Construction of cancer vaccines with carbohydrate and protein (peptide) tumor antigens.

Tumor vaccinology is as old as immunological thought and as young as our rapidly evolving understanding of antigen processing and presentation. The recent availability of carbohydrate and peptide tumor antigens suitable for vaccine construction, conjugate and recombinant vector technologies capable of augmenting helper and cytotoxic T cell activity and potent new immunological adjuvants have combined to produce considerable optimism for the future of tumor vaccines.

HLA-A, B, C and DR alloantigen expression on forty-six cultured human tumor cell lines.

Forty-six cultured cell lines of diverse human tumor origins, including 25 melanoma cell lines, were HLA allotyped with the use of a modified eosin complement-dependent cytotoxicity test in combination with absorption and two-color fluorescence techniques. In 10 cases (1 renal cell carcinoma line and 9 melanoma cell lines), the cell line donors had been HLA typed a few years before the cell line-typing project had started and in 13 cases (1 renal cell carcinoma line and 12 melanoma cell lines), the cell line donors were currently available for comparative typing of lymphocytes. HLA-typing results suggested that most cell lines expressed genetically appropriate HLA antigens, although 1 cell line had more than two HLA antigens for one HLA locus and 2 cell lines lacked expression of one or more HLA antigens in comparison with donor typing. One hepatoma cell line, 1 of 2 of the bladder carcinoma cell lines tested, and 17 of the 25 melanoma cell lines expressed DR alloantigens in addition to their HLA-A, B, and C locus antigens. For 9 of the melanoma cell lines, comparisons with donor DR alloantigens could be made, and all these cell lines had exactly the same DR allospecificities as those found on donor B-lymphocytes. HLA typing of cell lines can be used as an adjunct to polymorphic isoenzyme marker tests to verify their patient source and lack of contamination by another cell line, and HLA typing should be used to determine the antigen composition of cells used in the preparation of reagents for immunotherapy or in studies of tumor-specific immunity.

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.

Augmenting the immunogenicity of synthetic MUC1 peptide vaccines in mice.

Human mucin MUC1 is abundantly expressed in some cancers of epithelia] origin and is largely restricted to the apical surface of secretory cells in normal tissues. It is, therefore, a potential target for cancer immunotherapy. In preparation for clinical trials, vaccines containing synthetic MUC1 peptides of different lengths and sequences mixed with various adjuvants or covalently attached, using different linker methods, to protein carrier keyhole limpet hemocyanin (KLH) were studied in mice. MUC1 peptides (containing 30 amino acids), plus adjuvants QS-21 or Bacillus Calmette-Guerin, were incapable of inducing antibody. However, MUC1 peptides conjugated to KLH (MUCl-KLH), plus QS-21, induced high titer antibody against the immunizing peptides and against MUC1-expressing tumor cells. Although T-cell responses, including delayed-type hypersensitivity, lymphocyte proliferation, and CTL, were not observed in mice immunized with these vaccines, significant protection from MUC1-expressing tumor cell challenge in mice immunized with MUC1-KLH was observed. Based on these studies, a vaccine containing MUC1-KLH conjugate prepared with m-maleimidobenzoyl-N-hydroxysuccinimide ester linker, plus QS-21, has been constructed for testing in clinical trials.

Characterization of IgG and IgM antibodies induced in melanoma patients by immunization with purified GM2 ganglioside.

The ganglioside GM2 is a differentiation antigen expressed on the cell surface of human malignant melanomas and other cancers of neuroectodermal origin. We have previously reported that immunization with purified GM2 combined with Bacillus Calmette-Guérin as adjuvant and pretreatment with low-dose cyclophosphamide induced production of antibodies against GM2 in five of six patients. We have now extended our study and analyzed the induced antibodies against GM2 were not detected. After immunization, high-titer IgM antibodies were induced in 17 of 24 patients, and high-titer IgG antibodies in eight cases. Additional treatment of 12 patients with cimetidine, a histamine H2 receptor antagonist reported to have antisuppressor cell activity, had no effect on GM2 antibody titers. Antibodies against asialo-GM2 were present in all patients, and antibodies against GM1 were present in 33% of patients, before and after immunization. Antibodies induced by immunization were specific for GM2, though some reacted predominantly with N-acetylneuraminic acid GM2 (GM2), and some reacted with GM2 and N-glycolylneuraminic acid GM2(NeuGcGM2). The pattern of reactivity with GM2 is consistent with the response to T-cell-independent antigens: both IgM and IgG antibody responses against GM2 were short lived; peak titers seen after initial and secondary vaccinations were similar; and delayed-type hypersensitivity responses to skin test challenges with GM2 were not detected in any patients. However, the IgG response typed as predominantly IgG1 and IgG3, not IgG2 as might be expected for carbohydrate antigens (which are generally T-cell-independent antigens). Because IgG1 and IgG3 antibody responses are usually to T-cell-dependent antigens, the humoral immune response elicited by GM2 vaccination has both T-cell-dependent and T-cell-independent characteristics. These IgM and IgG responses against this neuroectodermal differentiation antigen expressed on melanoma cells have been induced without evidence of neurological or other toxicity.

GD3 vaccines for melanoma: superior immunogenicity of keyhole limpet hemocyanin conjugate vaccines.

Cell surface gangliosides show altered patterns of expression as a consequence of malignant transformation and have therefore been of interest as potential targets for immunotherapy, including vaccine construction. One obstacle has been that some of the gangliosides that are overexpressed in human cancers are poorly immunogenic in humans. A case in point is GD3, a prominent ganglioside of human malignant melanoma. Using an approach that has been effective in the construction of bacterial carbohydrate vaccines, we have succeeded in increasing the immunogenicity of GD3 in the mouse by conjugating the ganglioside with immunogenic carriers. Several conjugation methods were used. The optimal procedure involved ozone cleavage of the double bond of GD3 in the ceramide backbone, introducing an aldehyde group, and coupling to aminolysyl groups of proteins by reductive amination. Conjugates were constructed with a synthetic multiple antigenic peptide expressing repeats of a malarial T-cell epitope, outer membrane proteins of Neisseria meningitidis, cationized bovine serum albumin, keyhole limpet hemocyanin, and polylysine. Mice immunized with these conjugates showed a stronger antibody response to GD3 than mice immunized with unconjugated GD3. The strongest response was observed in mice immunized with the keyhole limpet hemocyanin conjugate of the GD3 aldehyde derivative and the adjuvant QS-21. These mice showed not only a long-lasting high-titer IgM response but also a consistent high-titer IgG response (predominantly IgG1), indicating recruitment of T-cell help, although the titers of IgM and IgG antibodies following booster immunizations were not as high as they are in the response to classical T-cell-dependent antigens. This method is applicable to other gangliosides, and it may be useful in the construction of immunogenic ganglioside vaccines for the immunotherapy of human cancers expressing gangliosides on their cell surface.

Biochemical and serological characteristics of natural 9-O-acetyl GD3 from human melanoma and bovine buttermilk and chemically O-acetylated GD3.

Because its expression appears to be largely restricted to human melanomas, 9-O-acetyl-GD3 is a candidate antigen for vaccine construction. Searching for potential sources, we compared chemically O-acetylated calf brain GD3 and 9-O-acetyl-GD3 extracted from bovine buttermilk with 9-O-acetyl-GD3 from human melanoma. Three fractions (F1-F3) of chemically O-acetylated GD3 differed in the number and position of O-acetyl groups. O-Acetylation sites were the lactose portion in F1 and lactose as well as sialic acid in F2 and F3. Natural (melanoma- or buttermilk-derived) 9-O-acetyl-GD3 was O-acetylated solely on the sialic acid moiety. While F1 was not reactive with monoclonal antibodies against 9-O-acetyl-GD3, F2 and F3 were as reactive as the natural products. Immunization with the natural products induced high-titer antibodies against natural 9-O-acetyl-GD3 as well as F2 and F3. In contrast, mice immunized with the synthetic fractions produced antibodies only against the immunogen but not against natural 9-O-acetyl-GD3. Only immunization with the natural products induced production of antibodies reactive with surface antigens of melanoma cells expressing 9-O-acetyl-GD3. The findings suggest (a) that C-9 of the subterminal sialic acid is the site of chemical O-acetylation in F2 and F3, as opposed to C-9 of the terminal sialic acid in the natural products; (b) that O-acetylation of both the terminal and subterminal sialic acid moieties of GD3 results in recognition by three murine monoclonal antibodies (D1.1, ME 311, and Jones) reactive with human melanoma cells; (c) that O-acetylation of the terminal sialic acid is critical, on the other hand, for inducing an immune response against melanoma 9-O-acetyl-GD3; and (d) that O-acetyl GD3 from bovine buttermilk can substitute as immunogen for inducing an immune response against human melanoma cell surface antigens in the mouse.

Immunization of colorectal cancer patients with modified ovine submaxillary gland mucin and adjuvants induces IgM and IgG antibodies to sialylated Tn.

Tn and sialylated Tn (sTn) are blood group-related epitopes expressed on mucins of colon carcinoma and other epithelial tumors and are, therefore, potential targets for immunological control. We have immunized 20 colorectal cancer patients at high risk for recurrence with a vaccine consisting of partially desialylated ovine submaxillary gland mucin (modified OSM) which contains both Tn and sTn determinants. Six patients were treated with modified OSM alone (group 1), eight patients were treated with modified OSM and the immunological adjuvant DETOX (group 2), and six patients were treated with modified OSM and Bacillus Calmette-Guérin (group 3). Pre- and postvaccination sera were tested by enzyme-linked immunosorbent assay and dot blot immune stains for antibodies reactive with modified OSM. Antibody titers increased in 4 of 8 patients immunized with modified OSM and DETOX, in 5 of 6 patients immunized with modified OSM and B. Calmette-Guérin, and in 0 of 6 patients receiving modified OSM without adjuvant. The specificity of induced IgM and IgG antibodies was confirmed by demonstrating reactivity with OSM, bovine submaxillary mucin, and synthetic glycoconjugates sTn-human serum albumin (HSA) and Tn-HSA in enzyme-linked immunosorbent assay and immune stains. Median IgM pre-postvaccination reciprocal titers were 20/80 for Tn-HSA and 10/320 for sTn-HSA. Low level IgG antibody titers against sTn-HSA were detected after vaccination in 7 patients. Toxicity was limited to inflammatory skin reactions at the site of vaccination resulting from the adjuvants. No inflammatory infiltrates were seen in the skin when the modified OSM vaccine was administered in the absence of an immunological adjuvant. These results demonstrate that sTn and Tn can be recognized by the human immune system and that vaccines containing these structures can be administered safely with immunological adjuvants. Attempts to augment the immunogenicity of these carbohydrate antigens by covalent attachment to immunogenic carrier proteins and the use of more potent immunological adjuvants are now being pursued.

A murine monoclonal antibody detecting N-acetyl- and N-glycolyl-GM2: characterization of cell surface reactivity.

Antisera reactive with the ganglioside GM2 were raised by immunizing C57BL/6 mice with the C57BL/6 melanoma JB-RH. Fusion with NS-1 was performed using splenic mononuclear cells from a mouse with high antibody titer. An immunoglobulin M monoclonal antibody (monoclonal antibody 5-3) was identified which was reactive with an antigen that was resistant to heat, trypsin, and Pronase. A panel of purified glycolipids was used to determine the specificity of monoclonal antibody 5-3. Reactivity was restricted to N-acetyl- and N-glycolyl-GM2. No reactivity was detected with asialo-GM2 or other gangliosides. Monoclonal antibody 5-3 was used to define the expression of GM2 on the cell surface of cultured human normal and malignant cells. Reactivity was seen with cell lines derived from 8 of 8 astrocytomas, 5 of 5 neuroblastomas, 7 of 9 sarcomas, 4 of 18 human melanomas, 2 of 4 murine melanomas, 4 of 37 epithelial cancers and with 0 of 6 skin fibroblast and 0 of 2 brain fibroblast lines. GM2, like GD2 and GD3, appears to be a differentiation antigen largely restricted to cells of neuroectodermal origin.

Immune sera and monoclonal antibodies define two configurations for the sialyl Tn tumor antigen.

Sialyl Tn (sTn) is a mucin-associated carbohydrate antigen expressed in most types of human adenocarcinoma. Defining the configuration of tumor cell surface sTn recognized by antibodies is important for understanding the basis for the cancer cell specificity of sTn-reactive mAbs, for the development of more effective mAbs, and for designing cancer vaccines against sTn. In this study, we compared the immunogenicity of synthetic single sTn disaccharide epitopes and clusters [sTn(C)] of 3 sTn epitopes covalently linked via serine to keyhole limpet hemocyanin [KLH; sTn-KLH and sTn(C)-KLH, respectively]. The cell surface sTn configurations were analyzed with the use of sera from mice immunized with these neoglycoproteins and a panel of sTn-reactive mAb. Sera from mice immunized with sTn-KLH reacted in direct and inhibition assays with sTn-human serum albumin (HSA) but only weakly with sTn(C)-HSA, whereas sera from mice immunized with sTn(C)-KLH reacted with sTn(C)-HSA but not with sTn-HSA. Both anti-sTn and anti-sTn(C) sera reacted with ovine submaxillary mucin (a natural source of sTn) and with sTn-positive human tumor cell line LS-C but not with sTn-negative LS-B cells. With regard to the sTn-reactive mAbs, B72.3 reacted exclusively with clustered sTn, whereas mAb B195.3R11 reacted preferentially with unclustered sTn. Results with mAbs TKH2, B239.1, and CC49 were less clear, although all reacted more strongly with clustered sTn than with unclustered sTn. These results suggest that sTn is recognized at the tumor cell surface in at least two quite distinct configurations, as clustered and nonclustered arrays.

GM2-KLH conjugate vaccine: increased immunogenicity in melanoma patients after administration with immunological adjuvant QS-21.

The cell surface gangliosides GM2, GD2, and GD3 are often overexpressed in malignant melanoma. We have shown previously that immunization of melanoma patients with GM2 and Bacillus Calmette-Guérin induced an IgM antibody response in most patients and that patients with high titer GM2 antibodies showed increased survival. As is commonly seen with carbohydrate antigens (which are T independent), the IgM response was short lived, and an IgG response was rarely observed. To increase immunogenicity, we conjugated GM2 covalently with keyhole limpet hemocyanin (KLH). GM2-KLH vaccine was given to melanoma patients alone or with one of the three adjuvants: Bacillus Calmette-Guérin, DETOX, or QS-21. The most effective vaccine was GM2-KLH with QS-21. It induced a much higher titer, a longer-lasting IgM GM2 antibody response, and a consistent IgG response (isotype IgG1 and IgG3). It also induced the highest titer anti-KLH response. The results suggest that the conjugate GM2-KLH plus QS-21 vaccine elicited significant T-cell help. Because there was no serious toxicity, this vaccine approach is attractive for augmenting the immunogenicity of other gangliosides, such as GD2 and GD3, and to determine the effects of ganglioside antibodies on the course of melanoma. In addition, the finding that QS-21 significantly increased the immunogenicity of GM2-KLH suggests that it may do the same for other conjugate vaccines, many of which are currently used without adjuvant.

Improved survival in stage III melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with GM2 ganglioside.

PURPOSE: To perform a double-blind randomized trial with American Joint Commission on Cancer (AJCC) stage III melanoma patients for the following reasons: (1) to confirm our previous finding that patients with antibodies against the melanoma differentiation antigen GM2 have an improved prognosis, and (2) to demonstrate clinical benefit from GM2 antibody induction. PATIENTS AND METHODS: One hundred twenty-two patients with AJCC stage III melanoma who were free of disease after surgery were randomized: 58 to receive treatment with the GM2/BCG vaccine, and 64 to receive treatment with bacille Calmette-Guèrin (BCG) alone. All patients were pretreated with low-dose cyclophosphamide (Cy). RESULTS: GM2 antibody was detected in 50 of 58 patients treated with GM2/BCG and seven of 64 patients treated with BCG alone. With a minimum follow-up period of 51 months, there was a highly significant increase in the disease-free interval (P = .004) and a 17% increase in overall survival (P = .02) in these 57 antibody-positive patients, confirming our earlier experience. Exclusion of all patients with preexisting GM2 antibodies (one in the GM2/BCG group and five in the BCG group) from statistical analysis resulted in a 23% increase in disease-free interval (P = .02) and a 14% increase in overall survival (P = .15) at 51 months for patients treated with the GM2/BCG vaccine. However, when all patients in the two treatment groups were compared as randomized, these increases were 18% for disease-free interval and 11% for survival in the GM2/BCG treatment group, with neither result showing statistical significance. CONCLUSION: (1) Vaccination with GM2/BCG induced immunoglobulin M (IgM) antibodies in most patients. (2) GM2 antibody production was associated with a prolonged disease-free interval and survival. (3) Comparison of the two arms of this trial as randomized fails to show a statistically significant improvement in disease-free interval or survival for patients treated with GM2/BCG vaccines.

Immunization of melanoma patients with BEC2 anti-idiotypic monoclonal antibody that mimics GD3 ganglioside: enhanced immunogenicity when combined with adjuvant.

Previous attempts to immunize melanoma patients against GD3 ganglioside have been unsuccessful because of the poor immunogenicity of GD3. BEC2, an anti-idiotypic monoclonal antibody that mimics GD3, can induce anti-GD3 IgG in rabbits. Since clinical trials with BEC2 in melanoma patients demonstrated that BEC2 alone is not highly immunogenic, we have carried out sequential clinical trials exploring the use of two immunological adjuvants, BCG and QS21, administered with BEC2. Melanoma patients free of disease after surgical resection but at high risk for recurrence were immunized either with BEC2/BCG (14 patients) or BEC2/QS21 (6 patients). All patients developed high-titer IgG antibodies against BEC2, demonstrating that both adjuvants effectively enhanced the immunogenicity of BEC2. Anti-GD3 antibodies were induced in 3 of 14 patients immunized with BEC2/BCG; no patient immunized with BEC2/QS21 developed detectable anti-GD3 antibodies. After a median follow-up of 2.4 years, 71% of the patients immunized with BEC2/BCG remain alive and 64% are free of disease. In patients immunized with BEC2/BCG, no apparent association was observed between class II HLA type and either development of anti-GD3 antibodies or survival. We are encouraged by the results with BEC2/BCG, which suggest that further enhancement of the immune response to BEC2 will result in more frequent anti-GD3 antibody responses among immunized patients.

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.

Immunization of melanoma patients with BEC2-keyhole limpet hemocyanin plus BCG intradermally followed by intravenous booster immunizations with BEC2 to induce anti-GD3 ganglioside antibodies.

BEC2 is an anti-idiotypic mouse monoclonal antibody that mimics GD3 ganglioside. Previous clinical trials demonstrated that intradermal immunization using 2.5 mg of BEC2 with BCG or i.v. immunization with 10 mg of BEC2 can induce anti-GD3 antibodies in a subset of patients. We hypothesized that combining these two immunization strategies might be more effective in inducing anti-GD3 antibodies and that conjugation of BEC2 to keyhole limpet hemocyanin (KLH) would further enhance the immunogenicity of BEC2. In this clinical trial, 18 melanoma patients who were free of disease after complete surgical resection within 1-6 months received intradermal immunizations on weeks 0, 2, 4, 6, and 10 with 2.5 mg of BEC2 conjugated to KLH and mixed with BCG (BEC2-KLH/BCG). Booster immunizations of 10 mg of unconjugated BEC2 were administered i.v. on weeks 24, 37, and 50. Four of 18 patients (22%) developed IgM anti-GD3 antibodies. No IgG anti-GD3 antibodies were detected. All four responding patients developed anti-GD3 IgM during immunization with BEC2-KLH/BCG; only one patient demonstrated a reboost of the IgM anti-GD3 titer during the i.v. immunizations. Thirteen of the patients are free of melanoma (3 after undergoing re-resection for local relapse); 14 patients (78%) remain alive with a median follow-up of 28 months. These results confirm our previous trial, showing that BEC2 with BCG can induce anti-GD3 antibodies in patients. The data do not provide evidence that conjugation to KLH increases the immunogenicity of BEC2 when it is administered with BCG.

Vaccination with a bivalent G(M2) and G(D2) ganglioside conjugate vaccine: a trial comparing doses of G(D2)-keyhole limpet hemocyanin.

Immunization with GMK vaccine (G(M2) ganglioside conjugated to keyhole limpet hemocyanin mixed with QS-21 adjuvant) induces anti-G(M2) antibodies in close to 100% of patients. We found previously that anti-G(D2) antibodies could be induced in some patients using G(D2)-keyhole limpet hemocyanin + QS-21 (GDK). In this trial, we wished: (a) to determine whether immunization with both GMK and GDK vaccines could induce antibodies against both G(M2) and G(D2); and (b) to determine the optimal dose of GDK. Thirty-one patients with melanoma or sarcoma who had no evidence of disease after complete surgical resection were immunized with both GMK (30 microg of G(M2)) and GDK on weeks 1, 2, 3, 4, 12, 24, and 36. Patients were assigned to one of five GDK dose levels (3, 10, 30, 70, or 130 microg of G(D2)). Anti-G(M2) IgM or IgG were induced in 97% of patients. The dose of GDK did not affect the anti-G(M2) response, although at the highest GDK dose level, 3 of 7 patients did not make anti-G(M2) IgG. GDK was less immunogenic; overall 45% of patients developed either IgM or IgG against G(D2). At GDK doses of 30 or 70 microg, 8 of 11 patients (73%) made either IgM or IgG anti-G(D2) antibodies. We conclude that both GMK and GDK vaccines can induce antibodies against G(M2) and G(D2) in a majority of patients and are safe. The optimal dose of GDK appears to be either 30 or 70 microg when administered with GMK vaccine.

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.

Induction of antibodies against GM2 ganglioside by immunizing melanoma patients using GM2-keyhole limpet hemocyanin + QS21 vaccine: a dose-response study.

In a previous randomized Phase III trial (P. O. Livingston et al, J. Clin. Oncol., 12: 1036-1044, 1994), we demonstrated that immunization with GM2 and bacille Calmette-Guerin reduced the risk of relapse in stage III melanoma patients who were free of disease after surgical resection and who had no preexisting anti-GM2 antibodies. That vaccine formulation induced IgM anti-GM2 antibodies in 74% but induced IgG anti-GM2 antibodies in only 10% of the patients. To optimize the immune response against GM2, a reformulated vaccine was produced conjugating GM2 to keyhole limpet hemocyanin (KLH) and using the adjuvant QS21 (GM2-KLH/QS21). In pilot studies, 70 microg of vaccine induced IgG anti-GM2 antibodies in 76% of the patients. We wished to define the lowest vaccine dose that induced consistent, high-titer IgM and IgG antibodies against GM2. Fifty-two melanoma patients who were free of disease after resection but at high risk for relapse were immunized with GM2-KLH/QS21 vaccine at GM2 doses of 1, 3, 10, 30, or 70 ILg on weeks 1, 2, 3, 4, 12, 24, and 36. Serum collected at frequent and defined intervals was tested for anti-GM2 antibodies. Overall, 88% of the patients developed IgM anti-GM2 antibodies; 71% also developed IgG anti-GM2 antibodies. GM2-KLH doses of 3-70 microg seemed to be equivalent in terms of peak titers and induction of anti-GM2 antibodies. At the 30-microg dose level, 50% of the patients developed complement fixing anti-GM2 antibodies detectable at a serum dilution of 1:10. We conclude that the GM2-KLH/QS21 formulation is more immunogenic than our previous formulation and that 3 microg is the lowest dose that induces consistent, high-titer IgM and IgG antibodies against GM2.

Expression of potential target antigens for immunotherapy on primary and metastatic prostate cancers.

Defining the expression of tumor-associated antigens on primary and metastatic prostate cancer is the crucial first step in selecting appropriate targets for immune attack. In this study, the distribution of the tumor-associated antigens GM2, Tn, sTn, Thompson-Friedenreich antigen (TF), Globo H, Le(y), MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, carcinoembryonic antigen, beta chain of human chorionic gonadotropin (hCG beta), HER2/neu, PSMA, and KSA on primary and metastatic prostate cancer and 16 types of normal tissues was compared by immunohistochemistry, using a panel of well-characterized monoclonal antibodies. Our results show that GM2, KSA, and MUC2 were strongly expressed on 8 or 9 of 9 metastatic prostate cancer biopsy specimens and, with PSMA, hCG beta, TF, Tn, and sTn, on 8 or more of 11 primary prostate cancer specimens. Tn, MUC1, and PSMA were expressed on 4-6 of 9 metastatic specimens. The remaining antigens were expressed on no more than three of nine metastatic specimens. Normal tissues were also tested with all antibodies. With regard to the eight antigens most widely expressed on prostate cancers, PSMA was not expressed significantly on any of the normal tissues except prostate epithelium. Tn, sTn, hCG beta, and MUC2 were detected on up to 3 of 10 types of normal epithelia. GM2, TF, MUC1, and KSA were more broadly distributed on normal epithelia, all primarily at the secretory borders. STn, KSA, and hCG beta were also detected in the testis, and GM2 was expressed on gray matter of brain. From the 30 antigens that we have screened, this study provides the basis for selecting GM2, TF, Tn, sTn, hCG beta, MUC1, MUC2, KSA, and PSMA as target antigens for specific immunotherapy of prostate cancer.

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.