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.     

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.     

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995     

Isolation and characterization of a carcinoma-associated antigen

GA733 is a murine IgG2a monoclonal antibody (MAb) against human gastric carcinoma and is highly tumoricidal in nude mice. The GA733 antigen is a cell surface protein with two subunits of 30,000 and 40,000 daltons. The antigen isolated by immunoaffinity chromatography consists mainly of the 30,000-dalton subunit which bears the GA733 epitope. This subunit displayed several isoelectric points between 6.9 and 7.7. Anti-colon carcinoma MAb 17-1A also detects this antigen, but probably binds to a different epitope.     

Neuronal autoantibody titers in the course of small-cell lung carcinoma and platinum-associated neuropathy

The aims of this study were to investigate, in patients with newly diagnosed small-cell lung carcinoma (SCLC), whether or not there may be a relationship between the presence, type or titer of circulating neuronal autoantibodies and (i) the extent of SCLC dissemination at presentation, (ii) the development of peripheral neuropathy during platinum chemotherapy, (iii) survival time. We studied stored serum from 58 patients with uncomplicated SCLC who had participated in two trials conducted by the North Central Cancer Treatment Group (NCCTG); 29 had extensive disease and 29 had limited disease. No patient had neuropathy or other neurological or paraneoplastic problems at the time of enrollment but each group included 14 or 15 patients respectively who developed peripheral neuropathy in the course of chemotherapy. We tested five consecutive serum specimens from each patient in blinded fashion by (i) an indirect immunofluorescence assay optimized to detect neuron-restricted nuclear and cytoplasmic antibodies (triple substrate of mouse cerebellum, gut and kidney), and (ii) immunoprecipitation assays to detect neuronal Ca²⁺-channel-binding antibodies (N-type and P/Q-type). Sera that were positive by immunofluorescence were analyzed further by Western blotting. Neuronal autoantibodies were significantly more frequent in patients who had limited SCLC at presentation (12/29 or 41% positive) than in those with extensive SCLC (5/29 or 17% positive, P = 0.02). Neuronal autoantibodies of nuclear or cytoplasmic specificity were found in 50% of the seropositive patients with limited SCLC (21% of the total group), but in no patient with extensive SCLC (P = 0.01). The frequency of neuronal autoantibodies did not differ significantly among patients who did and did not develop peripheral neuropathy. Titers fell progressively during chemotherapy and did not rise again when peripheral neuropathy became clinically evident. This argues against a synergism between drug toxicity and neuronal autoimmunity as the mechanism of platinum-associated peripheral neuropathy. Seropositivity for neuronal autoantibodies did not affect the survival of patients with either limited or extensive SCLC. It is conceivable that the immunosuppression attendant on combined cisplatin/etoposide therapy cancels a pre-existing protective antitumor immune response (presumably cytotoxic-T-cell-mediated) for which the nuclear and cytoplasmic paraneoplastic IgG autoantibodies serve as a surrogate marker. Testing of this hypothesis would require the survival of seropositive and seronegative patients to be compared in a larger trial, using a therapeutic modality that does not compromise immunocompetence. Received: 20 November 1998 / Accepted: 6 January 1999     

Detection of Autoantibodies to Annexin A11 in Different Types of Human Cancer

Introduction  Annexin A11 was previously identified as an autoantigen in 4.1–10.1% of patients with various systemic autoimmune diseases. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed to investigate the occurrence and features of anti-annexin A11 autoantibodies in sera from patients with different types of cancer. Methods  The recombinant protein of GST fused to the N-terminal domain (1–175 residues) of human annexin A11 was expressed and used as antigen in ELISA. A total of 246 serum specimens were analyzed, which includes sera from healthy women (77), patients with ovarian cancer (72), breast cancer (18), colon cancer (19), pancreatic cancer (20), prostate cancer (20), and diabetes (20). Results  The overall titer of anti-annexin A11 autoantibodies in ovarian cancer patients (or primary tumors only) was found much higher than that in healthy controls (P < 0.05). At the cut-off value designating positive reaction, anti-annexin A11 autoantibodies were detected in 12.5% (5/40) of primary ovarian cancer patients with a significant difference from 2.6% (2/77) of the healthy controls (P < 0.05), but only in 6.25% (2/32) of recurrent tumors. ROC curve demonstrated the potential diagnostic value of anti-annexin A11 autoantibodies in primary ovarian cancer patients with an AUC of 0.62 (0.52–0.73). Anti-annexin A11 autoantibodies were also detected in 5.26% (1/19) of colon cancer and 10% (2/20) of diabetes patients but without significant difference from the healthy controls. Conclusion  A convenient assay to detect anti-annexin A11 autoantibodies in patients was developed, and the experimental data are promising but need to be expanded to address their biological/clinical relevance.     

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     

Pharmacological administration of granulocyte/macrophage-colony-stimulating factor is of significant importance for the induction of a strong humoral and cellular response in patients immunized with recombinant carcinoembryonic antigen

Eighteen colorectal carcinoma patients without macroscopic disease after surgery were immunized using recombinant (r) human (h) carcinoembryonic antigen (CEA) with (n = 9) or without (n = 9) the addition of soluble granulocyte/macrophage-colony-stimulating factor (GM-CSF). The dose of rhCEA per immunization was 100 μg (n = 6), 316 μg (n = 6) or 1000 μg (n = 6). rhCEA was given s.c. on day 1 and 80 μg/day of GM-CSF s.c. on days 1–4. The schedule was repeated six times during a period of 9 months. All patients in the GM-CSF group developed a strong rhCEA-dose-dependent IgG antibody response while only one-third of the non-GM-CSF patients mounted a weak antibody response. All patients (9/9) in the GM-CSF group developed a strong rhCEA-specific proliferative T cell response as well as type I T cells (interferon γ secretion). In 45% of the patients also a weak type II T cell response (interleukin-4 secretion) was evoked. Both MHC-class-I- and -II restricted rhCEA-specific T cells were noted. A specific cellular response (proliferation and/or cytokine secretion) against native hCEA could be found in 8/9 patients in the GM-CSF group, although at a significantly lower level than against rhCEA. In the non-GM-CSF group a weak rhCEA-specific T cell response was induced. Three patients had a proliferative response, 4 patients type I T cells and 6 patients type II T cells. No signs of autoimmune reactions were noted. Local pharmacological administration of GM-CSF seemed to be a prerequisite for the induction of a strong immunity against baculovirus-produced hCEA protein. However, the cellular response against native CEA was of a significantly lower magnitude. Received: 13 November 1997 / Accepted: 21 May 1998     

A new type of anti-ganglioside antibodies present in neurological patients

High titers of anti-GA1 antibodies have been associated with neurological syndromes. In most cases, these antibodies cross-react with the structurally related glycolipids GM1 and GD1b, although specific anti-GA1 antibodies have also been reported. The role of specific anti-GA1 antibodies is uncertain since the presence of GA1 in the human nervous system has not been clarified. A rabbit was immunized with GD1a and its sera were screened for antibody reactivity by standard immunoassay methods (HPTLC-immunostaining and ELISA). Anti-GD1a antibodies were not detected but, unexpectedly, anti-GA1 IgG-antibodies were found. Antibody binding to GA1 was inhibited by soluble GA1 but also by GD1a. These results indicate that the rabbit produced antibodies that recognize epitopes present on the glycolipids, that are absent or not exposed on solid phase adsorbed GD1a. We investigated the presence of these unusual anti-ganglioside antibodies in normal and neurological patient sera. Approximately, 10% of normal human sera contained low titer of specific anti-GA1 IgG-antibodies but none of them recognized soluble GD1a. High titers of IgG-antibodies reacting only with GA1 were detected in 12 patient sera out of 325 analyzed. Of these, 6 sera showed binding that was inhibited by soluble GD1a and four of them also by GM1. This new type of anti-ganglioside antibodies should be considered important elements for understanding of the pathogenesis of these diseases as well as their diagnosis.     

An EGP-2/Ep-CAM-expressing transgenic rat model to evaluate antibody-mediated immunotherapy

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), also known as 17-1A or Ep-CAM, is a 38-kDa transmembrane antigen, commonly used for targeted immunotherapy of carcinomas. Although strongly expressed by most carcinomas, EGP-2 is also expressed in most simple epithelia. To evaluate treatment-associated effects and side-effects on tumor and normal tissue respectively, we generated an EGP-2-expressing transgenic Wistar rat. To express the cDNA of the EGP-2 in an epithelium-specific manner, the 5′ and 3′ distal flanking regions of the human keratin 18 (K18) gene were used. EGP-2 protein expression was observed in the liver and pancreas, whereas EGP-2 mRNA could also be detected in lung, intestine, stomach and kidney tissues. In this rat, EGP-2-positive tumors can be induced by injecting a rat-derived carcinoma cell line transfected with the GA733-2 cDNA encoding EGP-2. Transgenic rats were used to study specific in vivo localization of an i.v. anti-EGP-2 monoclonal antibody, MOC31, applied i.v. Immunohistochemical analyses showed the specific localization of MOC31 in s.c. induced EGP-2-positive tumors, as well as in the liver. In contrast, in EGP-2-transgenic rats, MOC31 did not bind to EGP-2-negative tumors, the pancreas, or other normal tissues in vivo. In conclusion, an EGP-2-transgenic rat model has been generated that serves as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities. Received: 9 March 1999 / Accepted: 6 May 1999     

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 of mice with fucosyl-GM1 conjugated with keyhole limpet hemocyanin results in antibodies against human small-cell lung cancer cells

Fucosyl-GM1 (Fuc-GM1) [Fucα1 → 2Galβ1 → 3GalNAcβ1 → 4(NeuAcα2-3)Galβ1 → 4Glcβ1 → O-Cer] is a small-cell-lung-cancer (SCLC)-associated ganglioside initially defined by the murine monoclonal antibody F12. On the basis of its known distribution, Fuc-GM1 is a potential target for active immunotherapy in SCLC patients. Fuc-GM1 has been extracted and purified from bovine thyroid. The immunogenicity of Fuc-GM1 was tested in mice either alone, mixed with carrier protein keyhole limpet hemocyanin (KLH) or covalently linked with KLH, plus immunological adjuvant QS-21. The Fuc-GM1-KLH conjugate plus QS-21 adjuvant was found to be optimal. It induced consistent IgM and IgG enzyme-linked immunosorbent assay (ELISA) titers against Fuc-GM1. These antibodies were strongly reactive with the strongly Fuc-GM1-positive rat hepatoma cell line H4-II-E, and they were moderately reactive with the moderately positive human SCLC cell line H146 by flow cytometry and complement-mediated lysis. Both ELISA and fluorescence-activated cell sorting (FACS) reactions were inhibited with Fuc-GM1or H4-II-E but not with the structurally related ganglioside GM1 or Fuc-GM1-negative colon cancer cell line LS-C. On the basis of these results, a vaccine comprising Fuc-GM1-KLH plus QS-21 is being prepared for testing in patients with SCLC. Received: 25 March 1999 / Accepted: 5 August 1999     

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.     

Anti-self antibodies selected from a human IgD heavy chain repertoire: a novel approach to generate therapeutic human antibodies against tumor-associated differentiation antigens

Human antibodies were isolated by phage display from a naturally expressed human antibody repertoire. Antibody selection was carried out against the epithelial cell adhesion molecule (EpCAM) or 17-1A antigen, that in a clinical trial had been successfully used as a target for antibody therapy of minimal residual colorectal cancer. VH chains were selected from the human IgD repertoire expressed on naive B2 and autoreactive B1 lymphocytes. By guiding the selection through a murine template antibody, two EpCAM-specific human antibodies, HD69 and HD70, were obtained that closely resembled the murine therapeutic 17-1A antibody in their binding properties when expressed as complete huIgG1 molecules in CHO cells. However, both human antibodies recruited human cytotoxic effector cells far more efficiently than the murine 17-1A antibody used for clinical trials. Therefore, and in view of the long in vivo half-life of human IgG1 antibodies, HD69 and HD70 are regarded as highly promising third generation versions of the murine therapeutic antibody. Because of their origin from an evolutionary conserved germline VH repertoire, they are expected to exhibit minimal immunogenicity in patients. Received: 16 November 2000 / Accepted: 11 January 2001     

Frequent loss of heterozygosity in the β2-microglobulin region of chromosome 15 in primary human tumors

Downregulation or total loss of HLA class I expression on tumor cells is known as a mechanism of cancer immune escape. Alterations of the HLA phenotype are frequently due to mutations affecting genes encoding the HLA class I heavy chains located on chromosome 6p21 or the β2-microglobulin (β2m) gene encoding the light chain of the HLA complex located on chromosome 15q21. Frequently irreversible total loss of HLA class I molecules is due to the coincidence of two molecular events, the mutation of one β2m gene and the loss of the second copy. The latter is detectable as loss of heterozygosity (LOH) of microsatellite markers in the β2m region on chromosome 15q21 (LOH-15q21). Thus, LOH-15q21 might be an important event in the processes of HLA class I downregulation and total loss. Here we studied the frequency of LOH-15q21 in tumor tissues of different entities. By determining the status of heterozygosity of two microsatellite markers we detected LOH-15q21 in 44% of bladder carcinomas (n = 69), in 35% of colon carcinomas (n = 95), in 16% of melanomas (n = 70) but only in 7% of renal cancers (n = 45). Moreover, we observed a frequent coincidence of LOH-15q21 and LOH-6p21 in colorectal carcinoma, bladder carcinoma and melanoma, but not for renal carcinoma. We believe that the high incidence of LOH-15q21 in some malignancies and especially the coincidence of LOH-15q21 and LOH-6p21 might have a strong impact on tumor immunogenicity and on the efficiency of cancer immunotherapy.     

The role of monocytes and natural killer cells in mediating antibody-dependent lysis of colorectal tumour cells

Monocytes and natural killer (NK) cells are known to be important effector cell populations in mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Purified monocyte and NK effector cell populations, from normal and colorectal cancer (CRC) patients, together with a number of murine (17-1A and 323/A3) and their chimaeric (c17-1A) or humanised (3622W94) equivalents, and chimaeric (c) SF25 were compared for their ability to mediate ADCC of colorectal tumour cells. The chimaeric and humanised antibodies were significantly better at mediating tumour lysis than their murine equivalents with all-effector populations. When effector cells from CRC patients were used the cSF25 antibody was significantly better than 3622W94 (P < 0.02) which, in turn, was significantly better than c17-1A (P < 0.03). Depletion of NK cells produced a decrease in specific tumour lysis with all antibodies. In addition a higher rate of NK cell death was observed in CRC patients during the assay than in normal controls. The chimaeric and humanised antibodies stained a similar percentage of the HT-29 target cells (>80%), but 3622W94 bound to significantly more cells from primary tumour biopsies than cSF-25 (P = 0.001). Together, the results suggest that NK cells are the most important effector cell type mediating ADCC in vitro, that there is some impairment of NK function in CRC patients, and that cSF25 is the most potent antibody. For use in vivo the anti-Ep-CAM antibody 3622W94 would appear to be the most suitable reagent for further study. Received: 3 June 1999 / Accepted: 22 July 1999     

Development of a new vaccine formulation that enhances the immunogenicity of tumor-associated antigen CaMBr1

Aberrant glycosylation is one of the most constant traits of malignant cells. The CaMBr1 hexasaccharide antigen, originally defined on the human breast carcinoma cell line MCF7, is expressed on some normal tissues but overexpressed in a high percentage of human breast, ovary, prostate and lung carcinomas. CaMBr1 overexpression is associated with poor prognosis. The epitope consists of the tetrasaccharide Fuc(α1-2)Gal(β1-3)GalNAc(β1-3)Galα-O-spacer, which has recently become available as a synthetic oligosaccharide. Here we report the CaMBr1 tetrasaccharide conjugation to two different carrier proteins (CRM197 and KLH) and the evaluation of conjugate immunogenicity in mice following their administration in various vaccine formulations with two adjuvants (MPL-SE and Detox-PC). Radioimmunoassay to determine the level and isotype of anti-tetrasaccharide antibodies in mouse sera, and cytofluorimetric analysis and ⁵¹Cr-release assay on human tumor cells, to evaluate specificity of binding and complement-dependent lysis respectively, identified CaMBr1-CRM197, in association with the MPL-SE adjuvant, as the best vaccine formulation. This combination induced (1) production of tetrasaccharide-specific antibodies, with negligible side-effects; (2) antibodies with complement-mediated cytotoxic activity on human CaMBr1-positive cells and (3) a high titer of IgG1 detected in sera obtained 3 months after the first injection, indicating that the anti-tetrasaccharide antibody response was mediated by T cell activation. The availability of CaMBr1-glycoconjugate in the minimal and functional antigenic structure and the identification of an efficacious vaccine formulation opens the way to exploring the activity of this glycoconjugate in a clinical setting. Received: 20 January 2000 / Accepted: 16 March 2000     

Preparation and application of anti-idiotypic antibody against anti-gibberellin A4 antibody.

A monoclonal anti-idiotypic antibody was raised against anti-gibberellin A4 (GA4) antibody, which recognizes biologically active gibberellins such as GA1 and GA4 specifically. Amino acid sequences of variable regions of both anti-GA4 and anti-idiotypic antibodies were analyzed. By using the property of the anti-idiotypic antibody to compete with GA1/4 in binding to the anti-GA4 antibody, we successfully applied the anti-idiotypic antibody to ELISA as a tracer for measuring GA1/4. The single-chain Fv (scFv) gene of the anti-idiotypic antibody was constructed, and scFv expressed in E. coli showed binding activity to anti-GA4 antibody. These results suggest the possible application of anti-idiotypic antibody as a handy and stable source of an enzymatic tracer for ELISA by production of fusion protein of the scFv and an appropriate enzyme.     

Autoantibodies against chaperonin CCT in human sera with rheumatic autoimmune diseases: comparison with antibodies against other Hsp60 family proteins

Chaperonin CCT containing t-complex polypeptide 1 is a cytosolic molecular chaperone that assists in the folding of actin, tubulin, and other proteins and is a member of the 60-kDa heat shock protein (Hsp60) family. We examined antibody titers against human CCT and other Hsp60 family members in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematodes, Sj?gren syndrome, and mixed connective tissue disease. Autoantibody titers against not only human mitochondrial Hsp60 but also CCT were significantly higher in the sera of patients with rheumatic autoimmune diseases than in healthy control sera. Although immunoglobulin G (IgG) titers against Escherichia coli GroEL were high in all the groups of sera tested, no significant differences in anti-GroEL responses were detected between patients and healthy controls. IgG titers against mycobacterial Hsp65 showed a similar pattern to titers of autoantibodies recognizing GroEL. Immunoabsorption experiments demonstrated that most of the autoantibodies recognizing CCT were cross-reactive with mitochondrial Hsp60, E coli GroEL, and mycobacterial Hsp65. Although most of the anti-Hsp60 IgG recognized CCT, anti-GroEL (or antimycobacterial Hsp65) IgG contained antibodies specific for GroEL (or mycobacterial Hsp65) in addition to antibodies cross-reactive with CCT and Hsp60. Results from immunoblot analyses, together with weak (15% to 20%) amino acid sequence identities between CCT and the other Hsp60 family members, suggested that CCT-reactive autoantibodies recognize conformational epitopes that are conserved among CCT and other Hsp60 family members.