monolayer was washed 3 times with PBS, and the cells were fixed with a freshly prepared solution of 2% paraformaldehyde in PBS for 30 min at room temperature. The monolayers were washed 3 times with culture media and blocked with 1% BSA in PBS for 30 min. Cells were then treated with 1 pg/ml of MAb COL- 1, a murine MAb which is reactive with the 180-kDa glycoprotein form of CEA and non-reactive with non-specific cross-reacting antigen (NCA), the 90-kDa glycoprotein member of the CEA gene family (Muraro er al., 1985; Kuroki et al., 1989). B72.3, a murine antibody previously shown to be unreactive with CEA (Kuroki et al., 1989) was used as a negative control. After being shaken at room temperature for 1 hr, the cells were washed 5 times in PBS, and fluorescine-conjugated goat antimouse second antibody at a dilution of 1:100 was added to the cells for 30 min. The cells were washed 5 times with PBS and visualized under a fluorescence microscope. In vivo studies C57BL/6 mice were inoculated by intraperitoneal injection with V-WR or rV-CEA. Ten mice were vaccinated per group, and each received lo8 pfu/lOO pl of virus on 3 separate days with a 2-week interval between vaccinations. Blood was drawn from each mouse at 2-week intervals, and the sera were used for ELISA determination as described below. Anti-vaccinia antibody, determined for each mouse over time, was correlated with anti-CEA antibody production. ELISA The preparation of the vaccinia plates involved coating a 96-well microtiter plate with 100 pl of antigen (1 X lo7 viral particles per 100 p1 of 0.1 M sodium carbonate buffer; PH 9.6) overnight. The plate was blocked with 1% BSA in Trisbuffered saline containing 0.1% glutaraldehyde. After a 1-hr incubation at room temperature with appropriately diluted mouse sera, antibodies were reacted with a secondary antibody containing biotinylated streptavidin-alkaline phosphatase. The resulting complex was detected with p-nitrophenyl phosphate

as the chromogen. The reaction was stopped by addition of sodium hydroxide, and sample absorbances at 405 nm were read by an ELISA reader (Bio-Tek Microplate Reader, Model EL 310). A rabbit polyclonal anti-vaccinia antibody (gift of Dr. M. Buller, NIAID, Bethesda, MD) was used as a positive control on the anti-vaccinia plates (Palumbo et al., 1989). The CEA plates were coated with 100 p1 (250 ng) purified CEA protein (International Enzyme, San Diego, CA) at 37°C overnight. The plates were blocked with 1% BSA in Trisbuffered saline. The ELISA procedure as described above for the vaccinia plates was then followed. MAb COL-1 was used as a positive control antibody. A standard curve was constructed to correlate A405 readings to the amount of MAb COL-1 added. The amount of CEA-specific antibody present in experimental samples was calculated in MAb equivalents from the A405 readings in the linear range obtained with the appropriate sample dilutions. Therapy studies Four- to 5-week-old female C57BL/6 mice obtained from Frederick Cancer Research Facility were injected S.C. with 2 x lo5 MCA38 murine adenocarcinoma cells transduced with the human CEA gene, and which express human CEA on their surface (Robbins et al., in press). These MCA38 transduced tumors grow in syngeneic C57BL/6 mice. The MCA38 cells were transduced with a retroviral construct containing cDNA encoding the human CEA gene. Individual transduced clones, isolated and characterized for CEA expression, were reactive with 7 anti-CEA MAbs including COL-1 (Robbins et al., in press). Ten animals per group were inoculated by tail scarification, 7 days post tumor transplant with 10 pl of crude lysate containing 1 X 10lo pfu of wild-type vaccinia, V-WR, or recombinant vaccinia, rV-CEA. The second and third immunizations were given at 14-day intervals. Animals were checked weekly for the presence of tumor. Tumors were measured by caliper in 2 dimensions, and the volume was calculated using the following formula: widthZ X length + 2. The MCA38 murine colon carcinoma was kindly provided by Dr. S. Rosenberg, NCI. RESULTS

Construction of recombinant virus The CEA cDNA was inserted into the PSC-11 plasmid, with the proper orientation determined by BamH 1 restriction endonuclease digestion. The chimeric plasmid construct was then inserted into the vaccinia virus genome by homologous recombination following the procedures outlined in “Material and Methods” and as reported previously (Mackett et al., 1984, 1982). The progeny viral particles were collected and plated on a confluent monolayer of TK- 143B cells in the presence of BUdR and X-Gal. The formation of blue plaques under these conditions indicated appropriate recombinant viruses (Fig. 2). Plaque formation in BUdR can be effected only by TKdeficient viruses, that is, recombinant or spontaneously mutated wild-type viruses. However, only true recombinant viruses contain the E . coli P-galactosidase gene and possess the ability to form blue plaques when X-Gal is present in the medium. The blue plaques were isolated as single recombinant clones and purified by several rounds of selection under similar conditions. High-titer lysates were prepared from purified isolates by successive passage in larger tissue-culture flasks. In general, titers of 1 X lo8 pfu/ml to 1 x lo9 pfuiml were obtained and stored at - 20°C. Southern blot analysis Figure 3 compares a Southern blot of V-WR DNA with an rV-CEA DNA (Fig. 3a) after digestion with HindIII. The blot was hybridized with a 32P-dCTP-labelled vaccinia DNA and

RECOMBINANT CEA VACCINIA VIRUS

FIGURE2 - Recombinant vaccinia-CEA-induced plaques. The above tissue-culture platcs show a confluent monolayer of TK- 143B cells infected with (a) wild-typc virus, V-WR, or (b) the recombinant vaccinia-CEA virus, rV-CEA. The viral infection was propagated in media supplemented with 25 p,g/ml BUdR and 300 pg/ml X-Gal. Recombinant viruses produce obvious blue plaques under these conditions.

903

probe. This hybridization revealed multiple bands in both V-WR and rV-CEA columns, consistent with the bands identified following HindIII digcstion of human genomic DNA (Tran et al., 1988). Because of the genomic DNA contamination in the viral preparation, wc could not identify a clear CEA band in the recombinant DNA preparation (data not shown). To further idcntify the recombinant fragment, the HindIIIdigested Southern blot was hybridized with a radiolabelled DNA probe to the E . coli P-galactosidase gene. This gene is present in the recombinant virus, absent in the wild-type vaccinia genome, and not found in human genomic DNA. Hybridization with the P-galactosidase probe revealed a distinct band at 9.2 kb in thc rV-CEA viral DNA blot, which is consistent with the expected size of the recombinant HindIII J fragment (Fig. 36). DNA hybridizution studies Viral DNA directly transferred to a nylon membrane from a plate of cells infected with eithcr wild-type vaccinia virus (Fig. 4a) or recombinant vaccinia-CEA virus (Fig. 46) was hybridized with a radiolabelled CEA probe and exposed to X-ray film overnight. The recombinant vaccinia-CEA plaques hybridized to the CEA probe, whereas the wild-type vaccinia virus plaques did not. Infection of a cell monolayer with the virus VSC 8 was performed as a further control. The VSC 8 construct is a recombinant vaccinia virus containing the E . coli LacZ gene inserted into the vaccinia TK gene (Chakrabarti et al., 1985). The VSC 8 viral DNA also failed to hybridize with the labelled CEA probe (Fig. 4c). These hybridization studies provide evidence that the CEA scquenccs inserted into the vaccinia virus are incorporated into the vaccinia genome of the rV-CEA virus. PCR analysis Several recombinant cloncs from the original transfection procedure were analyzed by PCR. DNA samples from areas of plaque formation were collected, treated with 30 cycles of amplification followed by gel electrophoresis, and transferred to a nylon membrane. The DNA was hybridized to the radiolabelled CEA probe; the results are shown in Figure 5. All recombinant clones (lanes 1-9) hybridized with the CEA gene probe, whereas a DNA sample from wild-type vaccinia virus (lane 10) failed to show any hybridization with the CEA probe. Thus, PCR was a useful method for identifying recombinant vaccinia clones. CEA protein expression The expression and cellular localization of CEA protein was determined by immunofluorescent staining with MAb COL- 1 directed against CEA. The results of the immunofluorescent staining were maximal within 5 hr of initial viral infection.

FIGURE3 - Southern blot analysis of recombinant vaccinia-CEA virus. V-WR and rV-CEA digested with HindIII hybridized with (a) a radiolabelled vaccinia virus DNA probe or (h) a radiolabelled betagalactosidase DNA probe. Southern blot (0)shows the absence of the 5.1-kb HindIII J fragment in the rV-CEA construct. Southern blot (b) shows the presence of a 9.2-kb HindIlI fragment containing the betagalactosidase gene representing the recombinant plasrnid construct in the vaccinia HindIII J fragment.

exposed to X-ray film for 4 hr. The recombinant blot clearly lacks the 5.1-kb Hind111 J fragment present in the wild-type DNA blot. To determine the size of the recombinant HindIII J fragment containing part of the viral TK gene and human CEA, the Southern blot was hybridized with a radiolabclled CEA DNA

FIGURE4 - Detection of recombinant virus by direct plaque hybridization. (a) V-WR, (b) rV-CEA, or ( c ) recombinant vaccinia-betagalactosidase virus were lifted from a ccll monolayer onto a nylon membrane and hybridized to a radiolabelled CEA probe.

904

KAUFMAN ET AL.

CEA. Cells infected with the wild-type vaccinia virus failed to show any immunofluorescent staining with COL-1 (Fig. 6c,d). Furthermore, immunofluorescent staining with the isotypematched negative control antibody €372.3 failed to elicit any imaging on cells infected with the rV-CEA virus (Fig. 6e,fi.

FIGURE5 - PCR analysis of recombinant vaccinia-CEA viral isolates. Individual recombinant vaccinia-CEA viral plaques were toothpicked and subjected to PCR analysis using primers constructed from the 5 ' - and 3'-end of the CEA gene. Aliquots of the PCR reaction were electrophoresed, blotted onto a nylon membrane, and hybridized to a radiolabelled CEA probe. Lane 1, CEA-positive control; lanes 2-9, individual rV-CEA viral isolates; lane 10, V-WR.

In vivo studies The recombinant vaccinia construct rV-CEA was further characterized by immunizing C57BL/6 mice 3 times with lo8 pfu of virus at 2-week intervals by intraperitoneal injection. The mice developed antibody titers to CEA within 14 days of inoculation; this response could be boosted by subsequent immunization (Table I and Fig. 7). The response of the mice to the vaccinia virus itself was also measured by assaying for anti-vaccinia antibodies. This was performed to ensure that the mice had been adequately immunized with the vaccinia virus. Table I shows the antibody response to the vaccinia virus and clearly demonstrates that the mice received an adequate inoculum of virus. A control group of mice received los pfu of wild-type vaccinia virus at 2-week intervals. These mice developed a similar antibody response to the vaccinia virus (Table I) when compared with the recombinant virus-treated animals. The control animals did not develop an antibody response to CEA (Table I and Fig. 7). These data suggest that mice immunized with the recombinant virus rV-CEA can recognize human CEA and mount a humoral immune response against this antigen. None of the vaccinated mice exhibited any evidence of toxicity for the 42-day observation period following immunization.

Effect of recombinant vaccinia (rV-CEA)administration on the growth of Jyngeneic tumors expressing human CEA in mice CEA is expressed on more than 90% of human gastrointestinal carcinomas but not on murine tumors. Passive immunotherapy studies using anti-CEA MAbs have utilized athymic mice bearing human colon carcinoma xenografts. The athymic mouse with its impaired immune system would not be appropriate for active immunotherapy studies. Thus, studies were undertaken, in which the human CEA was transduced into and expressed in the MCA38 murine colon adenocarcinoma cell line. These CEA-transduced MCA38 tumors grew in syngeneic C57BL/6 mice (data not shown). Pilot studies were conducted to determine if there is any biologic activity in the recombinant CEA vaccinia construct described here. Figure 8 shows the results of the growth of 7-day established S.C. tumors of individual mice (10 per group) given 3 administrations of either wild-type vaccinia virus (Fig. 8a) or recombinant vaccinia virus (Fig. 8b). Animals that had FIGURE6 - Immunologic detection of CEA in cells using anti-CEA been administered the recombinant vaccinia virus containing MAb COL-I. Immunofluorescent staining and the corresponding light human CEA experienced a substantial reduction in the growth photomicrographs of a cell monolayer infected with vaccinia virus. (a) of the tumors during the course of 42 days. Two animals that Light photomicrograph of rV-CEA-infected TK- 143B cells; (6) immunofluorescent staining of TK-143B cells infected with rV-CEA and treated with the MAb COL-1; (c) light photomicrograph of V-WRinfected TK- 143B cells; ( d ) immunofluorescent staining of V-WRinfected cells with MAb COL-I; ( e ) light photomicrograph of rVCEA-infected TK- 143B cells; c f ) immunofluorescent staining of rVCEA-infected TK-143B cells treated with the MAbB72.3.

Further incubation with virus led to cytolysis of infected cells and degradation of membrane protein. The cells infected with the recombinant vaccinia construct rV-CEA showed distinct cell-surface staining with the MAb COL-1 under fluorescence (Fig. 6a,b). The rV-CEA virus expresses CEA and is able to insert the molecule in the cellular membrane, consistent with the normal cellular localization of

TAB1.E: I - IMML'Kli RESPONSE TO HCMAK CI!A AN1 IGkN A N D VACCINIA VIRLS AN'l'l(il:hS 1Y MICE IMML'NIZED WITH rV-CI:A A N D V-WR

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CEA antibody'

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VV antibody2

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14 days

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35 days

14 days 35 days

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V-WR

A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA).

Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein expressed on most gastrointestinal carcinomas. A 2.4-kb cDNA clone, containing the complete c...
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