WO1995032286A2 - Immunogenic carcinoembryonic antigen and methods of use and production - Google Patents

Abstract

A recombinant carcinoembryonic antigen (CEA) and method of preparing highly purified recombinant carcinoembryonic antigen protein by expression in insect cells using a baculovirus expression system is provided. The resulting protein is useful as an immunogen when prepared in an antigenic formulation suitable for administration to human subjects. The protein may be combined with a carrier or as part of a fusion protein and may be administered by injection with an adjuvant.

Description

IMMUNOGENIC CARCINOEMBRYONIC ANTIGEN

AND METHODS OF USE AND PRODUCTION

Background of the Invention The present invention relates to cancer

immunotherapy and more particularly relates to a recombinant immunogen for the treatment or

prevention of cancer.

Cancer Immunotherapy

In 1909 Paul Ehrlich first proposed the idea of vaccinating against cancer by administering defined immunogens. Since then, scientists have been trying to heighten immunological recognition of tumor cells in order to augment anti-tumor attack by the host immune system. Investigators have explored a variety of cancer immunotherapies, including immunization with autologous or

allogeneic tumor extracts, stimulation with nonspecific immune enhancers, stimulation with

cytokines, use of lymphokine activated natural killer cells or tumor infiltrating lymphocytes, and more recently, targeting specific markers on the surface of the tumor cells. (Kantor, I. and Schlom, J., "Comparison of a CEA-recombinant vaccinia virus, purified CEA, and an anti-idiotype antibody bearing the image of a CEA epitope in the treatment and prevention of CEA-expressing tumors." Vaccine Res. 2:79-94 (1993))

Antigens have been identified that are expressed preferentially on the surface of certain tumors and not normal tissue. Administration of a purified preparation or recombinant form of the antigen may be used to activate the host immune system. This specific, active, immunotherapeutic treatment strategy offers the potential for highly selective destruction of target tumor cells with minimal side effects. Tumor specific antigens include surface idiotypic immunoglobulin on neoplastic B cells, melanoma antigens MAGE-1 and p97 on melanoma cells, gangliosides on melanoma cells, mucin derivatives on ovarian and breast tumors, and carcinoembryonic antigen (CEA) on colon, lung, and breast

carcinomas.

When administered to cancer patients, immune therapy has been shown to stimulate tumor-reactive T-lymphocytes, nonspecific effector cells, and humoral responses in animal and human studies

(Stevenson, F.K., "Tumor Vaccines." FASEB J.

5:2250-2257 (1991); Kedar, E. and Klein, E.,

"Cancer immunotherapy: Are the results

discouraging? Can they be improved?" Adv. Cancer Res . 59:245-322 (1992); Klein, G. and Boon, T.,

"Cancer: Tumor immunology: present perspectives." Current Opin . Immunol . 5:687-692 (1993); Masucci, M.G., "Viral immunopathology of human tumors." Curr. Opin . Immunol 5:693-700 (1993); Edgington, S.M., "Turning on tumor-fighting T-cells."

Biol . /Technology 11 : 1117-1119 (1993); Golumbek, P., et al., "The antitumor immune response as a problem of self-nonself discrimination: implications for immunotherapy." Immunol . Res . 12:183-192 (1993); Pardoll, D.M., "New strategies for enhancing the immunogenicity of tumors." Curr. Opin . Immunol . 5:719-725 (1993)).

Clinical trials of several cancer immunogens are being conducted, with promising preliminary

results. For example, a clinical trial using a preparation from material shed by a pool of four allogeneic human melanoma cell lines containing multiple melanoma-associated antigens demonstrated an immune response to melanoma, consisting of both cellular and humoral immunity, in more than 50% of the patients. (Bystryn, et al., Cancer 61:1065- 1070 (1988)). Melanoma patients treated with an immunogen prepared from irradiated melanoma cell lines that were screened for expression of

gangliosides and known protein antigens showed an increased survival rate over five years of 26%, compared with historical controls showing survival of 6% (Morton, D.L., et al., "Prolongation of survival in metastatic melanoma after active specific immunotherapy with a new polyvalent melanoma vaccine." Annals Surg. 216:463-482

(1992); Cohen, J. "Cancer vaccines get a shot in the arm." Science 262:841-843 (1993)). Other immunogens being tested in humans include the ganglioside GM2 for melanoma; two proteoglycan anti-idiotypes for melanoma; sialyl-Tn antigen, a mucin derivative, for breast, ovarian, colon and pancreatic cancers; synthetic peptides of

immunoglobulin epitope for lymphocytic leukemia; and vaccinia virus vectored CEA for colorectal, gastric, pancreatic and breast cancers. (Irvine K., et al., "Comparison of a CEA-recombinant vaccinia virus, purified CEA, and an anti-idiotype antibody bearing the image of a CEA epitope in the treatment and prevention of CEA-expressing tumors." Vaccine Res . , 2:79-94, (1993))

Cancer immunotherapy trials in humans have also been conducted with irradiated autologous tumor cells, tumor cell extracts, virally induced

oncolysates, anti-idiotypic antibodies, and tumor- associated carbohydrates coupled to keyhole limpet hemocyanin (KLH).

Carcinoembryonic antigen

Carcinoembryonic antigen (CEA) was first

described by Gold and Freedman in 1965 as a cancer- specific fetal antigen in adenocarcinoma of the human digestive tract. (Gold, P., and Freedman, S.O., "Demonstration of tumor-specific antigens in human colon carcinomata by immunological tolerance and adsorption techniques." Exp . Med. 121:439-462 (1965)). CEA is a member of a family of cell surface glycoproteins that are produced in excess in nearly all solid tumors of the human

gastrointestinal tract, 70% of lung adenocarcinoma, and 50% of breast carcinomas (Thompson, J.A., et al., "Carcinoembryonic antigen gene family:

molecular biology and clinical perspectives." J. Clin . Lab. Anal . 5:344-366, 1991). The cDNA encoding four members of the CEA family are

described in U.S. Patent No. 5,122,599 to Barnett et al.

The CEA gene family is a part of the

immunoglobulin superfamily, a large group of molecules involved in basic cell surface

recognition events. The main features of members of this superfamily are domains whose sequences resemble the variable (V) or constant (C) domains of immunoglobulin (lg). The lg V and C domains both consist of two β-sheets stabilized by a disulfide bond, forming an lg fold; the main structural difference is that the V domain has an extra loop in the middle. All known members of the CEA gene family possess one N-terminal domain, which is most similar in sequence to the V domain of immunoglobulin. The CEA family N-domain is followed by zero to six domains which resemble the lg C domain. The CEA family proteins possess a hydrophobic C-terminal domain which allows for insertion into the cell membrane or serves as a signal for linkage to a

glycosylphosphatidylinositol moiety.

The CEA gene family is divided into two

subgroups: the CEA subgroup, which includes CEA, nonspecific cross-reacting antigen (NCA) , and biliary glycoprotein (BGP), and the pregnancy- specific glycoprotein (PSG) subgroup. CEA subgroup members are expressed in a variety of tissues; for example, BGP is present in bile, while NCA is found in lung, spleen, liver, lymphocytes and

granulocytes. CEA, NCA, and BGP have been shown to function as intercellular adhesion molecules. PSG subgroup glycoproteins are expressed in the

placenta as well as in other tissues, such as liver, and are released to the maternal blood during pregnancy. Serologically, the members of the CEA and PSG subgroups are distinct . Proteins within each subgroup share antigenic determinants in common, but between subgroups there is no evidence for serological relatedness. Monoclonal antibodies have been characterized that cross-react with CEA, NCA, and BGP but not with PSG.

The function of CEA and its relevance to

malignant transformation is not well understood.

CEA has been shown to be a member of the family of intercellular adhesion molecules and mediates homotypic aggregation of cultured human colon adenocarcinoma cells and mouse cells transduced with human CEA (Benchimol, et al.,

"Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule." Cell 57:327-334, 1989). Heterotypic cell adhesion between cells expressing CEA and non-specific cross-reacting antigen (NCA) has also been reported (Oikawa, et al., "Cell adhesion activity of nonspecific cross-reacting antigen (NCA) and

carcinoembryonic antigen (CEA) expressed on CHO cell surface: hemophilic and heterophilic

adhesion." Biochem. Biophys . Res . Comm. 164:39-45 (1989)). CEA is anchored to cell membranes by covalent attachment to a glycolipid (Hefta, et al.,

"Carcinoembryonic antigen is anchored to membranes by covalent attachment to a

glycosylphosphatidylinositol moiety: identification of the ethanolamine linkage site." Proc . Natl . Acad. Sci . USA 85:4648-4652 (1988)) and is found on adjacent cell membranes in aberrant tumor

intestinal epithelium (Benchimol, et al.,

"Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule." Cell 57:327-334 (1989)). Thus, overproduction of CEA may contribute to colon carcinogenesis by interfering with the normal constraints of cell proliferation mediated by adhesion molecules.

CEA is generally considered to be weakly

immunogenic in humans. In spite of the antigen's high level expression on tumors, there is little evidence that antibodies or cellular immunity against this marker develop naturally.

Immunization of mice with purified CEA antigen in Freund's adjuvant reportedly prevented the growth of CEA-transduced tumor cells but was not effective in the treatment of established tumors (Irvine K., et al., "Comparison of a CEA-recombinant vaccinia virus, purified CEA, and an anti-idiotype antibody bearing the image of a CEA epitope in the treatment and prevention of CEA-expressing tumors." Vaccine Res . 2:79-94 (1993)). The quality of the tumor- derived CEA used in this study was not documented and it was only weakly immunogenic.

The human gene for CEA has been cloned both from cDNA and genomic DNA (Oikawa, et al., "Primary structure of human carcinoembryonic antigen (CEA) deduced from cDNA sequence." Biochem . Biophys . Res . Comm. 142:511-518 (1987); Schrewe, et al., "Cloning of the complete gene for carcinoembryonic antigen: analysis of its promoter indicates a region

conveying cell type-specific expression." Moi .

Cell . Biol . , 10:2738-2748 (1990)) The gene is also described in European Patent Application

Publication No. EP 0 346,710, published on December 20, 1989, entitled "cDNAs Coding for Members of the Carcinoembryonic Antigen Family." Sequence

analysis of the gene shows that CEA is synthesized from a precursor with a cleaved signal peptide at the N-terminus followed by 668 amino acids. The first 108 N-terminal residues (N domain) are followed by three nearly identical 178 residue repeats (domains I, II and III), as shown in

Sequence ID No. 3. At the C-terminus is a 26 amino acid hydrophobic region which is removed from the mature CEA with subsequent addition of glycosyl- phosphatidylinositol. Mature human CEA is a glycoprotein with a molecular weight of about

180,000, of which approximately 50% is

carbohydrate.

Recombinant CEA has been expressed in mammalian cell lines, including murine, rat and human cell lines (Robbins, P.F., et al., "Transduction and expression of the human carcinoembryonic antigen gene in a murine colon carcinoma cell line."

Cancer Res . 51:3657-3662 (1991); Terskikh, J.P., et al., "Marked increase in the secretion of a fully antigenic recombinant carcinoembryonic antigen obtained by deletion of its hydrophobic tail."

Moi . Immunol , 30:921-927 (1993)). Recombinant CEA has also been expressed using a vaccinia expression vector (Kaufman, H., et al., "A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA)." Int. J. Cancer 48:900-907 (1991) and in insect cells using baculovirus expression vectors (Salgaller, M.L., et al., "Baculovirus recombinants expressing the human carcinoembryonic antigen gene." Cancer Res . 53:2154-2161 (1993). However, expression of CEA by Salgaller et al.

using baculovirus vectors produced only low levels of mutant CEA, which were truncated and cell- associated, not secreted. Truncation of the CEA polypeptide was reportedly due to a genetic

instability of the baculovirus expression vectors.

The recombinant CEA proteins currently available fail to elicit a therapeutic immune response in mammals. These CEA proteins are unstable,

truncated, and inappropriately glycosylated.

Furthermore, the methods used to produce the recombinant CEA proteins result in low production levels and rely on growth in serum-containing medium. Production in serum-containing medium or using any blood products from animals or humans is greatly discouraged because of the potential for contamination. Animal sera can carry

microorganisms that cause disease in humans, such as, for example, scrapie and immunodeficiency viruses similar to HIV. The use of serum- containing medium is also very costly. In

addition, serum proteins, such as serum albumin, are contaminants that complicate and interfere with purification.

What is needed is a stable, immunogenic, CEA protein that can be produced at high levels in serum-free medium.

It is therefore an object of the present

invention to provide a stable, recombinant

immunogenic CEA protein.

It is a further object of the present invention to provide a full-length immunogenic recombinant CEA protein or immunogenic fragments of a

recombinant CEA protein.

It is a further object of the present invention to provide a recombinant CEA protein that is substantially glycosylated.

It is a further object of the present invention to provide a recombinant CEA vaccine for the prevention or treatment of cancer.

Summary of the Invention A recombinant carcinoembryonic antigen (CEA) and method of preparing highly purified recombinant carcinoembryonic antigen protein by expression in insect cells using a baculovirus expression system is provided. The resulting protein is useful as an immunogen when prepared in an antigenic formulation suitable for administration to human subjects. The protein may be combined with a carrier or as part of a fusion protein and may be administered by injection with an adjuvant.

The recombinant protein consists of an

immunogenic, full length CEA or an immunogenic fragments thereof. The full length CEA has a molecular weight of approximately 120,000, is substantially glycosylated, and is secreted into the culture fluid of insect cells. The protein can be purified under non-denaturing conditions to 95% or greater purity.

A method of producing the recombinant CEA at very high levels in insect cells, grown in serum- free medium, is described. Very high levels of production are defined herein as levels greater than 100 mg/L.

In accordance with the method, the cloned CEA gene is modified by deletion of the natural hydrophobic signal peptide sequences and replacement thereof with a 61 kDa baculovirus signal peptide and deletion of the hydrophobic signal sequence at the terminus to promote

secretion in insect cells. When introduced into a baculovirus expression vector, the baculovirus polyhedron promoter directs transcription and the baculovirus signal peptide directs translation of the recombinant CEA into the insect cell

glycosylation pathway.

A purification process suitable for large scale production of product suitable for administration to humans and a method for treating cancer patients are also provided. The CEA is administered to patients in an amount sufficient to elicit

antibodies or inhibit tumor development.

Brief Description of the Drawings

Figure 1 is a flow chart showing the strategy for cloning the human CEA gene from LS175T human colon adenocarcinoma cells into a baculovirus expression vector.

Figure 2 is a schematic representation showing the strategy for sequencing CEA in an MGS12

recombination plasmid (pA9080). The length and position of each sequencing run used in

constructing the sequence is shown. The direction of each run is indicated by an arrowhead. The scale above the primer map refers to length in nucleotides. The position of the CEA coding region within the region sequenced is shown at the top, the N-terminal domain and three repeated domains are indicated. S indicates the AcNPV 61K protein signal peptide.

Figure 3 is a graph of anti-CEA titer versus time in weeks after immunization showing the immunogenicity of recombinant and tumor-derived human CEA in outbred CD-I Mice. Mice were Groups of 8 CD-I outbred mice immunized with purified recombinant CEA (rCEA) in PBS or with Alum

adjuvant. Two additional groups of eight mice each were immunized with human CEA (huCEA) (Vitro

Diagnostics, Inc., Littleton, CO) in PBS or

Freunds' adjuvant. The mice were immunized

intraperitoneally on weeks 0, 1, and 12 with 5 μg antigen. Anti-CEA antibodies were measured by ELISA at the indicated time points and the

geometric mean antibody titers for each group of mice determined. The closed square symbol

represents rCEA in phosphate buffered saline (PBS); the open square symbol represents rCEA in Alum; the closed diamond symbol represents huCEA in PBS; and the open diamond symbol represents huCEA in

Freunds' adjuvant.

Figure 4A and Figure 4B are graphs of tumor volume in cubic millimeters versus time in days showing the antitumor activity of rCEA for the treatment of CEA-expressing tumors in mice. Groups of C57B mice were given 2 x 10⁵ nontransduced MC-38 or CEA transduced MC-32-CEA cells by subcutaneous injection. After seven days, the mice in each group were treated twice at two week intervals with 25μg purified rCEA in PBS, Alum, or STS-1 adjuvants (rCEA group) or with PBS, Alum, or STS-1 alone (adjuvant group). The size of the resulting tumors were measured twice weekly beginning on day 12. A student T test for independent variables and a two tailed analysis was used to measure the statistical significant (p) in tumor volume between the

adjuvant control and rCEA immunogen treatment groups. The closed square symbol represents mice treated with PBS or adjuvant; the open square symbol represents mice treated with the recombinant CEA in PBS or with adjuvant.

Figure 5 is a graph of the geometric mean anti- CEA titer versus weeks post immunization showing the immunogenicity of recombinant CEA and human CEA in mice. The black square symbol represents recombinant CEA. The open square symbol represents human CEA.

Figure 6 is a graph of tumor volume versus days post tumor challenge showing the antitumor activity of recombinant CEA immunogen for the treatment of CEA expressing tumors in mice. Groups of fifteen C57B mice were inoculated with 2 x 10⁵ CEA

transduced MC32-CEA cells by subcutaneous injection on day 0. One group was immunized with 25 μg doses of purified rCEA on day 7 then boosted with 25 μg rCEA on day 14. A second group was treated on the same schedule with a control preparation. The resulting tumors were measured twice weekly

beginning on day 12 and the geometric mean tumor volume determined. The black square symbol

represents the control. The open square symbol represents the mice treated with recombinant CEA.

Figure 7 is a graph of mean tumor volume versus days post tumor challenge showing prevention of CEA-expressing tumor growth by administration of recombinant CEA immunogen. Groups of ten C57B mice were immunized with 25 μg doses of purified

recombinant CEA immunogen or with a control

preparation on days 0 and 28. The mice were then inoculated with 5 x 10⁵ MC32-CEA cells on day 35. Any resulting tumors were measured twice weekly beginning on day 12 following the challenge with MC32-CEA cells and the geometric mean tumor volume determined. The black square symbol represents the control. The open square symbol represents the mice treated with recombinant CEA.

Detailed Description of the Invention

An immunogenic, soluble (i.e., not cell bound), recombinant carcinoembryonic antigen (rCEA) is provided. A method for the production of rCEA protein and method of treating cancer by

administration of the rCEA protein are also provided.

The recombinant CEA protein is an immunogenic, full length CEA glycoprotein having a molecular weight of approximately 120,000, or immunogenic fragments thereof. The recombinant CEA is

substantially glycosylated, and is soluble, or secretory, not membrane bound. The recombinant CEA is made soluble by deleting all or most of the transmembrane region so that the protein is not bound to the cell surface as it is in naturally occurring human CEA. The recombinant CEA is less glycosylated than the native, human CEA, which may allow more effective presentation of new epitopes to the immune system. As used herein,

"substantially glycosylated" refers to the extent of the glycans attached to the glycosylation sites on the amino acids, not to the number of sites which are glycosylated. Insect cells attach more simple glycans which do not have terminal sialic acids to the amino acids than do mammalian cells. Native or human CEA (huCEA) protein is defined herein as a protein having the amino acid sequence as described by Oikawa, et al. Biochem. Biophys . Res . Comm. 142:511-518 (1987), which includes a transmembrane domain and is glycosylated in mammalian cells. Preferably, the recombinant CEA is at least 95% pure. A method of producing rCEA at very high levels in insect cells, grown in serum-free medium, using a baculovirus expression system is described. Very high levels of production are defined herein as levels greater than 100 mg/L medium. A

purification process, in which the protein can be purified under non-denaturing conditions to 95% or greater purity, for large scale production of rCEA protein suitable for human administration is described. When produced in insect cells, the small quantities of impurities that may be present are baculovirus and insect cell proteins rather than tumor-derived contaminants. Accordingly, it may be possible to administer higher doses of this recombinant preparation without triggering adverse reactions.

The human CEA gene was cloned from tumor cells using specially designed oligonucleotide probes and polymerase chain reaction (PCR) methodology and modified for proper expression in insect cells as shown in Figure 1. The cloned CEA was modified by deletion of the natural hydrophobic signal peptide sequences, which is replaced with the following 18 amino acid baculovirus signal peptide:

Met Pro Leu Tyr Lys Leu Leu Asn Val Leu Trp Leu Val Ala Val Ser Asn Ala

The cloned CEA gene was also modified to remove all or substantially all of the transmembrane domain of the CEA protein by deleting most or all of the hydrophobic signal sequence at the terminus of the CEA gene so that the protein is not bound to the cell surface and allows secretion of the protein in infected insect cells. The chimeric gene is then introduced into a baculovirus

expression vector wherein the baculovirus

polyhedron promoter directs the transcription of the rCEA protein in infected insect cells. The baculovirus signal peptide directs translation of the rCEA into the insect cell glycosylation pathway and is not present on the mature rCEA glycoprotein. The recombinant CEA protein is then harvested and purified from cultures of insect cells, preferably Lepidopteran cells, following exposure to the recombinant baculovirus vector.

It will be understood by those skilled in the art that the method for producing full length CEA can be extended to produce immunogenic fragments of CEA and fusions of CEA with other peptides or proteins and is not restricted to the production of full length CEA.

A general approach for the efficient extraction and purification of recombinant CEA secreted from insect cells and formulations of recombinant CEA for administration to humans is described below. Immunity is measured using immunoassays that quantitate antibodies that bind to rCEA, human CEA extracted from tumors, and CEA expressed on the surface of human adenocarcinoma cells. Cellular immunity is measured using assays that measure specific T-cell responses such as delayed-type hypersensitivity (DTH) and lymphocyte

proliferation. Anti-tumor effects of immunizing with rCEA can be measured in an animal model using mouse tumor cells transformed with human CEA. A clinical protocol is provided to measure the safety, immunogenicity, and provide preliminary evidence of anti-tumor effects of administration of the rCEA immunogen to cancer patients.

A method for treating cancer patients by

administration of the recombinant CEA protein is described in more detail below. Baculovirus Expression System

Baculoviruses are DNA viruses in the family Baculoviridae. These viruses are known to have a narrow host-range limited primarily to Lepidopteran species of insects (butterflies and moths). The baculovirus Autographa calif ornica Nuclear

Polyhedrosis Virus (AcNPV), replicates efficiently in susceptible cultured insect cells. AcNPV has a double-stranded closed circular DNA genome of about 130,000 base-pairs and is well characterized with regard to host range, molecular biology, and genetics.

Baculoviruses form large protein crystalline occlusions within the nucleus of infected cells. A single polypeptide, referred to as polyhedrin, accounts for approximately 95% of the protein mass of these occlusion bodies. The gene for polyhedrin is present as a single copy in the AcNPV viral genome. Because the polyhedrin gene is not

essential for virus replication in cultured cells, it can be readily modified to express foreign genes. Recombinant baculoviruses that express foreign genes are constructed by way of homologous recombination between wild-type baculovirus DNA and chimeric plasmids containing the gene sequence of interest. Recombinant viruses can be detected by virtue of their distinct plaque morphology and plaque-purified to homogeneity.

Method of Producing Recombinant CEA

Cloning and Expression of CEA

Human Colon Adenocarcinoma (LS174T) cells (ATCC CL 188) were obtained from the American Type

Culture Collection (Rockville, MD). LS174T cells express CEA antigen. This cell line was used as a source for human CEA messenger RNA (mRNA). An overview of the cloning of the human CEA gene from the LS174T cells, modification of the gene and construction of an AcNPV expression vector is shown in Figure 1 and described below.

Isolation of mRNA from LS174T Cells

Cytoplasmic mRNA was extracted from the LS174T cells using standard procedures described in the well-known manual of Maniatis, T., E. Fritsch, and I. Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL. Cold Spring Harbor Laboratories, Cold Spring, N.Y. 1982. Purified mRNA was used as a template with an oligo dT primer to make cDNA utilizing Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase supplied in a First-Strand cDNA Synthesis Kit

(Pharmacia, Piscataway, NJ).

Double Stranded DNA PCR Amplification

The single stranded cDNA was used as a template for amplification of the CEA gene by the polymerase chain reaction (PCR) using the protocol recommended by the supplier (Gene Amp PCR cloning kit,

Cetus/Perkin Elmer, Norwalk, CT). The PCR reaction mixture (100 μl) contained 20 pmol of primers specific for 5' and 3' ends of the CEA gene

determined by sequences found in Genbank data files. The 5' and 3' primers were designed with restriction endonuclease sites at the ends that are not found within the CEA gene. The 5' PCR primer begins 81 base pairs from the 5' end of the gene, deleting the natural CEA signal peptide sequence. The 3' PCR primer was designed to delete the last 72 basepairs at the 3' end of the gene which codes for the hydrophobic C-terminal region sequence of CEA which is not present on mature CEA expressed in human tumor cells.

The primers are set forth below. The sequences of both primers are written in the 5' to 3'

direction. Restriction enzyme recognition sites for EcoRI, Smal , Kpnl, and BamRI are incorporated into the PCR product and are indicated below with single underlining. The region of the 5' primer which encodes the N-terminal end of mature CEA is indicated by an arrow. The 3' primer has a stop codon (reverse complement) at the location of the carboxyl terminus found in mature human CEA. The portion of each primer which anneals to the CEA cDNA is doubly underlined, which corresponds to nucleotides 81 (5' primer) through 2006 (3' primer) of the CEA sequence. The sequence of the 5' primer is set forth as Sequence Listing ID No. 1. The sequence of the 3' primer is set forth as Sequence Listing ID No. 2.

Amplification of the CEA gene fragment was carried out for 30 cycles each consisting of one minute of denaturation at 94°C, two minutes at 35°C for reanealing and two minutes at 72°C for

extension. The resulting amplified CEA gene fragment was electrophoresed, then purified from an agarose gel and cloned into a pTA plasmid cloning vector (Invitrogen, San Diego, CA) using standard cloning procedures described by Maniatis, et al., MOLECULAR CLONING: A LABORATORY MANUAL. Cold Spring Harbor Laboratories, Cold Spring, N.Y. 1982

Construction of a CEA Recombination Plasmid

The CEA gene was removed from the pTA plasmid with Sxπal and Kpnl restriction enzymes and then subcloned by standard procedures described by

Maniatis, et al. into a cloning vector pMGS12, as shown in Figure 1. The pMGS12 plasmid contains from 5' to 3' the AcNPV polyhedrin promoter, an ATG initiation codon, the sequence for a cleavable signal peptide from a 61,000 molecular weight baculovirus glycoprotein (61K), Smal and Kpnl restriction enzyme cloning sites, and a TAA

universal stop codon sequence. Flanking these regulatory regions is DNA from the EcoRII fragment from the AcNPV genome described by Summers, M.D. and Smith, G.E., "A manual of methods for

baculovirus vectors and insect cell culture

procedures." Texas Agricul tural Experimental

Station Bulletin No. 1555 (1987). The CEA PCR fragment was excised from the TA vector with Smal and Kpnl and cloned into Smal and Kpnl digested pMGS12. The resulting AcNPV recombination vector (pA9080) contained the coding region for mature CEA linked in frame with the cleavable baculovirus signal peptide from the 6IK gene and the polyhedrin promoter as shown in Figure 1. The 5' end of pA9080 was sequenced to confirm that the CEA gene fragment was in the proper location, orientation, and reading frame.

The CEA gene and flanking AcNPV DNA was

sequenced from the pA9080 AcNPV-CEA vector. The nucleic acid and amino acid sequences of the CEA- encoding portion pA9080 are set forth in Sequence ID Nos. 3 and 4 respectively. The twelve N- terminal residues of the mature recombinant CEA protein correspond to nucleotides 72 to 107 and have been confirmed by amino acid sequencing. The three N-terminal residues, which are encoded by the baculovirus vector (MGS12) and not present in human CEA, correspond to nucleotides 72 to 80. Other features indicated are: the seven-adenine motif in the polyhedrin mRNA leader (A₇, nucleotides 1-7); the portion of MGS12 encoding the AcNPV 61K protein signal peptide (nucleotides 18 to 71); restriction endonuclease sites at the 5' and 3' end of the CEA coding sequences (Smal (nucleotides 75 to 80), BamHI (nucleotides 2010 to 2015), Kpnl (nucleotides

2016 to 2021), Bglll (2022 to 2027); and the universal translation termination signal,

containing stop codons in all three reading frames, in the MGS12 vector (3-frame stop TAATTAATTAA).

Transfection and Selection of the

AcA9080 rCEA baculovirus Expression Vector

Wild type AcNPV DNA and recombination vector pA9080 containing the CEA gene were mixed, coprecipitated with calcium chloride, and Sf9 cells were transfected as described by Estes, M.K., et al., J. Virol . 61:1488-1494 (1987). Recombinant viruses were identified by their plaque morphology and several were plaque purified. A recombinant virus was identified that expressed rCEA in

infected Sf9 cells and the expression vector was purified by an additional round of plaque

purification. The resulting AcNPV expression vector, AcA9080, was used to produce recombinant

CEA in insect cells.

DNA Restriction Analysis and Southern Blot

Hybridization of AcA9080

DNA restriction analysis and Southern blot hybridization were used to demonstrate that the baculovirus expression vector AcA9080 contained the CEA gene in the correct location and orientation. The AcA9080 virions were purified by sucrose density centrifugation and the viral DNA was purified using standard methods (Summer and Smith, Texas Agricul tural Experimental Station Bulletin No. 1555 (1987).

Purified DNA was digested with the restriction enzymes BamHI, Bgrlll, Pstl, PvuzII and Sstl , electrophoresed on an 0.8% agarose gel, and then stained with ethidium bromide. The DNA fragments were transferred to nitrocellulose and hybridized to a ³² [P]-labeled Smal -Kpnl fragment containing the cloned CEA gene isolated from the plasmid pA8986.

The sizes of the expected CEA-gene fragments in the

AcA9080 expression vector are listed in Table 1 below. The size of CEA-gene fragments as measured from the Southern blot agree closely with the predicted sizes, demonstrating that the AcA9080 recombinant CEA expression vector contains a single copy of the CEA gene in the correct location and orientation.

DNA Sequence Analysis of rCEA

The identity of the cloned CEA gene was

determined by sequencing the gene excised from the recombination vector pAc9080. The CEA coding region contains three repeated domains which are highly similar at the DNA sequence level and care had to be taken when designing sequencing primers to avoid those which anneal at multiple sites within the gene. Using the primer design program included in a commercial software package

(MacVector, International Biotechnologies, Inc., New Haven, CT), primers were designed with the appropriate lengths, G-C contents, and melting temperatures for automated thermocycle DNA

sequencing. The primers were designed that annealed to sites of optimal mismatch between the CEA repeats and were approximately 200 bases apart, and staggered between the two DNA strands as shown above and set forth in Sequence Listing ID Nos. 1 and 2.

Sequencing primers were made with an

oligonucleotide synthesizer (Gene Assembler,

Pharmacia, Piscataway, NJ) according to the

instructions of the manufacturer, then purified using Oligonucleotide Purification Columns (Applied Biosystems, Inc., Foster City, CA). Sequencing reactions were performed using a Taq DyeDeoxy™ Terminator Cycle Sequencing kit (Applied

Biosystems, Inc.), and reaction products were analyzed and data was collected with an ABI Model

373™ DNA Sequencer (Applied Biosystems, Inc.). The resulting sequence data was assembled using the AssemblyLign™ software package (International

Biotechnologies, Inc., New Haven, CT). For the final consensus sequence, it was required that at least three sequence runs span all regions and including at least one run in each direction. The DNA sequence for the CEA gene in the baculovirus vector pAc9080 is identical to the sequence for human CEA set forth as Sequence ID No. 3, below.

The nucleic acid sequence encoding the recombinant CEA therefore is the sequence set forth in Sequence ID No. 3 or degenerate variations thereof. The nucleic acid sequence of the recombinant CEA protein is defined herein as a nucleic acid

sequence that will hybridize to the sequence set forth in Sequence ID No. 3, encodes the amino acid sequence set forth in Sequence ID No. 4, hybridizes to the nucleic acid sequence encoding the amino acid sequence set forth in Sequence ID No. 4, and a sequence having conservation amino acid

substitution.

Purification of Recombinant CEA

Recombinant CEA is purified from insect cell culture supernatant using a procedure involving a low-pH viral inactivation step followed by four chromatography columns. The chromatography resins employed are hydrophobic interaction (HIC) Ether 650M™, HIC Butyl 650M™, Lentil Lectin Sepharose 4B™, and DEAE Sepharose Fast Flow™. (Pharmacia, Piscataway, NJ). Separation is based on the solubility, hydrophobicity, glycosylation and ionic properties of rCEA. The two-day purification procedure is carried out at 12°C to 16°C and yields 95% or greater pure rCEA.

Step 1: Virus Inactivation:

Insect cells, Sf900+ are infected with the rCEA expression vector AcA9080. Culture supernatant is harvested 3 days post-infection. Baculovirus in the culture fluid is inactivated by acidifying the supernatant with acetic acid. After a one hour incubation at pH 3.5, the supernatant is

neutralized to pH 7.0 and filtered to remove precipitated viral and cellular proteins. rCEA remains soluble throughout this step and represents one of the major remaining polypeptides. The remainder of the purification process is carried out at neutral pH.

Step 2: Ether/Butyl Hydrophobic Interaction Chromatography:

The rCEA-containing supernatant is pH-adjusted with 1.4 M ammonium sulfate and applied directly to linked Ether/Butyl Hydrophobic Interaction

Chromatography columns equilibrated in a pH 7 sodium phosphate buffer containing 1.4 M ammonium sulfate. rCEA flows through the Ether column and binds to the Butyl matrix. The linked Ether/Butyl columns are washed for two bed volumes with the above phosphate/ammonium sulfate buffer; the Ether column is taken off-line; the Butyl column is washed with an additional six bed volumes; and then the Butyl column is eluted with 10 mM sodium phosphate buffer at pH 7.0. The 120 K rCEA is the major protein in the Butyl column eluate as

demonstrated by gel electrophoresis.

Step 3: Lentil Lectin Affinity Chromatography: The Butyl column eluate is applied directly to a Lentil Lectin Sepharose 4B™ column equilibrated in neutral phosphate buffer containing 500 mM NaCl. rCEA quantitatively binds to the column while nonglycosylated species pass through. To remove nonspecifically bound species, including nucleic acids, the column is washed with 10 column volumes of the above high-salt phosphate buffer. rCEA is recovered by eluting the column with neutral phosphate buffer containing 300 mM alpha-methyl-D- mannoside. The lectin column eluate is diverted directly onto a DEAE column equilibrated in 10 mM sodium phosphate buffer at pH 7.0.

Step 4: DEAE Ion Exchange Chromatography:

This final DEAE ion exchange polishing column removes residual lentil lectin and several minor contaminating proteins, effects buffer exchange, and concentrates the rCEA for bulk storage. rCEA binds to the DEAE matrix, which is then washed with ten bed volumes of neutral phosphate buffer. The column is eluted with 10 mM sodium phosphate buffer containing 150 mM NaCl, and rCEA is recovered in a single sharp peak at greater than 95% purity as shown by an electrophoretic gel. The purified bulk protein is stored at 2°C to 8°C in the absence of preservatives. Bulk CEA antigen is stable for at least six months as assessed by SDS-polyacrylamide gel electrophoresis.

Biochemical Characterization of rCEA

Molecular Weight and Size.

Purified rCEA migrates predominantly as a single major polypeptide of 120,000 molecular weight

(120K) on an SDS-polyacrylamide gel stained with the stain, Coomassie blue. The 120K rCEA is also the major antigen reactive on a Western blot using a commercial source of antisera against tumor- derived human CEA. The major polypeptide from commercial sources of human CEA migrates at 180,000 molecular weight. As described below, the

recombinant CEA is smaller due to attachment of more simple glycan to the CEA protein when produced in insect cells.

Purity Determination.

The rCEA is measured for purity on SDS

polyacrylamide gels stained with Coomassie blue using a scanning laser densitometry (Ultroscan XL™, Pharmacia-LKB Instruments, Piscataway, NJ) and peak integration analysis. With 10 μg of purified rCEA per lane and a sensitivity of detection of 0.1 μg protein, insect cellular and baculovirus protein contaminants present at greater than or equal to 1% can be detected.

Glycosylation of rCEA.

To characterize the sugars present on the glycosylated recombinant CEA, purified preparations of rCEA were analyzed with glycosidase. Peptide-N- glycosidase F (PNGase F) removes any N-linked oligosaccharide side chains and is useful for estimating the total extent of N-linked

glycosylation. Endoglycosidase H (endo H) is specific for the high-mannose oligosaccharides which are added in the endoplasmic reticulum. Trimming of Man₅GlcNAc₂ to Man₃GlcNAc₂, which is known to occur in a medial compartment of the Golgi apparatus of insect cells, confers resistance to digestion with endo H.

Purified recombinant CEA was compared to a commercial source of CEA (huCEA) purified from human liver metastasis of colon adenocarcinomas (Vitro Diagnostics, Inc., Littleton, CO). The rCEA and huCEA antigens were first denatured by boiling for three minutes at 5 mg/ml in 2% SDS, 1% β- mercaptoethanol. The denatured glycoproteins were treated with glycosidase as follows. For PNGase F digestion, aliquots of each protein were diluted 10-fold, using a concentrated buffer stock and distilled water, into 20 mM sodium phosphate, pH 7.5, 50 mM EDTA, 0.02% sodium azide, 2% Tween-20, and incubated for 18 hours at 37°C with 50 units/ml enzyme. A mock-digested control was prepared and incubated as above without enzyme. The reactions were stopped by mixing each sample with an equal volume of 2X SDS-PAGE sample disruption buffer and boiling for five minutes to denature the proteins. Endo H digestion was performed in the same manner, except that the incubation buffer contained 50 mM sodium citrate, pH 5.5, 0.02% sodium azide, and 2.5% Tween-20, and the enzyme was used at 0.1 units/ml. 2.5 micrograms of each protein were electrophoresed on 11% SDS-polyacrylamide gels. The proteins were then either stained with

Coomassie blue or transferred to nitrocellulose and blotted with rabbit anti-human CEA. Control glycoproteins, which had also been treated with each enzyme as described above, were

electrophoresed along with the CEA samples, and demonstrated that the incubation conditions used resulted in specific cleavage of the glycoprotein standards.

A Coomassie blue stained polyacrylamide gel and Western blot with rabbit anti-CEA antisera

comparing PNGase F and endo H digestion of rCEA and huCEA were prepared. When treated with PNGase F which removes any N-linked sugar, rCEA and huCEA deglycosylated polypeptides are both approximately 79,000 molecular weight. Thus, the difference in sizes between recombinant and native CEA can be attributed to differences in glycosylation. The slight difference in mobilities may be attributable to the phosphatidylinositol which is bound to the carboxyl terminus of huCEA but would be absent from the rCEA if the signal for glycolipid addition was removed as a part of the cloning strategy. Human CEA is known to contain complex carbohydrates and is, as expected, completely resistant to endo H cleavage. Recombinant CEA expressed in insect cells, migrates at an intermediate molecular weight after endo H treatment, indicating that while some of the carbohydrate on the molecule is endo H sensitive, a substantial proportion has been matured to the point of endo H resistance during the passage of this secreted protein through the Golgi.

As evident from both the stained gel and Western blot, the recombinant CEA antigen is more

homogeneous than CEA extracted from tumor tissues, making rCEA a better defined product than the natural CEA preparations. The rCEA is also free of contaminants that would be in CEA from tumor tissues. Thus, rCEA immunogen is a safer and a more consistent product for use as a cancer

immunotherapeutic. Amino Acid Analysis.

Purified rCEA was precipitated with

trichloroacetic acid, and hydrolyzed in 6 N HCl, 0.01% phenol in a nitrogen atmosphere at 110°C for 20 hours. Table 2 shows the expected amino acid composition of rCEA predicted from the DNA sequence of rCEA compared to measured values.

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The measured amino acid composition of rCEA is in good agreement with the composition predicted from the DNA sequence of the rCEA gene.

Amino Terminal Sequence of rCEA.

A sample of purified rCEA produced from the expression vector AcA9080 was precipitated with trichloroacetic acid and analyzed by gas phase sequence analysis. The single amino terminal sequence was obtained and the first 13 residues are:

NH₂-Ile-Pro-Gly-Lys-Leu-Thr-Ile-Glu-Ser-Thr-Pro-Phe-Asn

The first three residues are predicted from the cloning strategy and the remaining are identical to the amino acids at the amino terminus of mature CEA (Oikawa, et al., Biochem. Biophys. Res. Comm.

142:511-518 (1987)). This demonstrates that purified rCEA has a single, unblocked amino

terminus and that the baculovirus 61K signal peptide is, as expected, not present on mature rCEA produced in insect cells.

Safety, Immunogenicity and Anti-tumor

Properties of rCEA

Animal Safety Studies.

Data from animal studies indicate that rCEA is free of significant toxicity. There are no

detectable toxic agents in the product, and the cell line used for its production has been shown to be non-tumorigenic. The recombinant CEA immunogen has been well tolerated in mice, guinea pigs and rabbits. No serious adverse reactions or

hypersensitivity reactions attributable to the vaccine were observed in guinea pigs inoculated intraperitoneally with up to 5 mg of rCEA, or in mice inoculated intraperitoneally with up to 500 μg of rCEA.

Antigenic and Immunogenic Properties of rCEA. An effective rCEA immunogen would be expected to induce immune responses against CEA-expressing tumor cells. Studies were done to demonstrate that rCEA shares T and B-cell determinants in common with CEA expressed on tumor cells. The serological properties of rCEA were studied using Western blot and ELISA analyses, immunogenicity studies in mice, competitive binding studies using an

Immunofluorescence (IF) assay and human

adenocarcinoma cells expressing CEA, and a delayed type hypersensitivity (DTH) assay.

Western Blot Analysis of rCEA

A commercial preparation of rabbit antisera prepared against CEA extracted from human tumor tissue was highly reactive with rCEA on Western blots. Rabbit anti-human CEA reacted strongly with the major 120K rCEA glycoprotein and also with the 79K deglycosylated form of rCEA. Antisera prepared against the recombinant CEA made in mice also reacts strongly with both the recombinant and tumor-derived CEA. These results demonstrate that rCEA and huCEA have common B-cell epitopes.

Formulation and Packaging

The rCEA immunogen can be formulated and

packaged, alone or in combination with other antigens, using methods and materials known to those skilled in the art for vaccines.

In a preferred embodiment, the rCEA immunogen is administered without a compound, such as bovine serum albumin. It is believed that the lack of a carrier may reduce the antibody response and enhance the T cell response. The rCEA with or without a carrier is preferably combined with an adjuvant, in an amount effective to enhance the immunogenic response against the CEA protein. At this time, the only adjuvant widely used in humans has been alum (aluminum phosphate or aluminum hydroxide). Saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary

applications have toxicities which limit their potential use in human vaccines. However,

chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman- Snitkoff et al. J . Immunol. 147:410-415 (1991) and incorporated by reference herein, encapsulation of the protein within a proteoliposome as described by Miller et al., J. Exo. Med. 176:1739-1744 (1992) and incorporated by reference herein, and encapsulation of the protein in lipid vesicles such as Novasome™ lipid vesicles (Micro Vescular

Systems, Inc., Nashua, NH) should also be useful.

rCEA-Alum Immunogen Formulation.

A formulation of rCEA antigen at the specified concentration in a phosphate buffered saline solution and adsorbed onto aluminum phosphate

(Alum) is as follows:

rCEA-Alum Immunogen:

Recombinant CEA protein (at the protocol- specified concentration)

Aluminum phosphate (A1P04), 0.5 mg/l .0 ml dose

Sodium phosphate, 10 mM

Sodium chloride, 150 mM

The aluminum phosphate is prepared immediately prior to protein addition by mixing solutions of aluminum chloride, sodium acetate and sodium phosphate. After the alum precipitate forms, the mixture is adjusted to pH of 7.0 ± 0.2 with sodium hydroxide, and the required volume of purified rCEA is then added. The protein is allowed to adsorb to the alum with constant mixing to ensure

homogeneity.

Methods of administration

In the preferred embodiment, the vaccine is packaged in a single dosage for immunization by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal

(i.e., intranasal) administration. The immunogen is most preferably injected intramuscularly into the deltoid muscle. The immunogen is preferably combined with a pharmaceutically acceptable carrier to facilitate administration. The carrier is usually water or a buffered saline, with or without a preservative. The antigen may be lyophilized for resuspension at the time of administration or in solution. The carrier may also be a polymeric delayed release system. Synthetic polymers are

particularly useful in the formulation of a vaccine to effect the controlled release of antigens. An early example of this was the polymerization of methyl methacrylate into spheres having diameters less than one micron to form so-called nano

particles, reported by Kreuter, J., Microcapsules and Nanoparticles in Medicine and Pharmacology, M. Donbrow (Ed). CRC Press, p. 125-148. The antibody response as well as the protection against

infection with influenza virus was significantly better than when antigen was administered in combination with aluminum hydroxide. Experiments with other particles have demonstrated that the adjuvant effect of these polymers depends on particle size and hydrophobicity.

Microencapsulation has been applied to the injection of microencapsulated pharmaceuticals to give a controlled release. A number of factors contribute to the selection of a particular polymer for microencapsulation. The reproducibility of polymer synthesis and the microencapsulation process, the cost of the microencapsulation

materials and process, the toxicological profile, the requirements for variable release kinetics and the physicochemical compatibility of the polymer and the antigens are all factors that must be considered. Examples of useful polymers are polycarbonates, polyesters, polyurethanes,

polyorthoesters and polyamides, particularly those that are biodegradable.

A frequent choice of a carrier for

pharmaceuticals and more recently for antigens is poly (d,l-lactide-co-glycolide) (PLGA). This is a biodegradable polyester that has a long history of medical use in erodible sutures, bone plates and other temporary prostheses, where it has not exhibited any toxicity. A wide variety of

pharmaceuticals including peptides and antigens have been formulated into PLGA microcapsules. A body of data has accumulated on the adaptation of PLGA for the controlled release of antigen, for example, as reviewed by Eldridge, J.H., et al.

Current Topics in Microbiology and Immunology.

1989, 146: 59-66. Antigens entrapped in PLGA microspheres of 1 to 10 microns in diameter has been shown administered orally. The PLGA

microencapsulation process uses a phase separation of a water-in-oil emulsion. The compound of interest is prepared as an aqueous solution and the PLGA is dissolved in a suitable organic solvents such as methylene chloride and ethyl acetate.

These two immiscible solutions are co-emulsified by high-speed stirring. A non-solvent for the polymer is then added, causing precipitation of the polymer around the aqueous droplets to form embryonic microcapsules. The microcapsules are collected, and stabilized with one of an assortment of agents (polyvinyl alcohol (PVA), gelatin, alginates, polyvinylpyrrolidone (PVP), methyl cellulose) and the solvent removed by either drying in vacuo or solvent extraction.

The recombinant CEA protein is preferably administered to cancer patients, particularly those having CEA-expressing tumors, including those having breast cancer, lung cancer, and colon cancer. Most preferably,, the recombinant CEA protein is administered to post-surgical Duke's stage B2 or C colon cancer patients. An effective amount of the recombinant CEA is administered to the patients. An effective amount is an amount sufficient to induce an immune response and have an anti-tumor effect.

The preferred dose is a 1 ml injection

containing between 100 and 3000 μg of rCEA

immunogen on day 0, followed by booster injections of the same amount at months 1, 2, 4, 6, 8, 10, and 12. The immunogen preferably contains no

preservative. The immunogen should be refrigerated in sealed vials and used within 24 hours after opening. Vials of rCEA immunogen should be kept at 2°C to 8°C (35.6°F to 46.4°F). Storage above or below the recommended temperature may reduce potency. The rCEA immunogen should be mixed by gently agitating the vial in which the immunogen is stored prior to loading the syringe for

administering an injection.

The present invention will be further understood by reference to the following non-limiting

examples.

Example 1

Immunogenicity Studies

Antibody binding studies have demonstrated that the rCEA protein is correctly folded and presents native conformational epitopes to the host immune system. In these studies, antibodies were

generated against both rCEA and huCEA (human tumor- derived CEA, used as a control), and the ability of each of the proteins to block the homologously and heterologously stimulated antibodies from binding to CEA on the surface of human tumor cells (LST174 cells) was measured. Both tumor-derived huCEA and rCEa are able to block binding of both the anti- rCEA and the anti-huCEA antibodies to the LST174 cells, in a dose-dependent fashion. DTH studies reveal that rCEA induces a vigorous T cell response in mice. CD-1 mice received intraperitoneal injections of 5 μg doses of rCEA or huCEA on weeks 0, 4 and 12. Fourteen days after the last injection, the animals received by foot pad injection either rCEA or huCEA in one foot and a control glycoprotein (HIV-1, gp160) in the opposite foot. Specific DTH responses were

produced in the mice immunized with rCEA that were detected with either rCEA or huCEA.

An experimental animal tumor model has been developed based on murine colon carcinoma cells (MC-38) transduced with the human CEA gene (Irvine, K., et al., "Comparison of a CEA-recombinant vaccinia virus, purified CEA, and an anti-idiotype antibody bearing the image of a CEA epitope in the treatment and prevention of CEA-expressing tumors." Vaccine Res . , 2:79-94, 1993) . CEA is expressed on the surface of the transduced cells (MC32-CEA) at levels that are comparable to that of human colon carcinomas. The CEA-transduced and non-transduced parent cells produce subcutaneous tumors in C57BL/6 mice. Example 2

Immunofluorescence and Competitive Binding

CEA is expressed on the surface of the human adenocarcinoma tumor cells LST174. This experiment was performed to measure the ability of antisera prepared against purified recombinant and tumor- derived CEA to bind to LST174 cells and to measure the ability of each of the proteins to block binding.

Serial dilution of rabbit anti-human CEA and mouse anti-recombinant CEA were tested for binding against the CEA-positive LST174 cells. Cells were plated at a density of 4 x 10⁶ cells/chamber in 200 μl of tissue culture medium in 8-chamber microscope slides. Antisera prepared against rCEA and tumor- derived CEA bind to the LST174 cells.

Tumor-derived huCEA was able to block binding of both the anti-rCEA and the anti-huCEA antibodies to the LST174 cells in a dose-dependent fashion.

Also, rCEA blocked the binding of anti-rCEA and anti-huCEA to the cells in a dose-dependent

fashion.

These results demonstrated that rCEA immunogen induces antibodies that recognize CEA-positive human tumor cells and that the rCEA protein shares epitopes in common with CEA expressed on human tumor cells.

Example 3

Immunogenicity of rCEA in Small Animals

In this experiment, the ability of purified rCEA, alone or with Alum adjuvant, to elicit antibody responses was measured in mice, guinea pigs, and rabbits. Specific anti-CEA IgG antibody titers were measured using a standard dilutional ELISA.

Briefly, 96 well microtiter plates were coated with 100 ng/well of purified rCEA. Serial two-fold dilutions of serum samples beginning at 1:100 were made and the plates incubated for one hour at 37°C. The plates were washed, and anti-CEA IgG antibody detected with a goat anti-mouse IgG-HRP conjugate. The antibody titer is the dilution of serum that gives an OD₄₅₀ of greater than or equal to 0.100 and within the linear response of the assay.

An example of the immunogenicity of rCEA in CD-I outbred mice is shown in Figure 3. Groups of eight CD-I mice were immunized with purified rCEA in PBS or formulated with an aluminum phosphate adjuvant (Alum) containing 0.5 mg/ml Al⁺³ and adjusted to neutral pH. Two additional groups of eight CD-1 mice were immunized with a commercial source of purified human CEA (huCEA) derived from liver metastasis of colon carcinoma (Vitro Diagnostics, Inc., Littleton, CO). One group received huCEA in PBS and the second was given an initial injection of huCEA mixed with complete Freunds' adjuvant then boosted with huCEA in incomplete Freunds'. The protein concentration of rCEA and huCEA was

determined using a modified Lowry protein assay (BCA; Pierce Chemical Co., Rockford, IL). The protein concentration of huCEA was approximately half of what was claimed by the supplier, but all studies were done using the actual measured

concentration of huCEA. Immunizations were

performed intraperitoneally (IP) on weeks 0, 1 and 12 with 5 μg of rCEA or huCEA immunogens in PBS or with adjuvants. Serum specimens were taken at weeks 0, 4, 6, 8, 10, 12 and 13 for analysis.

Figure 3 shows the geometric mean titers of anti-CEA IgG antibody for each group of mice.

Recombinant CEA induced anti-CEA antibody titers in excess of 10⁶ after one immunization and one boost when mixed with Alum and after a second booster at week 12 in PBS. The tumor-derived CEA in PBS was not as immunogenic as rCEA in PBS and huCEA mixed with Freunds' adjuvant generated similar titers as the rCEA with Alum. Together these data

demonstrate that rCEA is more immunogenic in mice than commercial huCEA and that Alum adjuvant increases the immunogenicity of recombinant CEA. Example 4

Cellular Immunity of rCEA in Small Animals

Delayed-type hypersensitivity (DTH) studies were conducted in mice to demonstrate that rCEA can induce a vigorous T cell response.

CD-1 outbred mice received a primary and booster intraperitoneal injections of 5 μg doses of rCEA or huCEA, with and without alum or other adjuvants, on weeks 0, 4 and 12. Fourteen days after the last injection, the animals received by foot pad

injection either rCEA or huCEA in one foot and a control glycoprotein (HIV-1, gp160) in the opposite foot. Specific DTH responses were produced in the mice immunized with rCEA that were detected with either the recombinant or tumor-derived CEA

proteins. The adjuvants alum and Freund's

increased the magnitude of these responses. These data demonstrate that significant cellular immune responses are generated in rCEA immunized mice that recognize both the recombinant and human CEA antigens, and that these responses can be increased by using alum adjuvant.

Antitumor Effects of rCEA.

An experimental animal tumor model has been developed which is based on murine colon carcinoma cells (MC-38) transduced with human CEA gene

(Robbins, P.F., et al., Cancer Res . 51:3657:3662 (1991)). CEA is expressed on the surface of the transduced cells (MC-32-CEA) at levels that are comparable to that of human colon carcinomas. The CEA-transduced and non-transduced parent cells grow in C57BL/6 mice when administered subcutaneously and led to large tumors within 2-8 weeks. MC-38 and MC-32-CEA cells were supplied by the laboratory of Dr. Jeffery Schlom (National Cancer Institute, Bethesda, MD). The cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum.

The anti-tumor effects of rCEA were studied using a human CEA-expressing mouse tumors cell model following procedures developed by Irvine, et al., Vaccine Res . , 2:79-94, (1993). In a

preliminary study, sixty C57BL/6 mice were given by subcutaneous injection, 2 x 10⁵ MC-38 or MC-32-CEA cells. The thirty mice in each group were then randomized into six groups of five mice per group and immunized IP on days 7 and 21 with 25 μg rCEA in PBS, Alum, or an experimental oil-water emulsion adjuvant (STS-1); with PBS, Alum, or STS-1 alone. Tumors were measured by caliper in two dimensions, and the volumes were calculated using the formula (width² x length) /2. The growth of tumors in the mice have been followed for 26 days post tumor cell challenge and a summary of the results are shown in Figures 4A and 4B. The non-CEA transduced cells (MC-38) grow rapidly with mean tumor masses of greater than or equal to 3,500 mm³ in all the mice. Immunization of mice given the parent mouse tumor cells with rCEA had no significant anti-tumor effect relative to the adjuvant groups (p=0.677) and the animals were terminated on day 26 due to the excessive size of most tumors. The CEA- expressing carcinoma cells (MC-32-CEA) are slower growing and it has been reported that it takes 30- 40 days for tumors to reach greater than or equal to 1,000 mm³ (Irvine, et al., 1993). At 36 days the mean size of the MC-32-CEA tumors is 1032 mm³ in 14 mice given adjuvant alone and 76 mm³ in 15 mice immunized at day 7 and 21 with rCEA. The tumors in three of the 29 mice treated with rCEA immunogen disappeared and the mice have remained tumor free. This study demonstrates that immunization with rCEA induces an anti-tumor effect in mice with

established CEA-expressing tumors.

Example 5

Immunogenicity of rCEA in Mice

The immunogenicity of rCEA was studied in mice, guinea pigs and rabbits. Groups of eight outbred

CD-I mice were immunized with 5 μg doses of

purified recombinant CEA (rCEA) or purified human tumor-derived CEA (huCEA) on weeks 0, 4, and 12.

Serum samples were collected at the indicated times and the anti-CEA antibody titers measured by standard ELISA assay. The geometric mean titers were calculated for each time point.

CEA antibody titers of approximately 1:1,000,000 were elicited following three injections with the rCEA immunogen as shown in Figure 5.

Example 6

Effect of rCEA Immunization on Established Tumors

In a study to measure the effect of rCEA

immunization on established tumors, mice were first inoculated with 2 x 10⁵ MC-38 or MC32-CEA cells subcutaneously. The tumors were allowed to

establish for one week. Half the animals were immunized with 25 μg doses of rCEA immunogen (rCEA group) and the other half with a control

preparation (control group). Two weeks later the mice were given a booster immunization with 15μg doses of rCEA or control. Tumor volumes were measured by caliper in two dimensions beginning at day 12, then twice weekly. The volume of tumors in the mice immunized with control alone or rCEA through day 36 is summarized in Figure 6.

The MC38 mouse carcinoma cells grow rapidly and produced tumors with a mean volume of greater than or equal to 2,000 mm³ by day 26. The CEA-expressing MC32-CEA cells are slower growing and reportedly take 40-50 days to produce large tumors (Irvine, et al., 1993). Immunization with rCEA or the control preparation had no effect on the growth of the non- CEA expressing MC38 cells in the mice. In

contrast, there was a marked reduction in the growth of MC32-CEA tumors following immunization with purified rCEA (Figure 6). Example 7

Effect of rCEA Immunization on Prevention of

Carcinoma Cell Growth

A study was conducted to investigate the ability of the rCEA immunogen to prevent the growth of CEA- transduced carcinoma cells in C57BL/6 mice. Groups of 10 mice were immunized with the 25 μg doses of purified rCEA or with a control preparation on days 0 and 28. The mice were then inoculated with 5 x 10⁵ MC32-CEA cells on day 35. Tumors formed rapidly in the control group with a geometric mean tumor volume of greater than or equal to 1,000 mm³ 36 days following challenge with CEA-transformed tumor cells, as shown in Figure 7. The mice immunized with rCEA prior to challenge with MC32-CEA cells remained essentially free of measurable tumors.

Modifications and variations of the methods and compositions described herein will be obvious to those skilled in the art. Such modifications and variations are intended to come within the scope of the appended claims.

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Claims

We claim :

1. A soluble, immunogenic, recombinant

carcinoembryonic antigen protein, substantially free of the transmembrane domain.

2. The protein of claim 1 expressed in a baculovirus expression system.

3. The protein of claim 1 in a pharmaceutically acceptable carrier for administration to a patient.

4. The protein of claim 1 in a pharmaceutically acceptable carrier and further comprising an adjuvant for administration as a vaccine.

5. The protein of claim 1 having a molecular weight of approximately 120,000.

6. The protein of claim 2 wherein the protein glycosylated with more simple glycans than isolated human carcinoembryonic antigen.

7. The protein of claim 6 wherein a hydrophobic signal peptide sequence at a terminus of the carcinoembryonic antigen is deleted.

8. The protein of claim 1 wherein transcription of the carcinoembryonic antigen gene is directed by the baculovirus polyhedron promoter.

9. The protein of claim 1 encoded by a nucleic acid sequence hybridizing to a nucleic acid

sequence encoding the amino acid sequence of

Sequence ID No. 4.

10. The protein of claim 1 wherein the

carcinoembryonic antigen protein is human

carcinoembryonic antigen protein.

11. A method for making a recombinant

carcinoembryonic antigen protein comprising

infecting cultured insect cells with a vector containing the following 5'->3' sequences: a polyhedrin promoter from a baculovirus, a

baculovirus signal peptide, and the coding sequences for soluble carcinoembryonic antigen, and culturing the cells in serum-free media.

12. The method of claim 11 further comprising isolating the carcinoembryonic antigen protein from insect cells to a purity of at least 95%.

13. The method of claim 11 wherein the

carcinoembryonic antigen protein is human

carcinoembryonic antigen protein.

14. The method of claim 11 wherein the

carcinoembryonic antigen protein is not

glycosylated with sialic acid.

15. A method for inhibiting tumor growth comprising administering to a patient having cancer or having the potential to develop cancer an effective amount of a soluble, immunogenic, recombinant carcinoembryonic antigen protein, substantially free of the transmembrane domain.

16. The method of claim 15 wherein the protein is administered in a pharmaceutically acceptable carrier.

17. The method of claim 16 wherein the protein is administered in combination with an adjuvant.

18. The method of claim 15 wherein the

carcinoembryonic antigen protein is human

carcinoembryonic antigen protein.

19. The method of claim 15 wherein the

carcinoembryonic antigen protein is not

glycosylated with sialic acid.

20. The method of claim 16 wherein the cancer is a CEA-expressing tumor cancer selected from the group consisting of breast cancer, lung cancer and colon cancer.