Anti-idotype and recombinant antigen in immunotherapy of colorectal cancer

The CO17-1A/GA733 antigen (Ag), bound by monoclonal antibodies (MAb) CO17-1A and GA733 that define two different epitopes on the Ag, has proven a useful target in passive and active immunotherapy of colorectal carcinoma (CRC). Previous studies suggest that the antitumor effects demonstrated in MAb-treated patients may be mediated by idiotypic cascades. In approaches to active immunotherapy against the Ag, polyclonal goat and monoclonal rat anti-idiotypic antibodies (Ab2) directed against MAb CO17-1A or GA733 (Ab1) were administered as alum precipitates to 54 patients with CRC (stage Dukes' B, C, and D). The majority of the patients treated with the various Ab2 preparations developed anti-anti-idiotypic antibodies (Ab3) that specifically bound to the CO17-1A or GA733 epitope and shared idiotopes with the corresponding Ab1. Approximately 30% of the patients tested developed specific cellular immunity, i.e., Ag-specific T-cells mediating delayed-type hypersensitivity (DTH) reaction in vivo or proliferating on stimulation with the Ag in vitro. The humoral and cellular immune responses may underlie the clinical responses observed in some of the treated patients. Recently, the CO17-1A/GA733 Ag has been molecularly cloned and expressed in baculo-, adeno-, and vaccinia viruses. In preclinical studies, these recombinant Ag preparations elicited specific humoral immunity (cytotoxic antibodies) and cellular immunity (DTH-reactive and proliferative T-cells), similar to the native Ag. Antibody titers elicited in experimental animals by recombinant Ag were significantly higher than those elicited by Ab2, presumably because Ag expresses numerous epitopes, whereas Ab2 mimics a single epitope. Recombinant CO17-1A/GA733 Ag has potential as a vaccine for CRC patients.     

Autoantibodies against the tumour-associated antigen GA733-2 in patients with colorectal carcinoma

The tumour-associated antigen (TAA) GA733-2 is expressed as a non-secreted surface molecule on the majority of human colorectal carcinoma cells. The antigen has been used as a target for passive and active immunotherapy during the last decade. To determine the incidence of autoantibodies against this antigen, sera from 1068 patients with colorectal carcinoma were analysed for naturally occurring IgG antibodies against the baculovirus-produced GA733-2E protein. A total of 14.5% of the patients had IgG antibodies against the antigen. In 519 patients, sera were collected at the time of diagnosis and 15% of those patients had anti-GA733-2E IgG antibodies. There was a tendency to a higher frequency of patients with antibodies among those in the advanced Dukes stages: 11% in stage A and 32% in stage D respectively (P = 0.06). Antibodies could be detected for up to 10 years after the diagnosis. Patients with Crohn's disease or colitis ulcerosa (n = 20) did not elicit anti-GA733-2E antibodies. No healthy control donor (n = 45) had detectable antibodies against the antigen. The specificity of GA733-2E-reactive serum IgG was indicated by significant inhibition of mAb17-1A (originally used to define GA733-2) binding to the GA733-2E antigen. Sera of positive patients bound to the GA733-2-expressing human colorectal carcinoma cell line, SW948. No significant correlation was found between the presence of antibodies and survival in the present patient population. However, the high incidence of autoantibodies against this tumour antigen in colorectal carcinoma patients confirms its antigenicity in humans and supports the use of the GA733-2 antigen as a target for immunotherapy. Received: 25 May 1998 / Accepted: 26 November 1998     

Oligomeric state of the colon carcinoma-associated glycoprotein GA733-2 (Ep-CAM/EGP40) and its role in GA733-mediated homotypic cell-cell adhesion

The GA733-2 antigen (GA733) is a homotypic calcium-independent cell adhesion molecule (CAM) present in most normal human epithelial cells and gastrointestinal carcinomas. Because oligomerization of some CAMs regulates cell adhesion and signal transduction, the correlation between GA733 oligomeric state and cell-cell adhesion was investigated. Sedimentation equilibrium studies showed that full-length (-FL) GA733 exists as dimers and tetramers in solution, whereas the GA733 extracellular domain (-EC) is a monomer. The Kd of GA733-FL is less than 10 nm for the monomer-dimer association, whereas the dimer-tetramer association is about 1000-fold weaker (Kd approximately 10 microm). Chemical cross-linking of purified GA733-FL in solution resulted in a major product corresponding to GA733 dimers, and minor amounts of trimers and tetramers. However, GA733-EC cross-linked under the same conditions was consistently a monomer. Chemical cross-linking of dissociated colon carcinoma cells produced predominantly GA733 dimers, whereas cross-linking of cells in monolayers yielded some tetramers as well. GA733-FL retained its cell-cell adhesion function as shown by inhibition of cell aggregation, whereas monomeric GA733-EC was inactive. These data show that GA733 exists predominantly as high affinity noncovalent cis-dimers in solution and on dissociated colon carcinoma cells. The lower affinity association of dimers to form tetramers is most likely the head-to-head interaction between GA733 cis-dimers on opposing cells that represents its cell-cell adhesion activity.     

Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, and Ep-CAM)

The GA733-2 antigen is a cell surface glycoprotein highly expressed on most human gastrointestinal carcinoma and at a lower level on most normal epithelia. It is an unusual cell-cell adhesion protein that does not exhibit any obvious relationship to the four known classes of adhesion molecules. In this study, the disulfide-bonding pattern of the GA733-2 antigen was determined using matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of purified tryptic peptides treated with 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine or partially reduced and alkylated. Numbering GA733-2 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys4, Cys2-Cys6, and Cys3-Cys5, which is a novel pattern for a cysteine-rich domain instead of the expected epidermal growth factor-like disulfide structure. The next three disulfide linkages are Cys7-Cys8, Cys9-Cys10, and Cys11-Cys12, consistent with the recently determined disulfide pattern of the thyroglobulin type 1A domain of insulin-like growth factor-binding proteins 1 and 6. Analysis of glycosylation sites showed that GA733-2 antigen contained N-linked carbohydrate but that no O-linked carbohydrate groups were detected. Of the three potential N-linked glycosylation sites, Asn175 was not glycosylated, whereas Asn88 was completely glycosylated, and Asn51 was partially glycosylated. These data show that the extracellular domain of the GA733-2 antigen consists of three distinct domains; a novel cysteine-rich N-terminal domain (GA733 type 1 motif), a cysteine-rich thyroglobulin type 1A domain (GA733 type 2 motif), and a unique nonglycosylated domain without cysteines (GA733 type 3 motif).     

Retroposition in a family of carcinoma-associated antigen genes.

The gene encoding the carcinoma-associated antigen defined by the monoclonal antibody GA733 is a member of a family of at least two type I membrane proteins. This study describes the mechanism of evolution of the GA733-1 and GA733-2 genes. A full-length cDNA clone for GA733-1 was obtained by screening a human placental library with a genomic DNA probe. Comparative analysis of the cDNA sequence with the previously determined genomic sequence confirmed that GA733-1 is an intronless gene. The GA733-2 gene encoding the monoclonal antibody-defined antigen was molecularly cloned with a cDNA probe and partially sequenced. Comparison of GA733-2 gene sequences with the previously established cDNA sequence revealed that this gene consists of nine exons. The putative promoter regions of the GA733-1 and GA733-2 genes are unrelated. These findings suggest that the GA733-1 gene was formed by the retroposition of the GA733-2 gene via an mRNA intermediate. Prior to retroposition, the GA733-2 gene had been affected by exon shuffling. Analysis of GA733-2 exons revealed that many delineate structural motifs. The GA733-1 retroposon was localized either to chromosome region 1p32-1p31 or to 1p13-1q12, and the GA733-2 founder gene was localized to chromosome 4q.     

Murine response studies of insect cell (Sf9) expressed recombinant colorectal cancer vaccine candidate using surface plasmon resonance studies

The baculovirus vector expression system is considered a useful insect cell‐based platform for large‐scale production of recombinant biopharmaceutical glycoproteins. The GA733 antigenic protein is a 40 kDa cell surface glycoprotein that is overexpressed in human colorectal carcinoma. Three GA733‐expressing baculovirus vectors were constructed: (i) GA733 fused to the Fc fragment of human immunoglobulin G (GA733‐Fc); (ii) GA733‐Fc tagged with the endoplasmic reticulum (ER) retention signal KDEL (GA733‐FcK); and (iii) GA733‐FcK without the signal peptide [GA733‐FcK (w/o SP)]. These three recombinant proteins were expressed in Fall Armyworm (Spodoptera frugiperda) Sf9 insect cells. GA733‐Fc purified from insect culture medium (CM) and cell lysate (CL) (GA733^I‐Fc^CM and GA733^I‐Fc^CL, respectively), and GA733‐FcK and GA733‐FcK (w/o SP) purified from CL [GA733^I‐Fc^CL and GA733^I‐FcK^CL (w/o SP), respectively] were injected intraperitoneally into BALB/c mice [three times at 1 μg, with or without an adjuvant (aluminum hydroxide)]. Surface plasmon resonance (SPR) analysis (involving a chip with the immobilized GA733 antigen) showed that, in general, the serum samples from mice immunized with the adjuvant yielded stronger SPR signals than did those from mice immunized without an adjuvant. The serum samples from mice after the second injection yielded stronger signals than did those from mice before the second injection. The group immunized with GA733^I‐FcK^CL (glycosylated with an oligomannose) showed the strongest SPR signals. The SPR signals from the GA733^I‐FcK^CL group were stronger than or similar to those from mice immunized with mammalian‐cell‐expressed GA733‐Fc (GA733^M‐Fc). Thus, the baculovirus vector expression system can be used to produce immunogenic cancer glycoprotein.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Immunogenic regions of the GA733-2 tumour-associated antigen recognised by autoantibodies of patients with colorectal carcinoma

The tumour-associated antigen (TAA) GA733-2 is overexpressed by >90% of human colorectal carcinomas (CRC). The antigen has previously been shown to be recognised by B and T cells. The aim of the present study was to define B cell epitopes of GA733-2. Fifteen percent of CRC patients with no previous immunotherapy have recently been shown to elicit an anti-GA733-2 IgG antibody response. Sera of these patients ( n=136) were analysed by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies against 23 partly overlapping synthetic peptides (18 amino acids: aa) derived from the extracellular domain of GA733-2. An 18-aa long sequence at the N-terminal region of the antigen (peptide 2) was found to be an immunodominant B cell epitope. Fifty percent of the patients had antibodies against peptide 2, while 8% to 9% had antibodies against peptides 1, 4, 7, 8 or 20. In healthy donors ( n=30) antibodies against peptides 2 and 8 were also detected in 13% and 3% of cases respectively, while no antibodies were found against the other peptides and the complete protein. Thirteen percent of CRC patients ( n=30) with no IgG antibodies against the GA733-2 antigen elicited antibodies against peptide 2. The specificity of peptide-reactive sera was verified by inhibition ELISA. The binding of sera to GA733-2 was significantly inhibited by peptides to which CRC sera bound, but not by control peptides. Binding to peptide 2 of sera showing both peptide 2 and GA733-2 reactivity was specifically inhibited by the complete GA733-2 antigen, while binding of peptide 2-reactive sera showing no GA733-2 reactivity was not inhibited. CRC sera interfered with the binding of monoclonal antibody (mAb) 17-1A and mAb C215 that recognise distinct epitopes of GA733-2. No significant correlation was found between the presence of anti-peptide antibodies in CRC patients and clinical stage or overall survival. The results provide additional evidence for immune recognition of CRC by the host.     

Expression of a recombinant chimeric protein of human colorectal cancer antigen GA733-2 and Fc fragment of antibody using a replicating vector based on Beet curly top virus in infiltrated Nicotiana benthamiana leaves

We describe expression and characterization of recombinant human colorectal cancer antigen GA733-2 fused to Fc fragment of antibody (GA733-2-Fc) using a replicating vector based on Beet curly top virus in infiltrated Nicotiana benthamiana leaves. Recombinant GA733-2-Fc/KDEL with a molecular mass of ~68?kDa was transiently expressed. The level of expression of GA733-2-Fc with ER retention signal KDEL (GA733-2-Fc/KDEL) in the expression vector system was 0.96% of total soluble proteins. Recombinant GA733-2-Fc/KDEL was purified using an affinity chromatography. Mice immunized with recombinant GA733-2-Fc/KDEL mounted a strong GA733-2-Fc/KDEL-specific serum antibody response. Vaccination of plant-derived recombinant GA733-2-Fc/KDEL regressed tumor volumes in BALB/c mice. The population of activated-T and NK-T cells increased notably in lymph node, spleen, and tumor-infiltrating lymphocytes derived from the tumor-regressed mice. Taken together, recombinant GA733-2-Fc/KDEL expressed in plants can be used as an effective experimental immunogen for research in cancer vaccine development.     

Optimization of colorectal cancer vaccine candidate protein GA733‐Fc expression in a baculovirus–insect cell system

The baculovirus–insect cell expression system has been used to produce functional recombinant proteins. The antigen GA733 is a cell‐surface glycoprotein highly expressed on most human colorectal carcinoma cells. Conditions for the expression of GA733 fused to the human immunoglobulin IgG Fc fragment (GA733‐Fc) were optimized in the baculovirus expression system. Several variable factors were adjusted to optimize expression, including the cell line (Sf9 and High Five), multiplicity of infection (MOI) value (0.05, 0.1, 0.5, 1 and 3), post‐infection time (48, 72 and 96 h) and harvested sample (cell culture media (CM) or cell lysate (CL)). In addition, two pFastBac Dual vectors carrying the GA733‐Fc gene were constructed to express GA733‐Fc with or without an endoplasmic reticulum (ER) retention sequence KDEL and used to generate recombinant baculoviruses. Western blot showed that expression depended on the conditions used to express the recombinant proteins. The protein production level and secretion capability differed in each cell line. In Sf9 cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 96 h post‐infection at 0.1 MOI and with GA733‐FcK at 96 h post‐infection at 3 MOI, respectively. In High Five cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 48 h post‐infection at 1 MOI and with GA733‐FcK at 48 h post‐infection at 3 MOI, respectively. These results suggest that the MOI value, post‐infection time and subcellular localization affect expression, and that these conditions can be modified to optimize protein expression in the baculovirus–insect cell system.     

Immunization with a plant-produced colorectal cancer antigen

Cancer vaccination has become an important focus of oncology in recent years. Active immunization with tumor-associated antigens such as colorectal cancer antigen GA733-2 is thought to potentially overcome the reoccurrence of metastasis. As recombinant protein production in bioreactors is costly and subject to growing safety concerns, we tested plants as an alternative for the expression of a potential colorectal cancer vaccine. Comparing colorectal cancer antigen GA733-2 produced in tobacco plants with the same antigen produced in insect cell culture, we found a similar humoral immune response to injection of either of the two antigen preparations into mice. Some minor differences were observed in the cellular response that might be due to impurities. Our studies compare for the first time, immunization with the same antigen expressed in either plants or insect cell culture. This will provide important data for use of plants as production systems of therapeutics.     

Identical D region sequences expressed by murine monoclonal antibodies specific for a human tumor-associated antigen

Sequence analysis has revealed significant structural similarities among murine mAb specific for the human tumor-associated Ag GA733. Antibodies generated in independent immunizations, either with Ag-positive tumor cells or with anti-idiotypic antibodies produced by immunization of goats with a GA733-specific antibody, all use members of the same gene families; remarkably, they also express identical amino acid sequences in their H chain CDR3. Inasmuch as this region normally exhibits considerable sequence variability, the identity displayed by the antibodies indicates a requirement for this particular sequence in the generation of their specificity for the GA733 antigen. Moreover, this homology suggests that the antibodies recognize a common determinant on the Ag, and that the polyclonal anti-idiotypic antibodies can functionally mimic the Ag at the level of the structure of the Ag-specific antibody that is induced.     

Identification of a family of high molecular weight tumor-associated glycoproteins

The monoclonal antibody (MAb) designated DF3 was prepared against a human breast carcinoma metastatic to liver. This MAb reacts with a high molecular weight glycoprotein detectable in human breast carcinomas and human milk. In contrast, MAb F36/22 was prepared against the MCF-7 breast carcinoma cell line, MAb 115-D8 against human milk fat globule membrane (HMFGM) and MAb Ca1 against the HEp-2 human laryngeal carcinoma cell line. These MAb have similar patterns of reactivity with normal tissues and tumors based upon immunoperoxidase staining. In the present study we have monitored reactivity of these MAb against DF3 antigen purified from human breast carcinoma cell lines (MCF-7, BT-20) and HMFGM. Solid phase immunoassays and immunoblotting demonstrate that MAb DF3, F36/22, 115-D8, and Ca1 all react with the same purified DF3 antigen. Furthermore, immunoblot analysis indicates that the DF3 antigen reactive with these MAb differs structurally in preparations from breast carcinoma cells and HMFGM. We also demonstrate that MAb F36/22 completely inhibits MAb DF3 binding in competitive blocking assays. In contrast, the results indicate that MAb 115-D8 and Ca1 only partially block MAb DF3 reactivity and the extent of this inhibition varies with DF3 antigen purified from breast carcinoma cells and HMFGM. Taken together, these findings with multiple MAb prepared against a variety of immunogens suggest that existence of a family of related but not identical high molecular weight tumor-associated glycoproteins.     

Effects of maturational agents on expression and secretion of two partially characterized high molecular weight milk-related glycoproteins in MCF-7 breast carcinoma cells

We have previously defined a human mammary epithelial antigen using a murine monoclonal antibody (MAb), designated DF3, prepared against a membrane-enriched fraction of a human breast carcinoma. MAb DF3 detects a cell surface antigen with a molecular weight (mw) of approximately 300 Kd and a higher mw species also detectable in human milk. These findings and the demonstration that butyric acid (BA) increases DF3 antigen expression suggested that MAb DF3 reacts with a differentiation antigen detectable in human breast carcinoma cells. The results of the present study demonstrate that MAb DF3 reacts with two mucin-like high mw glycoproteins (330 and 450 Kd) present in MCF-7 breast carcinoma cells. The results also demonstrate that the intracellular content and secretion of DF3 antigen is increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-beta-D-arabinofuranosylcytosine (ara-C). Other known inducers of differentiation including retinoic acid (RA), hexamethylene bisacetamide (HMBA), 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) and certain polar solvents decrease DF3 antigen expression. Furthermore, the results demonstrate that DF3 antigen is secreted and that the extent coincides with changes in intracellular content. Finally, actinomycin D and cycloheximide inhibit the increases in DF3 antigen expression following TPA treatment thus suggesting that newly synthesized RNA and protein are required for induction of this antigen. Thus, the monitoring of DF3 antigen expression may provide a marker for studying maturation of human breast cancer cells.     

Baculovirus titration method based on MOI values for optimizing recombinant protein expression of the anti‐cancer vaccine candidate GA733‐Fc using Sf9 insect cells

The baculovirus expression system has been considered as a highly efficient tool for the production of recombinant biopharmaceutical proteins. The recombinant antigenic glycoprotein GA733 is a cell surface protein that is strongly expressed in human colorectal cancer. Efficient virus titration should be established to achieve optimal multiplicity of infection (MOI) conditions, which are in turn essential for strong expression of the recombinant GA733 fused to the human immunoglobulin IgG Fc fragment (GA733‐Fc) in the baculovirus‐insect system. In the present study, the Sf9 cell line was transfected with plasmid DNA containing the GA733‐Fc expression cassette under the control of the baculovirus polyhedron promoter. MOI values (0.05, 0.1, 0.5, 1, and 3) were calculated based on both microscope observations and results of titration assay and then used to determine the optimum recombinant expression and harvested sample [cell culture media (CM) or cell lysate (CL)]. The pFastBac dual vector carrying the GA733‐Fc gene was constructed to express GA733‐Fc and used to generate recombinant baculoviruses. Western blotting results showed that recombinant protein expression was dependent on the MOI. In addition, CM and CL showed significant differences in protein synthesis and protein secretion capacities. Our findings suggested that our proposed titration method can be used for reliable calculation of MOI values, which significantly influence recombinant GA733‐Fc protein expression in the baculovirus‐insect cell system.     

The use of monoclonal antibody B72.3 in the management of gynecologic malignancies

Monoclonal antibodies are currently used in the diagnosis of gynecologic malignancies by way of immunohistochemical assays, serum assays, and in situ radiolocalization of carcinoma lesions. Among them is MAb B72.3, generated against a human tumor-associated antigen (TAG-72). Using immunohistochemical techniques, MAb B72.3 has shown reactivity with 100 percent of common epithelial ovarian carcinomas and endometrial carcinomas and non-reactivity with normal adult tissues, with the exception of normal secretory endometrium. B72.3 appears to be a valuable immunocytologic adjunct, with greater than 90 percent of effusions and fine-needle aspiration biopsies from gynecologic carcinomas showing reactivity. Using a serum assay developed to detect the presence of the TAG-72 antigen, 48 percent of patients with ovarian carcinoma demonstrated TAG-72-positive sera versus 1 percent of control sera. 131I-labeled MAb B72.3 IgG and gamma scanning have been used for the in situ detection of metastatic carcinoma. Twelve of 15 patients with ovarian carcinoma showed positive gamma scans, and approximately 80 percent of the lesions demonstrated specific localization of the antibody. These studies indicate the potential utility of MAb B72.3 in the diagnosis of gynecologic carcinoma.     

Expression,glycosylation and function of recombinant anti‐colorectal cancer mAb CO17‐1A in SfSWT4 insect cells

Colorectal cancer is a commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17‐1A recognizes the tumor‐associated antigen GA733, a cell surface glycoprotein highly expressed in colorectal carcinoma cell, which is considered to be applicable for diagnosis and therapeutic treatment against colorectal cancer. In addition antibodies are glycoproteins, efficiently recognize and eliminate specific pathogenic and disease antigens. We have currently established baculovirus insect cell expression system to produce anti‐colorectal cancer mAb CO17‐1A. In this study, mAb CO17‐1A was expressed in the transgenic insect cell line SfSWT4, in which glycosylation processing pathway has been humanized. Immunoblot confirmed that mAb CO17‐1A properly expressed in SfSWT4 insect cells. mAb CO17‐1A was purified using protein G affinity column. In addition, MALDI‐TOF verified that the mAb CO17‐1A fused to KDEL, endoplasmic reticulum (ER) retention signal (mAb CO17‐1AK) had high mannose type of glycan structure. Migration assay showed that insect cell‐derived mAb CO17‐1AK (mAb^I CO17‐1AK) with high mannose type of glycan structure was effective as mammalian‐derived mAb CO17‐1A (mAb^M CO17‐1A) in inhibition of metastasis. Kinetic analysis of antigen‐antibody interaction using Surface Plasmon Resonance (SPR) confirmed that mAb^I CO17‐1AK is efficient to interact with antigen GA733 as mAb^M CO17‐1A. These results suggest that the insect cell expression system with the SfSWT4 possibly can be used as a useful alternative way to produce full‐size mAb for cancer immunotherapy.     

Differential localization within human kidney of five membrane proteins expressed on acute lymphoblastic leukemia cells

The reactivity of a panel of monoclonal antibodies (MAb) produced against non-T, non-B acute lymphoblastic leukemia cells was investigated by immunoperoxidase staining of sections of normal human kidney. The antigens of kidney reactive with the MAb were isolated by immunoaffinity chromatography and were purified further by immunoprecipitation. Two MAb, 44D7 and 44H9, reacted with determinants found exclusively on the basolateral membranes of proximal convoluted tubules. The 44D7 antigen isolated from kidney was biochemically similar to that isolated from leukemic cells. It was resolved as a multimeric complex with an apparent m.w. of 120,000 when analyzed by SDS-PAGE under nonreducing conditions. The 44H9 antigen has not yet been purified from kidney. MAb 50B4 reacted with components of the interstitium and with the mesangium of glomeruli. It immunoprecipitated a polypeptide chain of apparent m.w. 85,000, similar to that of the 50B4 antigen isolated from leukemic cells. MAb 44G4 also reacted with the mesangium of glomeruli and with the interstitium of the kidney. However, the endothelium of glomerular capillaries and of interstitial blood vessels has also reacted with MAb 44G4. The kidney antigen recognized by MAb 44G4 was characterized as a major polypeptide band, 95,000 m.w. (reduced) and 125,000 m.w. (nonreduced), a subunit structure analogous to the 44G4 antigen isolated from leukemic cells. MAb 44E3 reacted with all cellular elements of glomeruli, tubules, blood vessels, and interstitium. Two polypeptide chains of apparent m.w. 94,000 and 90,000 were immunoprecipitated from kidney by MAb 44E3, while a single polypeptide chain of 94,000 m.w. was precipitated from leukemic cells. Our results describe five new antigens with distinctive cellular distributions within kidney.     

Epitope Specificity of a Monoclonal Antibody Generated against the Dissociated CFA/I Fimbriae of Enterotoxigenic Escherichia coli

A monoclonal antibody (MAb 84) raised against the dissociated CFA/I fimbriae of enterotoxigenic Escherichia coli was characterized with regard to antigen binding and epitope specificity. Enzyme-linked immunosorbent assay (ELISA) showed that MAb 84 had higher affinity to CFA/I subunits than to intact CFA/I fimbriae and recognized a Salmonella flagellin carrying an insert corresponding to amino acids 32 to 45 of the CFA/I subunit. Fine epitope mapping based on the Pepscan technique showed that the peptide ³⁹TFESY⁴³, derived from the sequence of the mature CFA/I subunit, was specifically recognized by MAb 84. The ³⁹TFESY⁴³ sequence is probably not accessible on the surface of the native CFA/I fimbriae since MAb 84 did not bind to intact fimbriae as evaluated in inhibition ELISA tests. Moreover, MAb 84 did not agglutinate fimbriated ETEC cells nor inhibit CFA/I-mediated hemagglutination or the adhesion to Caco-2 cells.     

Plant-derived EpCAM antigen induces protective anti-cancer response

Immunotherapy holds great promise for treatment of infectious and malignant diseases and might help to prevent the occurrence and recurrence of cancer. We produced a plant-derived tumor-associated colorectal cancer antigen EpCAM (pGA733) at high yields using two modern plant expression systems. The full antigenic domain of EpCAM was efficiently purified to confirm its antigenic and immunogenic properties as compared to those of the antigen expressed in the baculovirus system (bGA733). Recombinant plant-derived antigen induced a humoral immune response in BALB/c mice. Sera from those mice efficiently inhibited the growth of SW948 colorectal carcinoma cells xenografted in nude mice, as compared to the EpCAM-specific mAb CO17-1A. Our results support the feasibility of producing anti-cancer recombinant vaccines using plant expression systems.