B. Michael Longenecker

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.

Immunogenicity of synthetic peptides related to the core peptide sequence encoded by the human MUC1 mucin gene: Effect of immunization on the growth of murine mammary adenocarcinoma cells transfected with the human MUC1 gene

The immune response of CAF1 mice to various synthetic peptides (SP) related to the amino acid sequence (PDTRPAPGSTAPPAHGVTSA) of the tandem repeat of the MUC1 human breast mucin core peptide was evaluated. The most immunogenic preparations of the synthetic peptides were those conjugated to keyhole limpet hemocyanin (KLH) or clustered in a dendritic multiple antigenic peptide (MAP-4) configuration. The mice were immunized subcutaneously with synthetic peptides emulsified in RIBI adjuvant, employing various immunization protocols. Equivalently high IgG responses were induced using SP-KLH conjugates (GVTSAPDTRPAPGSTA-KLH) or an SP — MAP-4 chimeric configuration (SP1-6), which also included a universal malarial CST-3 T-helper epitope (SP1-6 = SAPDTRPAEKKIAKMEKASSVFNVVNS — MAP-4). These IgG antibodies bound both the appropriate MUC1 synthetic peptides and the cell surface expressed MUC1 mucin on murine mammary cells that had been transfected with the human MUC1 gene and a human breast cancer cell line that expresses cell-surface MUC1. A MAP-4 molecule, which included the entire 20-aminoacid sequence of the MUC1 tandem repeat (SP1-5 = PDTRPAPGSTAPPAHGVTSA—MAP-4) induced a poor IgG response. In contrast, all three types of molecule: SP-KLH, SP1-6 and SP1-5, were found to be good immunogens for the induction of specific delayedtype hypersensitivity (DTH) reactions measured using either synthetic peptides or MUC1-transfected cells. In addition, immunization with irradiated MUC1-transfected cells induced strong DTH reactions measured using synthetic peptides that expressed the PDTRP sequence, which has been shown to be, or to overlap, a T cell epitope in humans and a B cell epitope in mice. Finally, it was demonstrated that synthetic MUC1 peptide “vaccines” could be used both prophylactically and therapeutically to inhibit the growth of MUC1-transfected tumor cells and prolong the survival of tumor-bearing mice.

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.

The biological role of mucins in cellular interactions and immune regulation: Prospects for cancer immunotherapy

Among the human mucins, MUC1 is unique in its cell-surface transmembrane expression and its apparent signal-transduction functions. The high expression of MUC1 on many human cancers makes it an attractive target for immunotherapy. Immunization of human cancer patients with MUC1 peptides has resulted in the generation of both anti-MUC1 antibody and cytotoxic T lymphocyte responses. Recently, a novel immunoregulatory role for MUC1 has been suggested by experiments demonstrating that soluble MUC1 induces T-cell unresponsiveness, and that T cells appear to express and secrete MUC1 following their activation. MUC1 is an apparent paradox, having both adhesive and antiadhesive functions, and immunostimulatory and immunosuppressive activities.

Mucin 1-specific immunotherapy in a mouse model of spontaneous breast cancer.

Human mucin 1 (MUC1) is an epithelial mucin glycoprotein that is overexpressed in 90% of all adenocarcinomas including breast, lung, pancreas, prostate, stomach, colon, and ovary. MUC1 is a target for immune intervention, because, in patients with solid adenocarcinomas, low-level cellular and humoral immune responses to MUC1 have been observed, which are not sufficiently strong to eradicate the growing tumor. The hypothesis for this study is that enhancing MUC1-specific immunity will result in antitumor immunity. To test this, the authors have developed a clinically relevant breast cancer model that demonstrates peripheral and central tolerance to MUC1 and develops spontaneous tumors of the mammary gland. In these mice, the authors tested a vaccine formulation comprised of liposomal-MUC1 lipopeptide and human recombinant interleukin-2. Results indicate that when compared with untreated mice, immunized mice develop T cells that express intracellular IFN-&ggr;, are reactive with MHC class I H-2Db/MUC1 tetramer, and are cytotoxic against MUC1-expressing tumor cells in vitro. The presence of MUC1-specific CTL did not translate into a clinical response as measured by time of tumor onset, tumor burden, and survival. The authors demonstrate that some of the immune-evasion mechanisms used by the tumor cells include downregulation of MHC-class I molecule, expression of TGF-&bgr;2, and decrease in IFN-&ggr; -expressing effector T cells as tumors progress. Finally, utilizing an injectable breast cancer model, the authors show that targeting a single tumor antigen may not be an effective antitumor treatment, but that immunization with dendritic cells fed with whole tumor lysate is effective in breaking tolerance and protecting mice from subsequent tumor challenge. A physiologically relevant spontaneous breast cancer model has been developed to test improved immunotherapeutic approaches.

Immunogenicity and antitumor activity of a liposomal MUC1 peptide-based vaccine

A human MUC1‐transfected mouse mammary adenocarcinoma cell line (GZHI) was used to develop both subcutaneous and intravenous tumor models. A vaccine formulation comprised of a 24 mer (human MUC1) synthetic peptide encapsulated with monophosphoryl lipid A adjuvant (MPLA) in multilamellar liposomes was tested for immunogenicity and anti‐tumor activity. A low dose of the human MUC1 peptide (5 μg) administered in liposomes provided excellent protection of mice in both tumor challenge models. The protective antitumor activity mediated by the liposome formulation correlated with anti‐MUC1‐specific T‐cell proliferation, gamma‐interferon (IFN‐γ) production and IgG2a anti‐MUC1 antibodies, suggesting a type 1 (T1) T‐cell response. In contrast, lack of protection in mice immunized with negative control vaccines correlated with IgG1 anti‐MUC1 antibody formation, low or no anti‐MUC1 IgG2a and low antigen‐specific T‐cell proliferation, consistent with a type 2 (T2) T‐cell response to the tumor. Int. J. Cancer 75:295–302, 1998.

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.

MUC1-specific CTLs are non-functional within a pancreatic tumor microenvironment

Pancreatic cancer is a highly aggressive, treatment refractory disease and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas over-express altered forms of a tumor-associated antigen, MUC1 (an epithelial mucin glycoprotein), which is a target for immunotherapy. Using a clinically relevant mouse model of pancreas cancer that demonstrates peripheral and central tolerance to human MUC1 and develops spontaneous tumors of the pancreas, we have previously reported the presence of functionally active, low affinity, MUC1-specific precursor cytotoxic T cells (pCTLs). Hypothesis for this study is that MUC1-based immunization may enhance the low level MUC1-specific immunity that may lead to an effective anti-tumor response. Data demonstrate that MUC1 peptide-based immunization elicits mature MUC1-specific CTLs in the peripheral lymphoid organs. The mature CTLs secrete IFN-γ and are cytolytic against MUC1-expressing tumor cells in vitro. However, active CTLs that infiltrate the pancreas tumor microenvironment become cytolytically anergic and are tolerized to MUC1 antigen, allowing the tumor to grow. We demonstrate that the CTL tolerance could be reversed at least in vitro with the use of anti-CD40 co-stimulation. The pancreas tumor cells secrete immunosuppressive cytokines, including IL-10 and TGF-ß that are partly responsible for the down-regulation of CTL activity. In addition, they down-regulate their MHC class I molecules to avoid immune recognition. CD4+CD25+ T regulatory cells, which secrete IL-10, were also found in the tumor environment. Together these data indicate the use of several immune evasion mechanisms by tumor cells to evade CTL killing. Thus altering the tumor microenvironment to make it more conducive to CTL killing may be key in developing a successful anti-cancer immunotherapy.

“Natural” Antibodies to chicken MHC antigens are present in mice, rats, humans, alligators and allogeneic chickens

In the absence of any deliberate immunization, mice, rats, humans and alligators all have detectable titers of antibody against chicken red blood cells (CRBCs). Remarkably, this antibody is directed predominantly against private or public determinants of MHC proteins on the CRBCs, and little or no antibody is directed against species-specific determinants on MHC or other proteins, including other polymorphic blood group antigens. In chickens, “natural” antibody can be detected against CRBCs from all chickens differing at the MHC locus, but “natural” antibodies against other polymorphic antigens are not detected. Using a rosette-forming cell (RFC) assay, we have also shown that a large percentage of mouse spleen cells will rosette with chicken erythrocytes, and that the majority of these RFCs also recognize polymorphic antigens.

Glycopeptide-and carbohydratebased synthetic vaccines for the immunotherapy of cancer

On cancer cells, MUC-1 mucin displays distinct carbohydrate structures, such as ThomsenFriedenreich (TF) and sialyl-Tn, the presence of which is attributed to reduced glycosylation activity. The core peptide is increasingly exposed and is recognized by the immune system. Vaccines based on both the exposed core protein, which contains major histocompatibility complex unrestricted epitopes, and carbohydrate structures are targets for the immunotherapy of cancers of epithelial origin. A vaccine formulated using synthetic sialyl-Tn has proven to be highly target-specific in human trials, and the induction of high anti-STn antibody titers correlated with prolonged survival of breast cancer patients. Peptides and glycopeptides formulated as liposomebased vaccines have been effective in animal models.