Selection of GM2, fucosyl GM1, globo H and polysialic acid as targets on small cell lung cancers for antibody mediated immunotherapy.

Glycolipids GM2, GD2, GD3, fucosyl GM1, sialyl Lewis a (sLe(a)) and globo H, and polysialic acid on embryonal NCAM, are cell-surface antigens expressed on small cell lung cancer (SCLC) biopsy specimens. They are all candidates for inclusion in a polyvalent, antibody-inducing vaccine or for adoptive therapy with monoclonal antibodies (mAbs) against SCLC. To identify the minimum optimal combination of target antigens on SCLC and to confirm that antibodies against this combination might be able to mediate complement activation and lysis in the majority of cases, we tested ten SCLC cell lines with fluorescence activated cell sorter (FACS) and complement dependent cytotoxicity (CDC) assays using mAbs against these seven target antigens individually or pooled in different combinations. We find that (1) none of these mAbs demonstrated strong FACS reactivity with more than 6 of the 10 cell lines, (2) no mAb had strong CDC reactivity with more than 4 of the cell lines, (3) when the mAbs were pooled, nine cell lines were strongly positive by FACS and nine cell lines were strongly positive by CDC, and (4) mAbs against GM2, FucGM1, globo H and polysialic acid was the minimum optimal combination for inducing FACS reactivity. The addition of mAbs against sLe(a), GD2 and GD3 had no additional impact by FACS and only minimal additional impact in CDC assays. H345, the only cell line that had less than 30% CDC with the four mAb pool was strongly positive by FACS. To understand the lack of correlation between FACS and CDC in the case of H345, the ten cell lines were screened for expression of complement resistance factors CD55 and CD59. Three cell lines were strongly positive for CD55 and eight were strongly positive for CD59. Overall, no correlation was seen between expression of either of these factors on the ten cell lines and sensitivity to CDC. In the case of H345 however, complement resistance of H345 is demonstrated to be mediated primarily by CD59, and in the presence of mAb against CD59, the four mAb MEM-43 pool induced strong (94%) CDC. CD59 inhibits membrane attack complex formation but not activation of earlier complement components. Consequently, all ten cell lines are good targets for complement activation by the four antibody pool and for elimination by effector mechanisms including complement mediated inflammation and opsonization. These findings support our plan to develop a tetravalent vaccine against SCLC targeting GM2, fucosyl GM1, globo H and polysialic acid.

Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: a phase I trial.

The carbohydrate antigen globo H commonly found on breast cancer cells is a potential target for vaccine therapy. The objectives of this trial were to determine the toxicity and immunogenicity of three synthetic globo H-keyhole limpet hemocyanin conjugates plus the immunologic adjuvant QS-21. Twenty-seven metastatic breast cancer patients received five vaccinations each. The vaccine was well tolerated, and no definite differences were observed among the three formulations. Serologic analyses demonstrated the generation of IgM antibody titers in most patients, with minimal IgG antibody stimulation. There was significant binding of IgM antibodies to MCF-7 tumor cells in 16 patients, whereas IgG antibody reactivity was observed in a few patients. There was evidence of complement-dependent cytotoxicity in several patients. Affinity column purification supported the specificity of IgM antibodies for globo H. On the basis of these data, globo H will constitute one component of a polyvalent vaccine for evaluation in high-risk breast cancer patients.

Keyhole limpet hemocyanin conjugate vaccines against cancer: the Memorial Sloan Kettering experience.

Passively administered and actively induced antibodies have been associated with the eradication of circulating tumor cells and micrometastases in mice and humans. We have identified a series of cell surface carbohydrate and peptide antigens on melanomas, sarcomas, and cancer of the breast, prostate. ovary, and lung tissues. We found that breaking tolerance toward these tumor antigens was best achieved using vaccines containing antigens chemically conjugated to keyhole limpet hemocyanin (KLH) plus a potent immunological adjuvant (QS-21). To date, by using this approach to vaccination. antibodies have been induced in patients against glycolipid antigens GM2, GD2, GD3, FucosylGM1, Globo H, and Lewis Y, and glycoprotein (mucin) antigens Tn, sialyl Tn. TF, and MUC1. More recently, in a comparative study we investigated the T cell response induced by MUCI-KLH conjugates. Although a MUC1-specific T cell response was not consistently detected in any patient, the role of KLH in orienting the cytokine environment was crucial. We were able to confirm that KLH in these conjugate vaccines induces a Th1 T cell response as demonstrated by the high anti-KLH INF-gamma secretion and the IgGI and IgG3 subclasses of this high titer IgG antibodies induced. Clinical trials using KLH conjugated glycolipid and glycoprotein vaccines, are currently ongoing. These range from phase I/II single antigens trials with glycosilated MUC1, polysialic acid, synthetic Fucosyl GMI and GD2, to phase II trials with a polyvalent vaccine containing six or seven antigens. Randomized phase II trials with polyvalent vaccines are planned for initiation in 2001-2002 in patients with ovarian, breast, and prostate cancer.

Effect of immunological adjuvant combinations on the antibody and T-cell response to vaccination with MUC1-KLH and GD3-KLH conjugates.

A year ago we described a comparison of 19 immunological adjuvants for their ability to augment antibody and T-cell responses against vaccines containing two cancer antigens, GD3 ganglioside and MUC1 peptide, covalently attached to keyhole limpet hemocyanin (KLH). As in our previous experience, the saponin fraction QS-21 was the most potent single adjuvant but several other adjuvants also had potent adjuvant activity. Induction of an immune response against cancer antigens is generally difficult because these antigens are autoantigens. To get maximal benefit from the adjuvant component of cancer vaccines we have now tested whether combinations of the optimal adjuvants induced an improved immune response compared to QS-21 alone. Since over the intervening year a new semi-synthetic saponin adjuvant (GPI-0100) containing the dodecylamide derivative of hydrolyzed naturally-occurring saponins had become available, this was tested as well. Twelve different adjuvant combinations and GPI-0100 were compared for their ability to augment (1) antibody responses against GD3 and MUC1 and (2) T-cell responses against GD3, MUC1 and KLH. GPI-0100 and five adjuvant combinations were superior to QS-21 alone for induction of IgM and IgG antibodies against MUC1 and/or GD3: QS-21 plus bacterial nucleotide CpG, QS-21 plus monophosphoryl lipid A (MPL), QS-21 plus non-ionic block copolymer CRL-1005, QS-21 plus Titermax and Titermax plus CpG. Antibody responses were documented both by ELISA against purified antigens and by FACS for cell surface reactivity. There was no evidence for T-cell immunity against GD3 or MUC1. The antibody responses against GD3 and MUC1 were, however, strongly correlated with IFN-gamma release and DTH against KLH. These results demonstrate that combinations of immunological adjuvants are able to augment antibody and T-cell responses to these conjugates beyond that attainable with QS-21 alone, and again confirm the absolute necessity of potent adjuvants or adjuvant combinations for optimal immunogenicity with conjugate vaccines.

A second generation synthesis of the MBr1 (globo-H) breast tumor antigen: new application of the n-pentenyl glycoside method for achieving complex carbohydrate protein linkages.

A new synthesis of the hexasaccharide MBr1 antigen (globo-H) is reported. A revised construction with improved efficiency was necessary because an anti-cancer vaccine containing this antigen is entering phase II and phase III clinical trials for prostate cancer. The key feature of this second generation synthesis is the preparation of globo-H as its n-pentenyl glycoside. This group serves as an anomeric protecting group and as a linker for bioconjugation to carrier protein. The resultant synthesis allows for the production of suitable quantities of globo-H for clinical trials.

Immunization of ovarian cancer patients with a synthetic Lewis(y)-protein conjugate vaccine: a phase 1 trial.

As the initial step in developing carbohydrate-based vaccines for the treatment of ovarian cancer patients in an adjuvant setting, 25 patients were immunized with a Lewis(y) pentasaccharide (Le(y))-keyhole limpet hemocyanin (KLH)-conjugate vaccine together with the immunological adjuvant QS-21. Four different doses of the vaccine, containing 3, 10, 30, and 60 microg of carbohydrate were administered s.c. at 0, 1, 2, 3, 7, and 19 weeks to groups of 6 patients. Sera taken from the patients at regular intervals were assayed by ELISA for reactivity with naturally occurring forms of Le(y) (Le(y)-ceramide and Le(y) mucin) and by flow cytometry and a complement-dependent cytoxicity assay for reactivity with Le(y)-expressing tumor cells. The majority of the patients (16/24) produced anti-Le(y) antibodies as assessed by ELISA, and a proportion of these had strong anti-tumor cell reactivity as assessed by flow cytometry and complement-dependent cytotoxicity. One serum, analyzed in detail, was shown to react with glycolipids but not with glycoproteins or mucins expressed by ovarian cancer cell line OVCAR-3. The vaccine was well tolerated and no gastrointestinal, hematologic, renal, or hepatic toxicity related to the vaccine was observed. On the basis of this study, Le(y)-KLH should be a suitable component for a polyvalent vaccine under consideration for the therapy of epithelial cancers.

Autoimmune and antitumor consequences of antibodies against antigens shared by normal and malignant tissues.

There is now a considerable body of information documenting the autoimmune consequences of antibodies induced by growing malignancies, or by passively administered and actively induced antibodies, in cancer patients against antigens shared by normal and malignant tissues. This provides a rich source of information addressing the consequences of autoantibodies against a range of antigens. Antibodies against cell-surface or intracellular antigens in the central nervous system (CNS) or on epithelial surfaces of normal tissues do not generally result in autoimmunity, but the same types and titers of antibodies against cell surface antigens in the subepidermal skin, peripheral nerves, blood, or vascular sites such as the spleen and bone marrow readily induce autoimmunity. The blood brain barrier of the CNS and apical antigen expression and the basement membrane in epithelial tissues, may protect these sites from antibody induced damage. Cancer cells, however, are protected by neither unidirectional antigen expression nor basement membranes. Vaccine induced antibodies against a variety of cancer cell surface antigens have been associated with prevention of tumor recurrence in preclinical models and in vaccinated cancer patients, in the absence of demonstrable autoimmunity. This forms the basis for a series of ongoing Phase III trials with single or polyvalent antigen cancer vaccines designed for optimal antibody induction.

Vaccination of high-risk breast cancer patients with mucin-1 (MUC1) keyhole limpet hemocyanin conjugate plus QS-21.

Our objective was to determine whether an immune response can be generated against MUC1 peptide and against tumor cell MUC1 after vaccination with MUC1-keyhole limpet hemocyanin (KLH) conjugate plus QS-21 in breast cancer patients. Nine patients with a history of breast cancer but without evidence of disease were treated with MUC1-KLH conjugate plus QS-21, containing 100 microg of MUC1 and 100 microg of QS-21. s.c. vaccinations were administered at weeks 1, 2, 3, 7, and 19. Peripheral blood was drawn at frequent intervals to assess antibody titers. Skin tests were placed at weeks 1, 3, 9, and 21 to determine delayed type hypersensitivity reactions. Common toxicities included a local skin reaction at the site of the vaccine, usually of 4-5 days' duration, and mild flu-like symptoms usually of 1-2 days' duration. High IgM and IgG antibody titers against synthetic MUC1 were detected. IgG antibody titers remain elevated from a minimum of 106-137 weeks after the first vaccination. Binding of IgM antibody to MCF-7 tumor cells was observed in seven patients, although there was minimal binding of IgG antibody. Two patients developed significant antibody titers post-high-dose chemotherapy and stem cell reinfusion. There was no evidence of T cell activation. This MUC1-KLH conjugate plus QS-21 was immunogenic and well tolerated in breast cancer patients. Additional trials are ongoing to determine the optimal MUC1 peptide for use in larger clinical trials. Further investigation of vaccine therapy in high-risk breast cancer is warranted.

Polyclonal antibodies from patients immunized with a globo H-keyhole limpet hemocyanin vaccine: isolation, quantification, and characterization of immune responses by using totally synthetic immobilized tumor antigens.

We have previously reported on a carbohydrate-based vaccine program for immunotherapy in cancer patients. One such vaccine, based on the globo H antigen conjugated to the protein keyhole limpet hemocyanin (KLH), has been in clinical evaluation. Although this and other carbohydrate vaccines have been shown to induce antibody responses, there are currently no quantitative data on the antibody levels achieved in immunized patients by these or other anti-cancer vaccines. We report herein an efficient route to complex synthetic oligosaccharides attached to an affinity matrix for identifying and isolating antibodies elicited against such a carbohydrate-based vaccine in humans. Pre- and postvaccination profiles from serum samples of patients immunized with globo H-KLH were compared. All anti-globo H antibody activity was efficiently separated from other serum constituents. The isolated antibodies were readily quantified, and their specificities were analyzed. Since no comparable data were available on antibodies resulting from the vaccination of other cancer patients, we compared the observed levels with those quoted in studies with bacterial polysaccharide vaccines that had been quantified. Remarkably, cancer patients immunized with globo H-KLH produce anti-globo H antibody levels often exceeding those formed by immunization with bacterial polysaccharides. In addition, substantial quantities of both IgG and IgM antibodies were elicited, clearly indicating a class switch to IgG. Taken together, these analyses serve to clarify several aspects of the immune response to the vaccine and give several new insights to the carbohydrate-based vaccination strategy. Furthermore, antibodies so isolated could well have applications in clinical therapy.

Induction of antibodies against GD3 ganglioside in melanoma patients by vaccination with GD3-lactone-KLH conjugate plus immunological adjuvant QS-21.

The gangliosides GD3, GD2 and GM2 are expressed on the cell surface of malignant melanomas, GD3 being the most abundant. We have shown that immunization of melanoma patients with GM2 adherent to Bacillus Calmette-Guerin (GM2/BCG) induced an IgM antibody response. Vaccines containing GM2-keyhole limpet hemocyanin (KLH) conjugate and the immunological adjuvant QS-21 induced a higher titer IgM response and consistent IgG antibodies. Patients with antibodies against GM2 survived longer than patients without antibody. On the other hand, our previous trials with GD3/BCG, GD3 derivatives including GD3-lactone (GD3-L)/BCG failed to induce antibodies against GD3. In our continuing efforts to induce antibody against GD3, we have immunized groups of 6 melanoma patients with GD3-KLH or GD3-L-KLH conjugates containing 30 microg of ganglioside plus 100 microg of QS-21 at 0, 1, 2, 3, 7 and 19 weeks. Prior to vaccination, no serological reactivity against GD3 or GD3-L was detected. After immunization, IgM and IgG antibodies were detected against both GD3 and GD3-L in the GD3-L group exclusively. The GD3-L-KLH vaccine induced IgM titers against GD3-L of 1:40-1/1,280 in all patients and IgG titers of 1/160-1/1,280 in 4 patients. These antibodies also strongly cross-reacted with GD3. ELISA reactivity was confirmed by immune thin-layer chromatography on GD3 and melanoma extracts. Sera obtained from 4 of these 6 patients showed cell surface reactivity by FACS and from 2 showed strong cell surface reactivity by immune adherence (IA) assay and complement lysis against the GD3 positive cell line SK-Mel-28.

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) [Fucalpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 4(NeuAcalpha2-3)Galbeta1 --> 4Glcbeta1 --> 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.

Comparison of the effect of different immunological adjuvants on the antibody and T-cell response to immunization with MUC1-KLH and GD3-KLH conjugate cancer vaccines.

While the importance of immunological adjuvants for optimal induction of antibody and T-cell responses against tumor antigens is clear, the relevant potency of different adjuvants is not clear. We have screened 19 different immunological adjuvants with KLH conjugate vaccines containing the two human cancer antigens (MUC1 peptide and GD3 ganglioside) in the mouse. ELISA assays for IgM and IgG antibody responses as well as proliferation and cytokine release (IFN-gamma and IL-4) for T-cell responses were performed. Six adjuvants stood out as being especially effective for induction of IgM and IgG antibodies against both MUC1 and GD3: QS-21, TiterMax, MoGM-CSF, MPL/DETOX and CpG ODN. Of these QS-21, MPL/DETOX and MoGM-CSF were uniformly effective at inducing potent proliferation and potent IFN-gamma and IL-4 responses against KLH while TiterMax and CpG ODN generated potent IFN-gamma responses but less potent proliferation or IL-4 release. Overall, as in our previous experience, QS-21 was the most effective adjuvant. There was no clear evidence for induction of T-cell immunity against either GD3 or MUC1 with any of the adjuvants. There was a strong correlation between the antibodies induced against MUC1 and GD3 with different immunological adjuvants and the strength of the IFN-gamma release against KLH. This suggests that the primary role of adjuvants in the context of these conjugate vaccines may be induction of higher levels of T-cell immunity against KLH, which then leads to higher levels antibody against the conjugated antigens.

Immunogenicity of a fucosyl-GM1-keyhole limpet hemocyanin conjugate vaccine in patients with small cell lung cancer.

Although small cell lung cancer (SCLC) is highly responsive to chemotherapy, relapses are common, and most patients die within 2 years of diagnosis. After initial therapy, standard treatment is observation alone. We have been investigating immunization against selected gangliosides as adjuvant therapy directed against residual and presumably resistant disease persisting after chemotherapy and irradiation. Previously, we reported that the presence of anti-GM2 ganglioside antibodies is associated with a prolonged disease-free survival in patients with melanoma, and that SCLC patients immunized with BEC2, an anti-idiotypic monoclonal antibody that mimics the ganglioside GD3, had a prolonged survival compared with historical controls. In the present trial, fucosyl-alpha1-2Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3) Galbeta1-4Glcbeta1-1Cer (Fuc-GM1), a ganglioside expressed on the SCLC cell surface, was selected as a target for active immunotherapy. Fuc-GM1 is present on most SCLCs but on few normal tissues. SCLC patients achieving a major response to initial therapy were vaccinated s.c. on weeks 1, 2, 3, 4, 8, and 16 with Fuc-GM1 (30 microg) conjugated to the carrier protein keyhole limpet hemocyanin and mixed with the adjuvant QS-21. Ten patients received at least five vaccinations and are evaluable for response. All patients demonstrated a serological response, with induction of both IgM and IgG antibodies against Fuc-GM1, despite prior treatment with chemotherapy with or without radiation. Posttreatment flow cytometry demonstrated binding of antibodies from patients' sera to tumor cells expressing Fuc-GM1. In the majority of cases, sera were also capable of complement-mediated cytotoxicity. Mild transient erythema and induration at injection sites were the only consistent toxicities. The Fuc-GM1-KLH + QS-21 vaccine is safe and immunogenic in patients with SCLC. Continued study of this and other ganglioside vaccines is ongoing.

Carbohydrate vaccines in cancer: immunogenicity of a fully synthetic globo H hexasaccharide conjugate in man.

The complex carbohydrate molecule globo H hexasaccharide has been synthesized, conjugated to keyhole limpet hemocyanin, and administered with the immunologic adjuvant QS-21 as a vaccine for patients with prostate cancer who have relapsed after primary therapies such as radiation or surgery. Globo H is one of several candidate antigens present on prostate cancer cells that can serve as targets for immune recognition and treatment strategies. The vaccine, given as five subcutaneous vaccinations over 26 weeks, has been shown to be safe and capable of inducing specific high-titer IgM antibodies against globo H. Its immunogenicity was confirmed in prostate cancer patients with a broad range of stages and tumor burdens. Observations of several patients who had evidence of disease relapse restricted to a rising biochemical marker, prostate-specific antigen (PSA), indicated that a treatment effect could occur within 3 months after completion of the vaccine therapy. This effect was manifested as a decline of the slope of the log of PSA concentration vs. time plot after treatment compared with values before treatment. Five patients continue to have stable PSA slope profiles in the absence of any radiographic evidence of disease for more than 2 years. The concept of using PSA slope profiles in assessing early treatment effects in biological therapies such as vaccines awaits further validation in phase II and III trials. The use of a variety of lesser known candidate glycoprotein and carbohydrate antigens in prostate cancer serves as a focus for the development of a multivalent vaccine of the treatment of relapsed prostate cancer in patients with minimal tumor burden.

Vaccines prepared with sialyl-Tn and sialyl-Tn trimers using the 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide linker group result in optimal antibody titers against ovine submaxillary mucin and sialyl-Tn-positive tumor cells.

Sialyl-Tn (STn) is an O-serine- or O-threonine-linked disaccharide [NeuAcalpha(2-->6)GalNAcalpha-O-Ser/Thr) expressed on mucins of most types of adenocarcinoma as single STn or clustered STn [STn(c)] epitopes. Though STn is expressed on some normal tissues it is relatively tumor-specific, especially in the clustered conformation. Clinical trials with STn-keyhole limpet hemocyanin (KLH) conjugate vaccines, prepared using reductive amination with a two-carbon linker group, have resulted in high titers against STn but lower titers against natural forms of STn (ovine submaxillary mucin, or tumor cells). To obtain antibodies of more appropriate specificity, we attempted to prepare STn(c)-KLH conjugates to establish their immunogenicity in mice in preparation for clinical trials; however, conjugation efficiency was poor when the same two-carbon linker was used, presumably because of steric hindrance. STn-KLH and STn(c)-KLH conjugates were prepared using the regular two-carbon or the recently developed more efficient longer heterobifunctional 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide (MMCCH) linkers, and the resulting immunogenicities in mice were compared. The highest titers against STn were seen with the STn-KLH conjugate with the two-carbon linker, and the highest titers against STn(c) were seen with STn(c)-KLH with the MMCCH linker. Conjugation with MMCCH resulted in the highest conjugation efficiency (yield) and the highest titers against ovine submaxillary mucin and STn-positive tumor cells, and is the method of choice for the preparation of STn(c) vaccine for clinical trials.

Specificity analysis of sera from breast cancer patients vaccinated with MUC1-KLH plus QS-21.

The mucin MUC1 is expressed on breast cancers in an underglycosylated form compared to normal tissues and is therefore a potential target for cancer immunotherapy. MUC1 contains multiple tandem repeats of the 20 amino acid (aa) peptide (VTSAPDTRPAPGSTAPPAHG). The APDTRPA epitope is particularly immunogenic since it is recognized by a variety of murine monoclonal antibodies and by some sera and cytotoxic T-cells from unimmunized patients with epithelial cancers. We have prepared a 30 aa peptide (C)VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA with cysteine at the N-terminal end, and used the cysteine for chemical conjugation to keyhole limpet haemocyanin (KLH). Six breast cancer patients immunized with this conjugate plus the immunological adjuvant QS-21 have all produced high titre (by ELISA) IgG and IgM antibodies against the 30 aa MUC1 peptide, but these sera reacted moderately, or not at all, with MUC1-positive tumour cells. To understand this specificity better, we prepared a series of smaller peptides to determine the epitopes recognized by these immune sera in inhibition assays. Only peptides containing APDTRPA at the C-terminal end were able to completely inhibit ELISA reactivity for the full 30 aa peptide. No sera were completely inhibited by APDTR, APDTRP, PDTRPA or any other peptides that did not contain the full APDTRPA epitope. Remarkably, sera from all six patients recognized this same epitope and were completely inhibited by only this epitope. The specificity of these sera (1) primarily for C-terminal APDTRPA, and the absence of this epitope at the C-terminal end of any tumour mucins, and (2) the N-terminal APDTRPA alanine, which is normally buried in the beta turn MUC1 assumes in its secondary structure may explain the moderate to weak reactivity of these high titer sera against MUC1-positive tumour cells.

Selection of tumor antigens as targets for immune attack using immunohistochemistry: protein antigens.

The relative expression of mucin antigens MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC7 and glycoprotein antigens KSA, carcinoembryonic antigen, prostate-specific membrane antigen (PSMA), HER-2/neu, and human chorionic gonadotropin-beta on different cancers and normal tissues is difficult to determine from available reports. We have compared the distribution of these antigens by immunohistology on a broad range of malignant and normal tissues. MUC1 expression was most intense in cancers of breast, lung, ovarian, and endometrial origin; MUC2 was most intense in cancers of colon and prostate origin; and MUC5AC was most intense in cancers of breast and gastric origin. MUC4 was intensely expressed in 50% of cancers of colon and pancreas origin, and MUC3, MUC5B, and MUC7 were expressed in a variety of epithelial cancers, but not so intensely. KSA was intensely and uniformly expressed on all epithelial cancers; carcinoembryonic antigen was expressed in most cancers of breast, lung, colon, pancreas, and gastric origin; and PSMA was expressed only in cancers of prostate origin. Human chorionic gonadotropin-beta was expressed on the majority of sarcomas and cancers of breast, lung, and pancreas origin, although intense staining was not seen. Staining on normal tissues was restricted to one or many normal epithelial tissues ranging from MUC3, MUC4, and PSMA, which were expressed only on epithelia of pancreas, stomach, and prostate origin, respectively, to MUC1 and KSA, which were expressed on most normal epithelia. Expression was restricted to the secretory borders of these epithelia while stroma and other normal tissues were completely negative. These results plus the results of the two previous papers (S. Zhang et al, Int. J. Cancer, 73: 42-49, 1997; S. Zhang et al., Int. J. Cancer, 73: 50-56, 1997) in this series provide the basis for selection of multiple cell surface antigens as targets for antibody-mediated attack against these cancers.

Antibodies against GD2 ganglioside can eradicate syngeneic cancer micrometastases.

After 10 years of clinical trials in patients with advanced cancer, monoclonal antibodies (mAbs) against cell surface antigens have not lived up to their initial promise. One such cell surface antigen is the ganglioside GD2. GD2 is richly expressed at the cell surfaces of human neuroblastomas, sarcomas, and melanomas. We have described a murine lymphoma (EL4) that is syngeneic in C57BL/6 mice and expresses GD2, a mAb against GD2 (mAb 3F8), and we have prepared a conjugate vaccine (GD2-keyhole limpet hemocyanin plus immunological adjuvant QS-21) that consistently induces antibodies against GD2. We demonstrate here, for the first time in a syngeneic murine model, that passively administered and vaccine-induced antiganglioside antibodies prevent outgrowth of micrometastases, and we use this model to establish some of the parameters of this protection. The level of protection was proportional to antibody titer. Treatment regimens resulting in the highest titer antibodies induced the most protection, and protection was demonstrated even when immunization was initiated after tumor challenge. Treatment with 3F8 1, 2, or 4 days after i.v. tumor challenge was highly protective, but waiting until 7 or 10 days after challenge resulted in minimal protection. The results were similar whether number of liver metastases or survival was used as the end point. These results suggest that unmodified mAbs or antibody-inducing vaccines against GD2 (and possibly other cancer cell surface antigens) should be used exclusively in the adjuvant setting, where circulating tumor cells and micrometastases are the primary targets.

A novel and efficient method for synthetic carbohydrate conjugate vaccine preparation: synthesis of sialyl Tn-KLH conjugate using a 4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazide (MMCCH) linker arm.

STn (NeuAcalpha2 --> 6GalNAc alpha-O-Ser/Thr) is a carbohydrate epitope overexpressed in various human carcinomas. Clinical trials are underway using synthetic STn or STn trimeric glycopeptides [STn, cluster; STn(c)] conjugated with keyhole limpet hemocyanin (KLH) as active specific immunotherapy for these cancers. These vaccines have been prepared by conjugating a crotyl ethyl amide derivative of STn or STn(c) to KLH by direct reductive amination after ozonolysis. In the case of STn(c) the conjugation efficiency and the resulting epitope ratios were low. This may be due to steric hinderance of the short spacer arm. To overcome these difficulties, without resynthesis, the STn(c) glycopeptide was modified by attachment of an MMCCH (4-(4-N-maieimidomethyl) cyclohexane-1-carboxyl hydrazide) spacer arm to the aldehyde derivative, and then conjugated with thiolated KLH. This method gave a higher epitope ratio and yield than the direct method. The STn(c)-MMCCH-KLH conjugate induced high titer antibodies in mice against STn(c). This method may be generally applicable for large synthetic oligosaccharides.

Characterization of a mouse monoclonal IgG3 antibody to the tumor-associated globo H structure produced by immunization with a synthetic glycoconjugate.

Globo H (Fuc alpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 3Gal alpha1 --> 4Galbeta1 --> 4Glc) is a carbohydrate structure that shows enhanced expression in many human carcinomas. From mice immunized with a globo H-KLH (keyhole limpet hemocyanin) synthetic conjugate an IgG3 monoclonal antibody (mAb VK-9) was derived that recognizes the globo H structure. Serological analysis showed that the minimal structure recognized by this mAb was the tetrasaccharide sequence Fuc alpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 3Gal. An isomeric structure with an internal alphaGalNAc linkage was also recognized but less efficiently. mAb VK-9 did not react with many related structures, such as galactosylgloboside, globoside, H type 1, H type 2 blood group structures or fucosyl-gangliotetraosyl ceramide, but did react weakly with globo A ceramide. Not only did mAb VK-9 react with carbohydrate-protein conjugates but it could also recognize globo H-ceramide and human tumor cells expressing globo H. These results suggest that globo H-KLH could be explored as a vaccine in the treatment of carcinoma patients.