Grant D. MacLean

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.

Enhancing the effect of THERATOPE STn-KLH cancer vaccine in patients with metastatic breast cancer by pretreatment with low-dose intravenous cyclophosphamide.

THERATOPE (Biomira Inc., Edmonton, AB, Canada) STn-KLH cancer vaccine induces strong antibody titers against both the synthetic STn epitope and against a natural mucin, OSM, which expresses STn-like epitopes. In prospective, randomized studies in patients with metastatic breast cancer treated at two cancer centers, the effect of different low-dose, immunomodulatory cyclophosphamide (cyclo) pretreatments on the response to THERATOPE STn-KLH was compared. Patients were randomized to receive either intravenous cyclo 300 mg/m2 on day -3, or oral cyclo 50 mg daily from days -14 to -3 inclusive, or no cyclo, before THERATOPE treatments. The anti-STn and anti-OSM antibody titers were higher in the patients who received cyclo intravenously before THERATOPE. Patients treated with cyclo intravenously and THERATOPE STn-KLH cancer vaccine lived significantly longer (projected median survival of 19.7 months versus actual median survival of 12.6 months, p = 0.0176) than those treated with the same STn vaccine with oral or no cyclo. Although it is not clear how the anti-STn antibody response modifies tumor biology, we noted that patients in the intravenously administered cyclo group had a lower percentage of patients showing progressive disease at 9 weeks, and that there was an inverse correlation between serum anti-STN antibody titer and growth of measurable tumors. There was no correlation between tumor growth and anti-KLH antibody titers. These data are consistent with a therapeutic effect of THERATOPE STn-KLH cancer vaccine and support development of a phase III study to explore this further.

Antibodies Against Mucin-Associated Sialyl-Tn Epitopes Correlate with Survival of Metastatic Adenocarcinoma Patients Undergoing Active Specific Immunotherapy with Synthetic STn Vaccine

The humoral immune response of 85 metastatic breast, ovarian, and colorectal cancer patients was analyzed after immunization with THERATOPE STn-KLH (KLH, keyhole limpet hemocyanin) cancer vaccine emulsified in DETOX adjuvant. Enzyme-linked immunosorbent assay (ELISA) antibody titers against the synthetic sialyl-Tn (STn) epitope were estimated by using solid phase STn-HSA and compared with antibody titers generated to the more biologically relevant natural mucin STn epitopes by using ovine submaxillary mucin (OSM) as a solid phase. Anti-KLH antibody titers were compared with anti-STn antibody titers as a specificity control. All but two patients generated increased anti-OSM antibody titers after immunization with STn-KLH. Breast and colorectal cancer patients who had the highest anti-OSM antibody titers, determined 4 weeks after the fourth immunization with STn-KLH (post-4 ASI), survived longer than the patients who had lower post-4 active specific immunotherapy (ASI) anti-OSM antibody titers. In contrast, there was no correlation of anti-KLH antibody titers with survival, demonstrating the specificity of the association of anti-OSM antibodies with survival. Cox multivariate survival analysis models were used to attempt to determine whether the induction of high-titer antibodies after immunization is a prognostic indicator independent of age, level of various tumor markers, extent of disease, lactate dehydrogenase (LDH) level, and route of administration of low-dose cyclophosphamide before ASI. Increased pre-ASI CA-125 serum levels in the ovarian cancer patients were predictors of poor survival, independent of all of the other prognostic factors. The postimmunization increase in anti-OSM immunoglobulin M (IgM) titer was independently associated with longer survival of the colorectal cancer patients. Increased anti-OSM IgG titers were associated with a marked increased survival of the breast cancer patients, which was independent of all other prognostic factors except the size of measurable metastatic lesions at trial entry and the route of administration of cyclophosphamide. In a randomized trial design, breast cancer patients who received low-dose intravenous cyclophosphamide just before ASI showed longer survival and generated higher anti-OSM antibody titers than did patients who received low-dose oral cyclophosphamide before ASI.

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.

Pre-immunotherapy serum CA27.29 (MUC-1) mucin level and CD69+ lymphocytes correlate with effects of Theratope® sialyl-Tn-KLH cancer vaccine in active specific immunotherapy

Abstract Patients with metastatic breast, colorectal or ovarian cancers received active specific immunotherapy (ASI) with Theratope® sialyl-Tn-KLH (keyhole limpet hemocyanin) cancer vaccine emulsified in Detox™ adjuvant. The median log2 anti-STn IgG titer generated by ASI, estimated by enzyme-linked immunosorbent assay with solid-phase ovine submaxillary mucin, was 5.322 (range = 0 – 9.322). Following ASI, 51 patients who generated titers higher than the median value for anti-STn+ mucin IgG survived longer than 46 patients who generated lower titers below the median. 38 of the patients were phenotyped for CD69 prior to ASI. The patients with lower numbers of CD69+ peripheral blood lymphocytes prior to immunotherapy (pre-ASI) also had low serum CA27.29 cancer antigen (MUC-1) levels, and had longer times to disease progression and improved survival following ASI. Elevated pre-ASI serum CA27.29 tumor antigen levels were associated with higher numbers of CD69+ PBL, with decreased anti-STn antibody production and decreased survival following ASI. The data are compatible with the hypothesis that elevated serum MUC-1 mucin is specifically immunosuppressive.

T cell recognition of a tumor-associated glycoprotein and its synthetic carbohydrate epitopes: stimulation of anticancer T cell immunity in vivo.

SummaryThe Thomsen, Friedenreich (TF) and Tn carbohydrate antigens are expressed on the vast majority of human adenocarcinomas and are associated with aggressive behavior of certain tumors. TF and Tn antigens are also expressed on certain murine cancer cell lines including TA3-Ha, a highly lethal, transplantable mammary adenocarcinoma. TF and Tn cancer-associated carbohydrate haptens were synthesized, conjugated to protein carriers and used to demonstrate that delayed-type hypersensitivity (DTH) effector T cells can specifically recognize and respond to carbohydrate determinants on the TA3-Ha tumor-associated glycoprotein, epiglycanin. The effector cells were shown to have the helper DTH phenotype (Lytl+, Lyt2−, Thyl+) and it was demonstrated that they respond to specific carbohydrate determinants in an MHC-restricted fashion. These experiments provide the rationale for the use of synthetic tumor-associated glycoconjugates (S-TAGs) to stimulate anticancer T cell immunity. In support of this hypothesis, it was shown that preimmunization with the appropriate S-TAGs could provide a degree of protection against a subsequent tumor transplant and that antitumor effector Lytl+, Lyt2− T cells could be generated in vitro using the appropriate S-TAGs as antigens.

The Potential of Synthetic Tumor-Associated Glycoconjugates (S-TAGs) for Generating Monoclonal Antibodies for Breast Cancer Imaging and for Specific Immunotherapy

The Thomsen Friedenreich (TF) antigen may be important for the detection and immunotherapy of a number of common cancers including breast cancer. Revealed on normal human erythrocytes by neuraminidase treatment, TF has been characterized as: β-D-Gal-(1–3)- α-GalNAc, attached to glycophorin or other glycoproteins through O-serine or O-threonine linkages (1). Tn, the TF precursor, is reported to be α-GalNAc-O-serine/ threonine. While TF is normally cryptic due to the presence of a terminal sialic acid residue, Tn is exposed in individuals with a recessive genetic disorder (2). Springer (1) has claimed expression of TF and Tn antigens on over 90% of cancers of the breast, lung and pancreas, although the nature of the molecules which bear these antigens and their exact structures has not been defined.

Prospects for an Immunological Approach to Therapy of Ovarian Cancer

Characterisation of changes in expression of carbohydrates and peptides on cancer mucins enables a reconsideration of immunotherapy of ovarian cancer. Natural immune responses to cancer mucins have been demonstrated. Some results with cell extracts have been encouraging, but there are inherent limitations to using such mixtures for immunotherapy. Successful animal model studies with synthetic vaccines have demonstrated that pretreatment with low dosage cyclophosphamide can be used to inhibit suppressor activity induced by natural cancer mucins. Preliminary results from clinical trials with synthetic vaccines challenge us to redefine ‘successful’ cancer therapy. Combination biomodulation strategies may become the therapy of the future for ovarian cancer.

Characterization and Selection Criteria of Monoclonal Antibodies for Tumor Imaging Studies

Evidence now exists that abnormalities of cell-surface molecular architecture are often associated with cancer. These subtle, yet dramatic changes, are the result of alterations in the gene regulation mechanism leading to either a blockage, stimulation or neosynthesis of cell surface molecules which, under normal conditions, would mediate cell-cell interaction and cellular differentiation (1,2). Thus, cells which have been oncologically transformed either in vitro or in vivo, will display sufficiently different profiles from their progenitor cells that they can be targeted by means of monoclonal antibodies (MAbs) which in turn could carry a number of cytotoxic agents or radionuclides. On the surface, this approach appears to be not only elegant but rather simple. In actual fact, the problems encountered in the design of such probes are predicated by the availability of the right MAb which recognizes not only a particularly well-defined chemical structure, but also an epitope of such structure consistent with the cell membrane environment of the tumor cell. Even if such a “magic bullet” were to be found, other dynamic characteristics of cell membrane constituents alter, to an extent, the degree of specificity of the selected MAb. While fundamental questions are often raised as to the importance of marker turnover as a function of cell differentiation, others are related to the relevance of the animal models so often used prior to immunoscintigraphic studies. Such diversities frequently pose considerable difficulties when selecting the appropriate antibody for clinical trials. An excellent review of this subject has been presented by Larson (3). In our laboratory, we have been interested in generating MAbs against cancer-associated carbohydrate markers and the use of such MAbs for the in vivo and in vitro detection of human tumors (4,5). Some of our experience in selecting and characterizing these MAbs is described in this paper.