A mutation in these tumor suppressor genes and/or proto oncogenes in the cell, result in an unusually high rate of cell proliferation leading to the eventual appearance of the tumor cell [1]. Prior to this however, constant mutations are continually occurring in a wide array of superficial cells such as mucosa of bowel. This occurrence represents what one usually considers a field effect within the bowel mucosa probably induced by a virus such as the polyoma [2]. While most if not all of the cells within the field induced by such viral transformation appear phenotypically normal, should one employ immunohistochemistry (IHC) to define expression of existing tumor protein, those altered cells can now easily be defined. Such alterations represent the appearance of post translational modifications in the existing oncofetal proteins that characterize that specific type of malignancy. Here, one can see for the first time, sites of the expression of tumor protein within these normal appearing cells [3].

As one site of focus in the mucosa proliferates somewhat more rapidly than other adjacent sites, inhibitory factors are produced in the more advanced cell group keeping the other foci normal in their phenotypic appearance. This is probably the reason one rarely sees more than one clinically malignant growth (lesion) within a large segment of bowel. Removal of this particular growth is now the initiating factor for allowing any residual focus of atypia, after colectomy has been performed, to possibly reappear. Such as new growth usually expresses itself in the form of an anastomotic recurrence. Knudson [4] described the resulting transforming cell as exhibiting early signs of aberrant growth such as altered morphology or unusually large size (hyperplasia). Developing tumor cells also tend to proliferate at a higher than usual rate to form that lesion characterized as either a benign tumor (dysplasia) or with the addition of further oncogene mutations, the malignant or cancerous growth. In later stages of cancer progression, such tumor cells proliferate at an unusually high rate, resulting in uncontrolled growth of the existing tumor.

In addition to progression and invasion of surrounding structures, the malignant lesion exhibits immortality as noted above. When we originally looked at the transcriptional protein FGARAT, it was apparent that this molecule, when overexpressed, is capable of activating the telomerase enzyme within the tumor system. Here, as the tumor grows, loss of a telomere at the time of cell division would normally shorten the chromosomal length until eventually the cell could  no  longer  divide  and  as  such  reach its  end state of development. In the presence of a functional enzyme system, induced by FGARAT, the chromosome cap is replaced and immortality of the cell is established. In looking for potential sites of telomerase replacement, a homologous sequence to FGARAT has been found in the capsule of the EBV virus. With this virus being almost universally found in most tissue of the body, it’s not difficult to see how the virus helps the cancer cell achieve what is necessary for long term survival or immortality not seen in the benign lesion which is unable to maintain its telomere length.

In addition to immunogenic expression of antigens in colorectal cancer depicted by the presence of a post translational modification of MUC5ac as the primary immunogenic antigen, the initial HPLC of Hollinshead [10] antigen revealed that there were at least 2 additional and important immunogenic proteins to consider for possible vaccine therapy. These are 16C3 (oncofetal) antigen which proved to be a variant of CEAcam-5,6 and 31.1 which was derived from the antigen present in colon cancer first noted by Olds, that is A33.

Nature of the 16C3 antigen

As previously seen in the HPLC analysis of pooled colon tumor membrane antigen, the second most common antigen that we could identify in the malignant lesion was a combination of both CEAcam-5 and CEAcam-6 which comprised additional examples of cancer-specific antigens (Tumor Immunogens) expressed in colorectal and pancreatic cancer. This particular carcinoembryonic antigen (CEA) gene family is a member of the Ig Cam superfamily which includes 29 related genes and pseudogenes. These CEA proteins function as intercellular hemophilic and lipid rafts in the apical membrane of the polarized epithelial cells. CEAcam5 and CEAcam6 bind to a variety of gram-negative bacteria and mediate internalization/phagocytosis, participating in innate immune defense within the intestine [19,20].

CEAcam5 and CEAcam6 are overexpressed in many cancers (e.g. breast, ovarian, colon, pancreatic, lung and prostate). CEACAM5 and CEACAM6 are believed to be involved in cell adhesion, cellular invasiveness, resistance to anoikis (a form of programmed cell death) and metastatic behavior of tumor cells [21,22].

The 16C3 antibody (Neo-201) that we developed to target the corresponding colon immunogen is defined in US Patent Application No. 2009/0162931, but the nature of the antigen targeted by the mAb was not described at the time. To identify the 16C3 antigen sequence, several protein purification processes were utilized using either murine or humanized 16C3 mAb. The tumor antigen sources for these protein extracts were tumor cell lines, including LS174 (human colorectal tumor), CFPAC-1 (human pancreatic tumor) and cancer vaccine from the Hollinshead [23] library of cancer vaccines.

16C3 antigen, the second most commonly identified oncofetal protein, acts at a suboptimal level as one of the common tumor antigens in colon cancer. It was defined by coupling the antibody derived from the crude form of 16C3 to resins for needed antigen purification. This included magnetic beads for simple adsorption, washing and then elution from the beads. Proteins obtained from the beads were studied for determination of antigen presence, followed by their characterization and further identification.

Those proteins extracted from colon tumor tissue (operative specimens) or derived from the malignant cell lines LS-174T, HT-29, AsPC-l and CFPAC-I and their pellet extracts, were separated by SDS-PAGE, transferred to PVDF membrane and then stained with the 16C3 antibody. The results demonstrated two distinct molecular mass species cross-reactive with the 16C3 antibody. These were estimated to be 100 kDa and 200 kDa in size. The relative ratios of the two immunoreactive bands have however, generally been observed to be different among colorectal and pancreatic tumor cell lines. In colorectal tumor cells, the 200 kd band is found to be the predominant species, whereas in pancreatic tumor cells the 100 kd band is the predominant species.

The 16C3 antigen was prepared for identification by mass spectrometry by running immunopurified antigen preparations from several different tumor sources on SDS-PAGE and then excising the 16C3 immunoreactive bands from the polyacrylamide gel to define a protein with MW 300 kDa. A second band from HT-29 corresponded to a protein with MW-200 kDa, a third band from CFPAC-I corresponding to a MW-100 kDa. The proteins were then subjected to trypsin digestion followed by LC/MS/MS on a LTQ ORBITRAP@ XL mass spectrometer (Thermo Scientific).

Production data were searched against the forward and reverse International Protein Identification (IPI) human database using the Mascot search engine (Matrix Scientific, Ltd.). The database was appended with commonly observed background proteins to prevent false assignments of peptides derived from those proteins. Mascot output files were parsed into the Scaffold program for filtering to assess false discovery rates and allow only correct protein identifications.

Considered together, the three mass spectrometry experiments demonstrated the presence of CEAcam5- and/or CEAcam6-derived peptides in the 16C3 immunopurified preparations. These preparations were made from human colorectal (LS 174T, HT•29) and pancreatic (CFPAC-I) tumor cell lines. The CEAcam5 and CEAcam6 derived peptides appeared to be most relevant since the molecular mass of these CEACAM species are 100 kDa (CEAcam6) and 200 kDa (CEAcam5) and are expressed in colon tissue and have been shown to be over-expressed in many colon cancer tissue samples. Thus, these experiments suggested that the tumor associated antigen recognized by 16C3 antibody is an epitope of the CEAcam6 glycoprotein. The identity of CEAcam5 and CEAcam6 as the target antigens of the 16C3 antibody was confirmed by comparing the immunoreactivity of 16C3 antibody with commercially-available antibodies against CEAcam5 and CEAcam6. The flow cytometry results demonstrate that 16C3 staining of LS-174T, CFPAC-I HT-29 and H226 cells is similar to that observed with other antibodies known to react with CEAcam5 and CEAcam6. The H226 cell line was included as a cell specificity control since these squamous lung tumor cells do not react with the 16C3 antibody.

The identity of CEAcam5 and CEAcam6 as including the 16C3 antigens recognized by the 16C3 antibody was tested by cloning the genes encoding CEAcam5 and CEAcam6 into mammalian expression plasmids, transfected into human 293T cells (293T cells do not to express either CEAcam, 5 or CEAcam6.) After the DNA plasmids encoding CEAcam5 and CEAcam6 were transfected into 293T cells, the recombinant expression of the antigen targets was tested in western blots using 16C3 antibody and commercially available antibodies against CEAcam5 and CEAcam6. They included clone CB30 against CEA/CD66e (#2383, Cell Signaling Including 9A6 against CEAcam6 (#ab78029 Abcam) or MUS against CEAcam5/6 (#ab4539, Abcam). These results show that the 16C3 antibody detects 16C3 in both CEAcam5 and CEAcam6 expressed as recombinant antigenic molecules. The control anti-CEAcam5 and anti-CEAcam6 antibodies demonstrated that 16C3 detects proteins of the same approximate molecular mass.

The 16C3 antigen was defined by coupling the antibody to resins for needed antigen purification; this included magnetic beads for simple adsorption, washing and then elution from the beads. Proteins obtained from the beads were studied for determination of antigen presence, characterization and identification.

Proteins extracted from colon tumor tissue, or derived from LS 174T, HT-29, ASPC-l and CFPAC-I tumor cell pellet extracts, were separated by SDS-PAGE, transferred to PVDF membrane and then stained with the 16C3 antibody. The results demonstrate two distinct molecular mass species cross-reactive with the 16C3 antibody, estimated to be 100 kDa and 200 kDa. The relative ratios of the two immunoreactive bands have generally been observed to be different among colorectal and pancreatic tumor cell lines. In colorectal tumor cells, the 200 kd band is the predominant species whereas in pancreatic tumor cells the 100 kd band is predominant.

Production data were searched against the concatenated forward and reverse International Protein Identification (IPI) human database using the Mascot search engine (Matrix Scientific, Ltd.). The database was appended with commonly observed background proteins to prevent false assignments of peptides derived from those proteins. Mascot output files were parsed into the Scaffold program for filtering to assess false discovery rates and allow only correct protein identifications.

The siRNA ID#:S2885 was transfected into human colorectal LS 174T whereas siRNA ID#:S9285 was transfected into human pancreatic CFPAC-I tumor cells. Following transfection of the siRNA into tumor cells, the CEAcam5 and CEAcam6 expressed by the cells was measured by specific PCR to measure the levels of CEAcam5 and CEAcam6 mRNAs. Quantitative Western blot analysis (CEAcam5) or quantitative flow cytometry (CEAcam6), each using a 6C3 antibody measured the levels of 16C3-immunoreactive protein.

The quantitative western blot data demonstrates that siRNA specific for the CEAcam5 molecule reduced the amount of 16C3-reactive protein following transfection into LSI 74T cells. A commercially available anti-CEAcam5 antibody was used as a positive control in these experiments. The reduction of CEAcam5 expression, as detected by both the commercial and J6C3 antibodies was dependent on the amount of siRNA transfected into the cells. Approximately 70% of CEAcam5 expression was inhibited in LSI 74T cells at 100 pmol of the siRNA. These results confirmed that CEAcam5 comprises an epitope bound by the 16C3 antibody.

These results confirmed that the 16C3 antigen is likely present in the CEAcam6 protein. This second target antigen, the immunogenic protein characterizing colorectal and pancreatic cancer has a wider spectrum for attacking tumors. The immunogen is also found in adenocarcinoma of ovary, lung and triple negative metastatic breast cancer. By itself it can play an important role in the managing a malignancy defined by this immunogen. Should this target protein exist in combination with the MUC5ac oncofetal protein, the combination of both mAbs may prove in experimental studies and later in the clinical situation that an improved efficacy has been characterized by this approach.

Target protein

The third antibody developed as a byproduct of the Hollinshead vaccine was mAb 31.1 [23]. Of the three, this appeared to demonostrate a higher level of ADCC than the other two mAbs. While we initially spent the most effort in producing and testing the mAb first isolated as Neo 101, the one targeting Mutated MUC5ac because of its activity in metastatic colon and pancreas cancer we then shifted to 16C3 because of initiate activity in targeting colon, lung, ovary and triple negative metastatic breast Ca. In the time alloted to 31.1 it appeared that this mAb was equivalent to a post translational modification of the colon cancer protein described by Old, that is A33 [24,25].

The mAb to A33 detects a membrane antigen that is expressed in normal human colonic and small bowel epithelium and in >95% of human colon cancers. It is absent from most other human tissues and tumor types. The murine A33 mAb has been shown to target colon cancer in clinical trials and the therapeutic potential of a humanized antibody is currently being evaluated. The true immunogen of course has been shown to be a post translational modification of A33 as has been shown to be true for MUC5ac and CEAcam5,6. Using detergent extracts of the human colon carcinoma cell lines LIM1215 and SW1222, in which the antigen is highly expressed, the molecule was purified, yielding a 43 kDa protein. The N-terminal sequence was determined and further an internal peptide sequence was obtained following enzymatic cleavage. Degenerate primers were used in PCRs to produce a probe to screen a LIM1215 cDNA library, yielding clones that enabled us to deduce the complete amino acid sequence of the A33 antigen and express the protein. The available data bases have been searched and reveal no overall sequence similarities with known proteins. Based on a hydrophilicity plot, the A33 protein has three distinct structural domains: an extracellular region of 213 amino acids (which, by sequence alignment of conserved residues, contains two putative immunoglobulin-like domains), a single hydrophobic transmembrane domain and a highly polar intracellular tail containing four consecutive cysteine residues. These data indicate that the A33 antigen is a novel cell surface receptor or cell adhesion molecule in the immunoglobulin superfamily. It may be expressed alone or with additional immunogenic targets (Figure 11).

Cloning of A33 antigen cDNA

A LIM1215 cDNA library was custom-synthesized in the λZAPII expression vector by CLONTECH using poly(A)+ RNA prepared by us from confluent LIM1215 cells by two rounds of enrichment on columns of oligo(dT) cellulose. A cDNA probe encoding the A33 antigen N-terminal sequence was generated using PCR. Antisense primers were designed to hybridize to the carboxyl region of the N-terminal sequence obtained (unpublished data). Specifically, the six pools of 17-mer antisense oligonucleotides, each with 8-fold degeneracy, corresponding to amino acid residues 34-39 (LIQWDK) were as follows: primer 1477, 5′-A(AG)(CT)TT(AG)TCCCACTGAAT-3′; primer 1478, 5′-A(AG)(CT)TT(AG)TCCCATTGAAT-3′; primer 1479, 5′-A(AG)(CT)TT(AG)TCCCACTGGAT-3′; primer 1480, 5′-A(AG)(CT)TT(AG)TCCCATTGGAT-3′; primer 5915, 5′-A(AG)(CT)TT(AG)TCCCACTGTAT-3′; and primer 5916, 5′-A(AG)(CT)TT(AG)TCCCATTGTAT-3′.

These were paired with sense primers designed to hybridize to a primer sequence (KS; 5′-TCGAGGTCGACGGTATC) present in the backbone of the λZAPII vector. A 0.3 kb product obtained with primers KS and 1478 in a “touchdown” PCR (11) with an initial primer-annealing temperature of 55°C was gel-purified, sequenced and found to encode a portion of the A33 N-terminal protein sequence. Precise primers for this A33 antigen cDNA sequence were then used (sense primer, 5′-CCTGTCTGGAGGCTGCCAGT; antisense primer, 5′-AGGTGCAGGGCAGGGTGACA) to amplify a 189-bp PCR product that was radiolabeled with [α-32P]ATP and [α-32P] CTP [both at 3000 Ci/mM (1 Ci=37 GBq); Bresatec, Adelaide, Australia] to a specific activity of >10⁷ dpm/μg DNA using a Megaprime DNA labeling kit (Amersham). Clones (0.8 × 10⁶) were screened according to the Stratagene instruction manual for λZAPII and 16 positive cDNA clones, ranging in size from 0.4 to 2.8 kb, were automatically excised from the λZAPII vector in the Bluescript plasmid, pBS SK(±), using the Lambda Zap Automatic Excision Process (Stratagene). Both strands of five independent clones were sequenced using an Applied Biosytems automated DNA sequencer. Initially, DNA sequence data were obtained using primers directed to the pBS backbone (KS, SK, T3, T7); the internal DNA sequence was then established by the specific primer-directed method. To establish the relationship of the A33 antigen sequence to known DNA sequences, four data bases [EMBL, GenBank, DDBJ (DNA Data Bank of Japan) and dbEST (data base of expressed sequence tags)] were searched using the data base similarity search algorithms, blast and fasta.

Northern blot analysis

For studies of A33 antigen mRNA expression, specimens of colorectal carcinoma (obtained during surgical resection) and confluent layers of colon carcinoma-derived cell lines (LIM1215, LIM1863, LIM1899, LIM2099, LIM2405 and LIM2437) [14] were directly solubilized in denaturing solution (4 M guanidinium isothiocyanate/0.5% Sarkosyl NL30 [BDH]/25 mM sodium citrate/0.1 M 2-mercaptoethanol) and total RNA was prepared according to the method of Kocer et al. [15]. Samples of normal human colon, counterparts of the tumor samples above and inflamed colon (from patients with Crohn disease) were first enriched for epithelial cells by incubating in PBS containing 3 mM EDTA and 0.5 mM DTT to release the crypts from the underlying stroma prior to solubilization in denaturing solution. Samples (20 μg) of total cytoplasmic RNA were electrophoresed in 0.4 M formaldehyde and 1% agarose gels and transferred overnight by capillary action to nylon filters (Hybond N; Amersham) and immobilized by exposure to UV light. Filters were prehybridized in a buffer containing 50% formamide, 5x Denhardt’s solution (0.1% Ficoll/0.1% polyvinylpyrrolidone/0.1% BSA), 5x SSPE (1x SSPE=0.15 M NaCl/0.01 M NaH₂PO₄•2H₂O/1.2 mM EDTA, pH 7.4), 0.5% SDS and 1% (wt/vol) skimmed milk powder for at least 6 h at 42°C. The filters were then incubated at 42°C overnight in fresh hybridization solution with a 2.6 kb cDNA clone of A33 antigen, gel-purified and labeled with [α-32P]ATP (3000 Ci/mM; Bresatec) using a Megaprime DNA labeling kit. Filters were washed in 2x SSPE, 0.1% SDS followed by 1x SSPE and 0.1% SDS at 42°C and signals were visualized by autoradiography. To permit quantification of the mRNA signals, the filters were reprobed with an oligonucleotide specific for 18 S rRNA (5′-CGGCATGTATTAGCTCTAGAATTACCACAG), labeled with dATP[γ-32P] (3000 Ci/mmol; Bresatec) using T4 polynucleotide kinase.

Cos cell expression

A 2.6 kb putative A33 antigen cDNA clone was excised from the pBS (SK ±) plasmid using EcoRI and sub cloned in the sense orientation into the mammalian expression vector, pcDNA3 (InVitrogen, Leek, The Netherlands). Cos cells were seeded into 15 cm Petri dishes (Nunc) to achieve 50% confluency 24 h later. The cells were transfected over a 4 h incubation at 37°C with 15 μg of either pcDNA3/A33 or pcDNA3 (parental vector) using DEAE-dextran in the presence of chloroquine [17]. This was followed by dimethyl sulfoxide (DMSO) shock (10% DMSO in PBS) for 90 s; the cells were then returned to RPMI 1640 medium containing 10% fetal calf serum (FCS), 2 mM glutamine and 50 μg/ml gentamycin for 3-5 days. The cells were harvested and analyzed for A33 antigen expression by Western blot analysis, flow cytometry and immunocytochemistry.

Western blot analysis

Transfected cells were solubilized for 30 min at 4°C with 1% (vol/vol) Triton X-100 in 15 mM Tris•HCl (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM pepstatin, 0.1 mM leupeptin and 0.01 units per ml of aprotinin. The resulting extracts were centrifuged twice at 4°C for 20 min at 14,000x g and 2 μl aliquots were electrophoresed under nonreducing conditions in 8-25% SDS/PAGE Phastgels (Pharmacia) before they were transferred to poly(vinylidene difluoride) membranes and incubated with humanized mAb A33 (2 μg/ml). A33 signals were detected with anti-human IgG conjugated with horseradish peroxidase and visualized by enhanced chemiluminescence (Amersham).

Flow cytometry and immunochemistry

Transfected cells were detached using 10 mM EDTA in PBS for 10 min at 37°C and aspirated gently to produce a single cell suspension. They were then pelleted by centrifugation at 1500 rpm for 5 min and resuspended in 0.5 ml PBS containing 10 mM EDTA and 5% FCS and kept on ice for all remaining procedures to prevent internalization of antigen-antibody complexes. Murine A33 mAb was added to a final concentration of 20 μg/ml for 30 min. The cells were washed twice in PBS/EDTA/FCS and incubated on ice for a further 30 min in 0.5 ml PBS/EDTA/FCS containing fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (Silenus, Hawthorn, Australia) diluted 1:50. Cells were then washed twice more and resuspended in 1 ml PBS/EDTA/FCS for fluorescence-activated cell sorting (FACS) analysis in a Becton Dickinson FACScan and cytospin preparation. Cytospins were prepared in a Shandon cytospin 2 centrifuges (Shandon, Pittsburgh), allowed to air-dry, mounted in glycerol containing antifade and examined using a Nikon Fluorophot microscope.

RESULTS

Sixteen overlapping putative A33 antigen cDNA clones were obtained, the longest of which (clone 18) was 2793 bp prior to the addition of the poly(A)+ tail. The longest open reading frame in the cDNA sequence predicts a protein of 319 amino acids. Beginning at amino acid 22 of the predicted translation product, 40 contiguous residues are identical to the established amino-terminal sequence of native A33 antigen (GR, et al. unpublished data) [10]. The predicted sequence also contains regions (hatched boxes) identical with the amino acid sequences of several internal peptides released from the native molecule by enzymatic digestion

cDNA sequence and deduced amino acid sequence of the longest cDNA clone (clone 18; 2.8 kb) encoding the human A33 antigen. The longest open reading frame, encompassing nucleotides 345-1301, contains the known N-terminal sequence of the native A33 antigen and predicts a protein of 319 amino acids. The stop codon at 1302-1304 is boxed and the amino acid sequences of the internal peptides identified by digestion of the native molecule are shown (shaded areas). A putative signal sequence (bold underline), three potential N-linked glycosylation sites (overline) and a transmembrane domain (second bold underline) are indicated. Adjacent to the transmembrane domain, four consecutive cysteine residues are observed. The spans of the two putative Ig-like domains are enclosed by square brackets with the specific residues conserved in Ig superfamily members shown in circles. Other features of the DNA sequence include a tandem repeat of 25 bp in the 5′ untranslated region (bold overline) and a polyadenylylation signal (AATAAA) 11 bp upstream from the poly(A) tail. The asterisk above the C at position 294 denotes the fact that a C was found in this position in 2 out of 5 independent clones sequenced (including clone 18) and an A in the 3 other clones.

The predicted translation product of the human A33 antigen mRNA is initiated at the ATG positioned 345 bp from the 5′ end of clone 18. This ATG leads off the longest open reading frame and is in a favorable context for initiation of translation by reference to the Kozak consensus sequence, GCC(A/G)CCATGG. Following this, a sequence encoding 21 amino acids resembles a hydrophobic signal peptide. The putative cleavage site between alanine and isoleucine, which is required to produce the mature protein with the N-terminal sequence of the native molecule, is consistent with the (−3, −1) rule for signal peptide cleavage. The position of the first in-frame stop codon predicts a mature polypeptide chain comprising 298 amino acid residues, Mr 33,276. This Mr is not inconsistent with data demonstrating that the native A33 antigen is a glycoprotein of approximate Mr 40,000-45,000 because the sequence contains three potential N-linked glycosylation sites, one of which (N91) was strongly suspected from the initial sequence analysis of the peptide fragments. Each of these could be predicted to accommodate an average oligosaccharide chain length of 2.5-3 kDa. Indeed, in experiments where enzymes were used to remove sialic acid and both N- and O-linked glycosides from the A33 antigen polypeptide, there was a reduction of approximately 8000 in the Mr of the A33 antigen (unpublished data). Based on a Kyte-Doolittle hydrophilicity plot of the sequence, the molecule appears to have three structural domains: an extracellular region of 213 amino acids containing 6 cysteine residues, a hydrophobic transmembrane domain of 23 amino acids and a highly polar intracellular tail of 62 amino acids. Searches of available DNA and protein data bases revealed no direct sequence similarities with any known protein. However, relatively short spans of human A33 antigen nucleotide sequence could be matched with expressed sequence tags derived from the human colonic epithelial-derived cell line, T-84 (92% identity with GenBank accession no. AA055862; length, 344 nt) and the murine teratocarcinoma-derived cell line, F9 (74% identity with EMBL accession no. D28657D28657; length, 249 nt).

Kyte–Doolittle hydrophilicity plot of the deduced amino acid sequence of the A33 antigen. Several structural features are indicated: the presence of an N-terminal hydrophobic region likely to correspond to a signal sequence, a more distal highly hydrophobic sequence consistent with the presence of a single-span transmembrane domain, and a highly polar C-terminal region consistent with an intracellular location for this part of the molecule.

Manual inspection of the extracellular domain revealed the presence of several residues that are conserved in members of the Ig superfamily. Specifically, we noted a V-type Ig-like domain at the N terminus, in which the presence of a disulfide bond between the two conserved cysteines (C22, C96) was predicted from the microsequence analysis of the peptide fragments generated by enzymatic digestion. The V-type domain was followed by an Ig-like domain of the C2-type. This combination of a V-type and a C2-type domain is characteristic of the CD2 subgroup of Ig superfamily members. Sequence alignment to proteins in this subgroup with known three-dimensional structure revealed that the A33 antigen shares the highest sequence identity with the D1D2 fragment of human CD4 (16% over 208 residues) [22]. The V-type domain is most similar to the VL domains of antibodies (up to 22% sequence identity over 117 residues) and the C2-type domain to the D4 domain of rat CD4 (18% over 85 residues). The overall similarity with the CD2 group of molecules suggests that the A33 antigen may participate in cell-cell recognition processes, perhaps further implying the existence of a soluble or cell-associated binding partner. The identification of non-antibody binding partners could be addressed using the same biosensor-based technology used recently to identify and purify both the A33 antigen and the ligand for the orphan receptor, HEK.

The N terminus of the predicted intracellular region of 62 amino acids begins with four consecutive cysteine residues. Data base analysis has identified 14 mature proteins containing the CCCC motif. Interestingly, three of these, G protein-coupled receptor 3, endothelial-1 receptor and the tachykinin-like peptide receptor, are members of the seven transmembrane G protein-coupled receptor family in which palmitoylation of the cysteine residues near the carboxyl terminus has been implicated in receptor coupling to G proteins and in the down-regulation of receptor activity by influencing receptor removal from the cell surface. Thus the presence of a CCCC motif adjacent to the transmembrane domain of the A33 antigen may indicate further membrane tethering of the molecule via palmitoylation and this could play a role in the trafficking of the protein to vesicles.

To verify that the clones we had isolated encoded the A33 antigen, we expressed a 2.6 kb cDNA transiently in Cos cells. The cells were then assayed for A33 antigen expression by Western blot analysis, flow cytometry and immunocytochemistry (data not shown). Only the Cos cells transfected with the expression vector containing a putative A33 antigen cDNA clone produced a protein that was recognized by the mAb A33. Furthermore, the Western blot analysis demonstrated that the recombinant A33 protein expressed by Cos cells was approximately the same Mr (40,000-45,000) as endogenously produced A33 antigen in LIM1215 cells. As expected, FACScan and immunocytochemical analysis of Cos cells transfected with the A33 antigen construct indicated that a large proportion of the A33 antigen was displayed on the cell surface.

Expression of recombinant A33 antigen by transfected Cos cells. Cos cells were transfected either with the parental vector, pcDNA3 or with pcDNA3 into which a 2.6 kb A33 antigen cDNA had been subcloned. Cells were harvested in this experiment 5 days after transfection and subjected to Western blot analysis (A) and flow cytometry (B). (A) Transfected cells were solubilized in Triton X-100 and electrophoresed into SDS/polyacrylamide gels without reduction and processed for Western blot analysis as described. The signal obtained with Cos cells transfected with pcDNA3 containing A33 antigen cDNA (lane 1) corresponds to approximately the same Mr (43,000) as the signal obtained with LIM1215 cells expressing high endogenous levels of A33 antigen (lane 3). Cells transfected with the parental vector gave no signal (lane 2). The positions of blue pre-stained standards (lane M) (NOVEX, San Diego) are indicated on the right. (B) FACScan profiles of control and A33 antigen-expressing Cos cells. The profile obtained with A33 antigen-expressing Cos cells (shown in bold) has been superimposed on the profile obtained with Cos cells transfected with pcDNA3 alone to show the shift to the right in fluorescence intensity of cells expressing A33 antigen.

Northern blot analysis demonstrated a strong A33 antigen signal (2.8 kb) in total RNA prepared from A33 antigen positive human colon carcinoma-derived cell lines (LIM1215, LIM1899 and LIM1863). No signal was obtained with total RNA from A33 antigen negative cell lines (LIM2099, LIM2405 and LIM2537; Figure 4A). Strong expression of A33 antigen mRNA in purified epithelial cells from normal human colon was always observed. In comparison, the A33 antigen mRNA signals obtained with RNA extracted from the adjacent tumor tissue were consistently weaker. The Northern blot analysis has been extended to more than 20 paired samples of normal and transformed colonic tissue with the same results. One explanation for this could be the higher fibroblast content of the tumor samples compared with the normal colonic crypt preparations, which are essentially pure epithelial cells.

Six colorectal carcinoma cell lines had been previously analyzed for A33 antigen expression using immunocytochemistry and flow cytometry (unpublished data). Half (LIM1215, LIM1863 and LIM1899) were found to be positive. The remaining half (LIM2099, LIM2405 and LIM2537) were negative. The pattern of A33 antigen mRNA expression was consistent with protein expression data. A33 antigen mRNA expression was found in samples of normal and diseased human colorectal tissue obtained from patients during surgical resection. In colonic mucosa, strong hybridization signals do correspond to relatively high expression of A33 antigen compared with that in the corresponding tumor preparations and the RNA extracted from the large polyp which all produced weaker A33 antigen mRNA signals. Crohn disease was found to produce an A33 antigen signal similar to that of its normal counterpart.

Immunohistochemical studies have demonstrated that the expression of the A33 antigen is essentially restricted to normal intestinal epithelium and 95% of colorectal tumors. Although several other mAbs capable of recognizing determinants on colon cancer cells exist, none of them matches the restricted tissue specificity of the A33 mAb. Ep-CAM (epithelial cell adhesion molecule) is a cell adhesion molecule expressed by most simple epithelia and its expression is maintained by a large proportion of colorectal, breast, lung, pancreatic and gastric tumors. Several mAbs to this tumor marker have been used in clinical trials and recently chimeric and single-chain versions of two of them, 17-1A and 323/A3, were developed and evaluated in cell-killing experiments. Similarly, members of the carcinoembryonic antigen family and the pancarcinoma antigen, TAG-72, have attracted attention as suitable targets for immunotherapy of colorectal, breast and lung tumors. Though their relatively wide expression offers the opportunity to develop antibodies that can target multiple tumors, experience suggests that optimal localization of antibody to tumors may be impeded by the presence of antigen in a variety of normal tissues as well as by the presence of shed antigen in the circulation. In this respect, it might also have been expected that the strong expression of the A33 antigen in normal colon would have compromised its usefulness as a target in immunotherapy. However, these concerns have been allayed by phase I/II studies that have shown prolonged accumulation of 131I- and 125I-labeled A33 at sites of tumor metastasis for up to 6 weeks after administration, while normal colon was antibody-free by 7-8 days and minimal toxicity to the colonic mucosa was observed. The rapid clearance of the antibody from normal colon is not fully understood, but it may be due to rapid transcytosis of radiolabeled A33 mAb from colonocytes and/or the relatively rapid shedding of colonocytes from the top of the crypts. A basic understanding of the mechanisms underlying the tightly regulated, tissue-specific nature of A33 antigen expression as well as insights into its biological function is eagerly awaited. Such insights are likely to advance our understanding of cell-cell interactions and differentiation in the colon. In particular, knowledge of the characteristics of the A33 antigen gene promoter that confers such tissue-specific expression should allow us to develop transgenic mouse models of colon cancer, which could cast light on the role of certain oncogenes and tumor suppressor genes in the tumorigenic process. Indeed, it is conceivable that the tissue specificity of the A33 antigen promoter could also be exploited to target therapeutic genes to the colon.

Clearly the A33 antigen in its post translational form is an exciting target for immunotherapeutic approaches to colon cancer and our identification of the A33 antigen with its receptor-like structure points toward a new signaling system in the colon. The availability of the A33 antigen should facilitate the development of new immunotherapeutic and ligand-directed approaches to the treatment of metastatic colon cancer.

CONCLUSION

With all three of the oncofetal proteins defining colorectal cancer having been characterized, a more rational approach to the immunotherapy of advanced and metastatic colorectal cancer becomes defined. Depending on what is determined from immunohistochemistry studies of the primary lesion, a protocol of one mAb or combinations of those mAbs defining the 3 Immunogens can be devised (Figure 11). As noted previously, when one defines the existence of a metastatic lesion at a time interval following treatment of the primary, it is not necessary to have a biopsy of the metastasis since the original primary contains unaltered immunogenic proteins expanding up the ladder from the original premalignant cell line, to the primary tumor in-situ, to the appearance of the functional lesion and under certain circumstances, to the metastatic lesion. Thus immunotherapy of the metastasis is based on the nature of the antigen expressed in the primary growth which can be employed as a vaccine [26]. The underlying immunogen that characterizes the primary lesion does not mutate as the lesion progresses. This is in contrast to many surface antigens that are eventually unstable and eventually result in a mutated variant.