The fully synthetic glycopeptide MAG-Tn3 therapeutic vaccine induces tumor-specific cytotoxic antibodies in breast cancer patients.

Cancer is one of the main causes of mortality worldwide and a major public health concern. Among various strategies, therapeutic vaccines have been developed to stimulate anti-tumoral immune responses. However, in spite of extensive studies, this approach suffers from a lack of efficacy. Recently, we designed the MAG-Tn3 vaccine, aiming to induce antibody responses against Tn, a tumor-associated carbohydrate antigen. The Tn antigen is of interest because it is expressed by several adenocarcinomas, but not normal cells. The fully synthetic glycopeptide vaccine MAG-Tn3 is composed of four arms built on three adjacent Tn moieties associated with the tetanus toxin-derived peptide TT830-844 CD4+ T-cell epitope. This promiscuous CD4+ T-cell epitope can bind to a wide range of HLA-DRB molecules and is thus expected to activate CD4+ T-cell responses in a large part of the human population. The MAG-Tn3 vaccine was formulated with the GSK-proprietary immunostimulant AS15, composed of CpG7909, MPL, and QS21, which has been shown to stimulate both innate and humoral responses, in addition to being well tolerated. Here, seven patients with localized breast cancer with a high-risk of relapse were immunized with the MAG-Tn3 vaccine formulated with AS15. The first results of phase I clinical trial demonstrated that all vaccinated patients developed high levels of Tn-specific antibodies. Moreover, these antibodies specifically recognized Tn-expressing human tumor cells and killed them through a complement-dependent cytotoxicity mechanism. Overall, this study establishes, for the first time, the capacity of a fully synthetic glycopeptide cancer vaccine to induce specific immune responses in humans.

A vision for vaccines built from fully synthetic tumor-associated antigens: from the laboratory to the clinic.

Cancer cells may be distinguished from normal cells by cell surface displays of aberrant levels and types of carbohydrate domains. Accordingly, these tumor-associated carbohydrate antigens (TACAs) represent promising target structures for the design of anticancer vaccines. Over the past 20 years, our laboratory has sought to use the tools of chemical synthesis to develop TACA-based anticancer vaccine candidates. We provide herein a personal accounting of our laboratory's progress toward the long-standing goal of developing clinically viable fully synthetic carbohydrate-based anticancer vaccines.

Boosting immunity to small tumor-associated carbohydrates with bacteriophage qß capsids.

The development of an effective immunotherapy is an attractive strategy toward cancer treatment. Tumor associated carbohydrate antigens (TACAs) are overexpressed on a variety of cancer cell surfaces, which present tempting targets for anticancer vaccine development. However, such carbohydrates are often poorly immunogenic. To overcome this challenge, we show here that the display of a very weak TACA, the monomeric Tn antigen, on bacteriophage Qß virus-like particles elicits powerful humoral responses to the carbohydrate. The effects of adjuvants, antigen display pattern, and vaccine dose on the strength and subclasses of antibody responses were established. The local density of antigen rather than the total amount of antigen administered was found to be crucial for induction of high Tn-specific IgG titers. The ability to display antigens in an organized and high density manner is a key advantage of virus-like particles such as Qß as vaccine carriers. Glycan microarray analysis showed that the antibodies generated were highly selective toward Tn antigens. Furthermore, Qß elicited much higher levels of IgG antibodies than other types of virus-like particles, and the IgG antibodies produced reacted strongly with the native Tn antigens on human leukemia cells. Thus, Qß presents a highly attractive platform for the development of carbohydrate-based anticancer vaccines.

Carbohydrate-based vaccines with a glycolipid adjuvant for breast cancer.

Globo H (GH) is a hexasaccharide specifically overexpressed on a variety of cancer cells and therefore, a good candidate for cancer vaccine development. To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked GH to a carrier protein, including keyhole limpet hemocyanion, diphtheria toxoid cross-reactive material (CRM) 197 (DT), tetanus toxoid, and BSA, and combined with an adjuvant, and it was administered to mice for the study of immune response. Glycan microarray analysis of the antiserum obtained indicated that the combination of GH-DT adjuvanted with the α-galactosylceramide C34 has the highest enhancement of anti-GH IgG. Compared with the phase III clinical trial vaccine, GH-keyhole limpet hemocyanion/QS21, the GH-DT/C34 vaccine elicited more IgG antibodies, which are more selective for GH and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) and SSEA4, all of which were specifically overexpressed on breast cancer cells and breast cancer stem cells with SSEA4 at the highest level (>90%). We, therefore, further developed SSEA4-DT/C34 as a vaccine candidate, and after immunization, it was found that the elicited antibodies are also IgG-dominant and very specific for SSEA4.

Cancer immunotherapy by intratumoral injection of α-gal glycolipids.

UNLABELLED: AIM/ BACKGROUND: To determine the feasibility and safety of intratumoral α-gal glycolipids injection for conversion of human tumors into autologous Tumor Associated Antigens (TAA) vaccine. α-Gal glycolipids bind anti-Gal--the most abundant antibody in humans. Pre-clinical studies indicated that injected α-gal glycolipids insert into tumor cell membranes, bind anti-Gal and target tumor cells to Antigen Presenting Cells, thereby converting tumors into autologous TAA vaccines. We hypothesized that α-gal glycolipids might have similar utility in humans. PATIENTS AND METHODS: Eleven patients with advanced solid tumors received one intratumoral injection of 0.1 mg, 1 mg, or 10 mg α-gal glycolipids. The primary endpoint was dose-limiting toxicity (DLT) within 4 weeks. Secondary endpoints included long-term toxicity, autoimmunity, radiological tumor response and survival. RESULTS: There were no DLT and no clinical or laboratory evidence of autoimmunity, or any other toxicity. Few patients had an unexpectedly long survival. CONCLUSION: Intratumoral injection of α-gal glycolipids is feasible and safe for inducing a protective anti-tumor immune response.

Conversion of tumors into autologous vaccines by intratumoral injection of α-Gal glycolipids that induce anti-Gal/α-Gal epitope interaction.

Anti-Gal is the most abundant antibody in humans, constituting 1% of immunoglobulins. Anti-Gal binds specifically α-gal epitopes (Galα1-3Galß1-4GlcNAc-R). Immunogenicity of autologous tumor associated antigens (TAA) is greatly increased by manipulating tumor cells to express α-gal epitopes and bind anti-Gal. Glycolipids with αgal epitopes (α-gal glycolipids) injected into tumors insert into the tumor cell membrane. Anti-Gal binding to the multiple α-gal epitopes de novo presented on the tumor cells results in targeting of these cells to APC via the interaction between the Fc portion of the bound anti-Gal and Fcγ; receptors on APC. The APC process and present immunogenic TAA peptides and thus, effectively activate tumor specific CD4+ helper T cells and CD8+ cytotoxic T cells which destroy tumor cells in micrometastases. The induced immune response is potent enough to overcome immunosuppression by Treg cells. A phase I clinical trial indicated that α-gal glycolipid treatment has no adverse effects. In addition to achieving destruction of micrometastases in cancer patients with advance disease, α-gal glycolipid treatment may be effective as neo-adjuvant immunotherapy. Injection of α-gal glycolipids into primary tumors few weeks prior to resection can induce a protective immune response capable of destroying micrometastases expressing autologous TAA, long after primary tumor resection.

Preclinical studies of carbohydrate mimetic peptide vaccines for breast cancer and melanoma.

Limited immune responses to tumor-associated carbohydrate antigens (TACA) are due in part to their being self-antigens. Immunization with xenoantigens of TACA provides an approach to break tolerance and augment responses to TACA. Carbohydrate mimetic peptides (CMPs) as xenoantigens can induce serum antibodies that target shared carbohydrate residues on differing carbohydrate structures. In preclinical studies, we observe that CMP immunization in mice induce immune responses that are effective in inhibiting the in vitro and in vivo growth of breast cancer and melanoma tumor cells expressing self-target antigens. CMPs of TACA can be further defined that induce IgM antibodies with broadened responses to both breast and melanoma cells. Consequently, CMPs are effective at generating a multifaceted carbohydrate-reactive immune response that should be clinically evaluated for their ability to amplify carbohydrate immune responses against circulating or disseminated tumor cells.

SialylTn-mAb17-1A carbohydrate-protein conjugate vaccine: effect of coupling density and presentation of SialylTn.

Carbohydrate antigens resulting from aberrant glycosylation of tumor cells, such as SialylTn, represent attractive targets for cancer vaccination. However, T-cell-independent carbohydrate antigens are poorly immunogenic and fail to induce memory and IgG class switch. Clustered expression patterns of some carbohydrates on the cell surface add further complexity to the design of carbohydrate-based vaccines. We describe here a vaccine consisting of SialylTn carbohydrate epitopes coupled to a highly immunogenic carrier molecule, mAb17-1A, adsorbed on alhydrogel and coformulated with a strong adjuvant, QS-21. The SialylTn-mAb17-1A conjugate vaccine was administered in Rhesus monkeys, and the immune responses against mAb17-1A, SialylTn, ovine submaxillary mucin, and tumor cells were analyzed. The data demonstrate that the density of carbohydrate epitopes on the carrier is an essential parameter for induction of anti-carbohydrate specific memory IgG immune responses. Furthermore, the influence of different types of presentation of SialylTn (monomeric vs trimers vs clustered via a branched polyethylenimine linker) on antibody titers and specificity was studied. High-density coupling of SialylTn epitopes to mAb17-1A induced the strongest immune response against synthetic SialylTn and showed also the highest reactivity against natural targets, such as OSM and tumor cells.

Rationale for the clinical development of STn-KLH (Theratope) and anti-MUC-1 vaccines in breast cancer.

Mucin-1 (MUC-1) is a high-molecular-weight glycoprotein rich in serine and threonine residues that are O-glycosylated. Expression of MUC-1 is increased in breast, ovarian, and other adenocarcinomas, and altered glycosylation results in exposure of novel peptide epitopes and the expression of tumor-associated carbohydrate residues, such as Thomsen-Freidenreich and sialyl-Tn (STn) antigens. Preclinical studies suggested that induction of immune response to tumor-associated carbohydrate moieties results in inhibition of tumor growth. A synthetic STn-keyhole limpet hemocyanin (KLH) vaccine (Theratope) is currently being evaluated in clinical trials as active specific immunotherapy in the treatment of advanced breast cancer. Two phase II trials in 50 breast cancer patients compared the STn-KLH vaccine with and without a single low-dose infusion of cyclophosphamide used as an immunomodulator prior to initiation of treatment. Humoral immune responses were higher in patients who had received low-dose cyclophosphamide intravenously (I.V.) compared with patients who had received no cyclophosphamide or oral cyclophosphamide. There was a statistically significant survival difference between all patients treated with the STn-KLH vaccine (overall median survival, 19.1 months; n = 50) and the retrospective control patients (overall median survival, 9.2 months; n = 104). Furthermore, patients who received cyclophosphamide I.V. prior to the STn-KLH vaccine had median survival rates close to 3 times that of patients in a retrospective, frequency-matched, control group who received conventional therapies (cyclophosphamide-I.V. group, 26.5 months vs. 9.2 months, control group). The trials reported minimal toxicity profile with local reactions in the injection site and some flu-like symptoms. On the basis of the phase II trial results, a phase III clinical trial of the STn-KLH vaccine is underway. The trial was closed to enrollment in March 2001 with the accrual of 1030 women. The final analysis is event driven and is expected to commence mid 2003.

Prospects for immunotherapy of malignant disease.

The majority of T cell-recognized tumour antigens in humans are encoded by genes that are also present in normal tissues. Low levels of gene expression in normal cells can lead to the inactivation of high-avidity T cells by immunological tolerance mechanisms. As a consequence, low-avidity T cell responses in patients are often inadequate in providing tumour protection. Recently, several technologies have been developed to overcome tolerance, allowing the isolation of high-affinity, HLA-restricted receptors specific for tumour-associated peptide epitopes. Furthermore, transfer of HLA-restricted antigen receptors provides an opportunity to empower patient T cells with new tumour-reactive specificities that cannot be retrieved from the autologous T cell repertoire.

Therapeutic effects of idiotype vaccination can be enhanced by the combination of granulocyte-macrophage colony-stimulating factor and interleukin 2 in a myeloma model.

Idiotype (Id) vaccination provides an interesting immunotherapeutic strategy against B-cell lymphomas. In multiple myeloma (MM), however, the therapeutic efficacy of Id vaccination has been disappointing. In an attempt to improve the antitumoral potential, we added granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 2 (IL-2) to the protocol. Balb/c mice were inoculated i.p. (d 2) with different doses (1-5 x 10(5)) of HOPC myeloma cells secreting the Ig(HOPC) Id protein. Two days later, animals were injected with 10,000 U GM-CSF i.p. for 6 d consecutively (d 0-5). On d 5 and 11, myeloma-specific immunoglobulin (Ig(HOPC)) was administered i.p. together with incomplete Freund adjuvans followed by IL-2 (2 x 10,000 U/d; i.p) for 10 d (d 5-14). In animals inoculated with 10(5) myeloma cells, treatment with IL-2 given as a single agent prolonged the median survival time (MST, 67 d) when compared with the tumour control group (MST 48 d), whereas GM-CSF did not elicit any survival benefit (MST 49 d). Complete tumour rejection could be achieved in 27% (4/15) by the combination of Id vaccination and GM-CSF. Additional treatment with IL-2 further increased antimyeloma activity. In this case, 59% of the animals showed no signs of tumour recurrence. In mice with high tumour burden (5 x 10(5)), no treatment modality achieved long-term survivors. Both natural killer (NK) cells and CD8+ T cells may be involved in the anti-tumoural immune response. These data provide evidence for the combined use of GM-CSF and IL-2 to enhance the therapeutic effectiveness of clinical cancer vaccination protocols.

Tumor growth enhances cross-presentation leading to limited T cell activation without tolerance.

Using a tumor model of spontaneously arising insulinomas expressing a defined tumor-associated antigen, we investigated whether tumor growth promotes cross-presentation and tolerance of tumor-specific T cells. We found that an advanced tumor burden enhanced cross-presentation of tumor-associated antigens to high avidity tumor-specific T cells, inducing T cell proliferation and limited effector function in vivo. However, contrary to other models, tumor-specific T cells were not tolerized despite a high tumor burden. In fact, in tumor-bearing mice, persistence and responsiveness of adoptively transferred tumor-specific T cells were enhanced. Accordingly, a potent T cell-mediated antitumor response could be elicited by intravenous administration of tumor-derived peptide and agonistic anti-CD40 antibody or viral immunization and reimmunization. Thus, in this model, tumor growth promotes activation of high avidity tumor-specific T cells instead of tolerance. Therefore, the host remains responsive to T cell immunotherapy.

Immunization with a carbohydrate mimicking peptide augments tumor-specific cellular responses.

The metastatic potential of some tumor cells is associated with the expression of the neolactoseries antigens sialyl-Lewis x (sLex) and sialyl-Lewis a (sLea) as they are ligands for selectins. We have recently shown that peptide mimetics of these antigens can potentiate IgG2a antibodies, which are associated with a Th1-type cellular response. As L-selectin is preferentially expressed on CD4+ Th1 and CD8+ T cell populations, specific induction of these phenotypes could augment a response to L-selectin ligand-expressing tumor cells. Here we demonstrate that immunization with a multiple antigen peptide (MAP) mimetic of sugar constituents of neolactoseries antigens induces a MHC-dependent peptide-specific cellular response that triggers IFN-gamma production upon peptide stimulation, correlating with IgG2a induction. Surprisingly, T lymphocytes from peptide-immunized animals were activated in vitro by sLex, also triggering IFN-gamma production in a MHC-dependent manner. Stimulation by peptide or carbohydrate resulted in loss of L-selectin on CD4+ T cells confirming a Th1 phenotype. We also observed an enhancement in cytotoxic T lymphocyte (CTL) activity in vitro against sLex-expressing Meth A cells using effector cells from Meth A-primed/peptide-boosted animals. CTL activity was inhibited by both anti-MHC class I and anti-L-selectin antibodies. These results further support a role for L-selectin in tumor rejection along with the engagement by the TCR for most likely processed tumor-associated glycopeptides, focusing on peptide mimetics as a means to induce carbohydrate reactive cellular responses.

Anti-tumor immunity provided by a synthetic multiple antigenic glycopeptide displaying a tri-Tn glycotope.

In many cancer cells the alteration of glycosylation processes leads to the expression of cryptic carbohydrate moieties, which make them good targets for immune intervention. Identification of cancer-associated glycotopes as well as progress in chemical synthesis have opened up the way for the development of fully synthetic immunogens that can induce anti-saccharide immune responses. Here, we synthesized a dendrimeric multiple antigenic glycopeptide (MAG) containing the Tn Ag O:-linked to a CD4(+) T cell epitope. This MAG is based on three consecutive Tn moieties (tri-Tn) corresponding to the glycotope recognized by an mAb (MLS 128) produced against the LS180 colon carcinoma cell line. The Abs induced by this MAG recognized murine and human tumor cell lines expressing the Tn Ag. Prophylactic vaccination using MAG provided protection of mice against tumor challenge. When used in active specific immunotherapy, the MAG carrying the tri-Tn glycotope was much more efficient than the mono-Tn analogue in promoting the survival of tumor-bearing mice. Furthermore, in active specific immunotherapy, a linear glycopeptide carrying two copies of the tri-Tn glycotope was shown to be poorly efficient compared with the dendrimeric MAG. Therefore, both the clustering of carbohydrate Ags and the way they are displayed seem to be important parameters for stimulating efficient anti-saccharide immune responses.

Clinical outcome of breast and ovarian cancer patients treated with high-dose chemotherapy, autologous stem cell rescue and THERATOPE STn-KLH cancer vaccine.

The purpose of this study was to evaluate the toxicity and potential efficacy of administering the THERATOPE STn-KLH cancer vaccine to ovarian and breast cancer patients after an autologous stem cell transplant. Forty patients (11 high-risk stage II/III breast cancer, 22 stage IV breast cancer, and seven stage III/IV ovarian cancer patients) were treated with high-dose chemotherapy followed by autologous/syngeneic stem cell rescue and vaccination with THERATOPE STn-KLH (Sialyl-Tn-KLH with Detox-B Stable Emulsion). Each patient was scheduled to receive a total of five vaccinations beginning on days 30-151 after stem cell infusion. The vaccine was well tolerated. Induration and erythema at the site of injection were the most common side-effects. When one compares the outcome of patients vaccinated with 66 breast and ovarian cancer patients who were not, following risk-adjustment analysis, vaccinated patients appeared more likely to survive (P = 0.07) and less likely to relapse (P = 0. 10). Vaccinated patients with the greatest specific lytic activity against STn+OVCAR tumor cells relative to nonspecific killing of Daudi cells tended to remain in remission longer than patients who displayed less specific immune activity (P = 0.057). We conclude that the THERATOPE STn-KLH cancer vaccine is well tolerated in breast and ovarian cancer patients after autologous transplant and, while not statistically significant, the trends in data support the concept that THERATOPE vaccine may decrease the risk for relapse and death and thus warrants further study. Bone Marrow Transplantation (2000) 25, 1233-1241.

Evidence of a cellular immune response against sialyl-Tn in breast and ovarian cancer patients after high-dose chemotherapy, stem cell rescue, and immunization with Theratope STn-KLH cancer vaccine.

Seven ovarian and 33 breast high-risk stage II/III and stage IV cancer patients received high-dose chemotherapy followed by stem cell rescue. Thirty to 151 days after stem cell transplantation, the patients received their first immunotherapy treatment with Theratope STn-KLH cancer vaccine. Most patients developed increasing IgG anti-STn titers to a sustained peak after the fourth or fifth immunizations. Only one patient had elevated CA27.29 (MUC1 mucin) serum levels at trial entry. Five of the seven patients with preimmunotherapy elevated serum CA125 levels demonstrated decreasing CA125 levels during immunotherapy, consistent with an antitumor response. Evidence of STn antigen-specific T-cell proliferation was obtained from 17 of the 27 evaluable patients who received at least three immunotherapy treatments. Eleven of the 26 patients tested had evidence of an anti-STn TH1 antigen-specific T-cell response as determined by interferon-gamma, but not interleukin (IL)-4, production. After immunization, lytic activity of peripheral blood lymphocytes (PBLs) tested against a lymphokine activated killer (LAK)-sensitive cell line, a natural killer (NK)-sensitive cell line, and an STn-expressing cancer cell line (OVCAR) increased significantly. In vitro IL-2 treatment of the PBLs after vaccination greatly enhanced killing of the STn+ cancer cell line. Evidence of the development of OVCAR specific killing activity, over and above that seen due to LAK or NK killing, is presented. These studies provide the strongest evidence in humans of the development of an antitumor T-cell response after immunization with a cancer-associated carbohydrate antigen.

T/Tn antigen vaccine is effective and safe in preventing recurrence of advanced breast carcinoma.

Since 1974, and as of March, 1993, we have used T/Tn antigen vaccine in safe, specific, effective, long-term intradermal vaccination against recurrence of advanced breast carcinoma (CA). Staging is by the pathologic TNM system. Treatment is ad infinitum. Of 19 consecutive breast carcinoma patients vaccinated, six Stage IV, six Stage III, and seven Stage II all survived > 5 years postoperatively. Three Stage III, three Stage IV, and five Stage II patients (i.e., 11) survived > 10 to > 18 years. Five others are alive but have not reached 10 years; three of them have no evidence of disease (NED). Three patients died of CA before reaching 10 years. An additional three breast CA patients are being treated for > 2 years, but, < 5 years postoperatively, they are NED. The vaccination are presented as a delayed-type hypersensitivity reaction with significant inflammation with increase of helper T lymphocytes and decrease of T suppressor/cytotoxic cell ratio.

Avaliaçåo da dosagem do CA-125 sérico no diagnóstico da endometriose / Assessement of serum concentration CA-125 in the diagnosis of endometriosis

A endometriose é a doença decorrente da presença de implantes heterotópicos com a mesma funçäo e estrutura histológica da mucosa de revestimento uterina. Possui etiologia desconhecida e diagnóstico dispendioso e invasivo. O presente estudo teve como objetivo a verificaçäo da eficácia da dosagem da concentraçäo sérica do CA-125, um marcador tumoral, no diagnóstico da endometriose nos seus diversos estágios, seguindo a classificaçä da Sociedade de Fertilidade Americana de 1985 e nos diversos valores de referência para a normalidade desse marcador. Com a finalidade de obtermos um método que facilitaria eu diagnóstico, avaliamos 41 pacientes inférteis, em idade reprodutiva, as quais submeteram à laparoscopia propedêutica no período de janeiro de 1992 a junho de 1993 e as dividimos em dois grupos: grupo das portadoras de endometriose, que foram subdivididas de acordo com o estágio da doença, e grupo das näo portadoras de endometriose. A dosagem do CA-125 foi realizada pelo método ELISA e teve como referência o ponto de corte predominante na literatura de 35 U/ml. Resultados similares foram obtidos em todos os níveis de comparaçäo, näo se encontrando associaçäo entre os dois parâmetros, com valores baixos para a detecçäo de pacientes portadores e näo portadores de endometriose (sensibilidade, especificidde, valores de prediçäo positivo e negativo). Por fim, realizamos diversos pontos de corte para os valores do marcador e observamos que ocorreu uma diminuiçäo importante da especificidade em detrimento da sensibilidade o que nos levou a considerar o teste sem poder de discriminaçäo

Therapeutic effect of intradermal injections with difucosyl lactosamine (dimeric Lex) on patients with rheumatoid arthritis.

As reported by us, a new myeloid cell population with an oncofetal membrane marker, dimeric Lex (di-Lex; III3FucV3 FucnLc6), was found in the epiphyseal bone marrow adjacent to the involved joints of patients with severe rheumatoid arthritis (RA). Patients with RA received intradermal (id) injections of di-Lex incorporated in liposome or of high molecular weight glycoprotein, or tumor associated carbohydrate antigen (TCA), containing the same carbohydrate epitope as di-Lex. The epiphyseal myeloid cells were reduced or sometimes eliminated during id injection. In random trials of id injection, observation under clinical and laboratory conditions showed improvement in 63% (17/27) of the patients treated for 6 months with appropriate doses of di-Lex (III3FucnLc4), and in 72% (31/43) of those treated with an identical protocol for TCA. However, id injection with monomeric Lex had no effect.

Immunization of breast cancer patients using a synthetic sialyl-Tn glycoconjugate plus Detox adjuvant.

We have synthesized various formulations that have potential for active specific immunotherapy (ASI) of human cancers. Sialyl-Tn (STn) is a potentially important target structure for ASI because its expression on mucins is a strong, independent predictor of poor prognosis, suggesting that it may have functional significance in the metastatic process. In this first pilot study of synthetic sialyl-Tn hapten conjugated to keyhole limpet hemocyanin (STn-KLH), with Detox adjuvant, toxicity and humoral immunogenicity were assessed in 12 patients with metastatic breast cancer. Toxicity was minimal, restricted to local cutaneous reactions (apart from transient nausea and vomiting following single low-dose cyclophosphamide treatment). Using STn-conjugated human serum albumin in a solid-phase enzyme-linked immunosorbent assay, it was shown that all patients developed IgM and IgG specific for the synthetic STn hapten. Following immunization, most patients were shown to develop increased titres of complement-mediated cytotoxic antibodies, partially inhibited by synthetic STn hapten, but not by the related TF hapten. We also detected IgM and IgG antibodies reactive with natural STn determinants expressed on ovine submaxillary mucin, the STn specificity of this reactivity being confirmed by hapten inhibition. Evaluation of clinical efficacy in a small pilot study is difficult. Five patients are alive 12 or more months after entry, and another 4 patients are alive 6 or more months after entry into the study. All 3 patients with known widespread bulky disease progressed despite ASI, 2 having died from widespread cancer. Two patients had partial responses, each lasting 6 months. While several patients had disease stability for 3-10 months, 1 patient with pulmonary metastases remains stable 15 months after entry into the program.