MVA-MUC1-IL2 vaccine immunotherapy (TG4010) improves PSA doubling time in patients with prostate cancer with biochemical failure.

PURPOSE: TG4010 is a recombinant MVA vector expressing the tumor-associated antigen MUC1 and IL2. We explored the effect two schedules of TG4010 on PSA in men with PSA progression. PATIENTS AND METHODS: A randomized phase II trial was conducted in 40 patients with PSA progression. Patients had PSA doubling times less than 10 months, with no overt evidence of disease. Patients received either weekly subcutaneous injection (sc) of TG4010 10(8) pfu for 6 weeks, then one injection every 3 weeks or sc injection of TG4010 10(8) pfu every 3 weeks. RESULTS: The primary endpoint of a 50% decrease in PSA values from baseline was not observed. Nevertheless, 13 of 40 patients had a more than two fold improvement in PSA doubling time. Ten patients had their PSA stabilized for over 8 months. Therapy was well tolerated. CONCLUSIONS: Although the primary endpoint was not achieved, there is evidence of biologic activity of TG4010 in patients with PSA progression, further investigation in prostate cancer is warranted.

Recombinant vaccinia virus encoding human MUC1 and IL2 as immunotherapy in patients with breast cancer.

Polymorphic epithelial mucin, encoded by the MUC1 gene, is present at the apical surface of glandular epithelial cells. It is over-expressed and aberrantly glycosylated in most breast tumors, resulting in an antigenically distinct molecule and a potential target for immunotherapy. This transmembrane protein, when produced by tumor cells, is often cleaved into the circulation, where it is detectable as a tumor marker (CA 15.3) by various antibodies, allowing for early detection of recurrences and evaluation of treatment efficacy. The objective of the current study was to examine the clinical and environmental safety and immunogenicity of a live recombinant vaccinia virus expressing the human MUC1 and IL2 genes (VV TG5058), referred to here as TG1031. The study was an open-label phase 1 and 2 trial in nine patients with advanced inoperable breast cancer recurrences to the chest wall. The patients were vaccinated intramuscularly with a single dose of TG1031; three patients were treated at each of three progressive dose levels ranging from 5x10(5) to 5x10(7) plaque-forming units. A boost injection at their original dose level was administered in patients responding immunologically, clinically, or both. Vaccination resulted in no significant clinical adverse effects, and there was no environmental contamination by live TG1031. All patients had been vaccinated as children, and patients treated at the highest dose level mounted a significant anti-vaccinia antibody response. None of the nine patients had a significant increase in MUC1-specific antibody titers after one single injection, whereas five patients had a detectable increase in vaccinia virus antibody titers. Peripheral blood mononuclear cells of one patient at the intermediate dose level showed a proliferative response to in vitro culture with vaccinia virus, with a stimulation index of 6. A second patient treated at the intermediate dose level had a stimulation index of 7 to MUC1 peptide and of 14 after a boost injection. This patient had a concomitant decrease in carcinoembryonic antigen serum levels and remained clinically stable for 10 weeks. Evidence of MUC1-specific cytotoxic T lymphocytes was detected in two patients. Immunohistochemical analysis revealed an increase in T memory cells (CD45RO) in tumor biopsies after vaccination. The absence of serious adverse events, together with the documentation of immune stimulations in vivo, warrant the further use of TG1031 in immunotherapy trials of breast cancer.

Targeted macrophage cytotoxicity using a nonreplicative live vector expressing a tumor-specific single-chain variable region fragment.

Antigen-specific recognition and subsequent destruction of tumor cells is the goal of vaccine-based immunotherapy of cancer. Often, however, tumor antigen-specific cytotoxic T lymphocytes (CTLs) are either not available or in a state of anergy. In addition, MHCI expression on tumor cells is often downregulated. Either or both of these situations can allow tumor growth to proceed unchecked by CTL control. We have shown previously that tumor antigen-specific monoclonal antibodies can be expressed in vaccinia virus and that activated macrophages infected with this virus acquire the ability to kill tumor cells expressing that antigen. Here we show that a membrane-anchored form of the scFv portion of the MUC1 tumor antigen-specific monoclonal antibody, SM3, can be expressed on activated macrophages with the highly attenuated poxvirus, modified vaccinia Ankara (MVA), as a gene transfer vector. Cells infected with the MVA-scFv construct were shown to express the membrane-bound scFv by Western blot and FACS analysis. That cells expressing the membrane-anchored scFv specifically bind antigen was shown by FACS and by BIAcore analysis. GM-CSF-activated macrophages were infected with the construct and shown to recognize specifically MUC1-expressing tumor cells as measured by IL-12 release. Furthermore, activated macrophages expressing the membrane-bound scFv specifically lyse target cells expressing the MUC1 antigen but not cells that do not express MUC1.

Redirected cellular cytotoxicity by infection of effector cells with a recombinant vaccinia virus encoding a tumor-specific monoclonal antibody.

Cytotoxicity is an important function of the immune system that results in the destruction of cellular targets by humoral and/or cellular mechanisms. We wanted to assess the possibility of targeting the lytic function of immune cells toward cancer cells, which express the gene coding for a known tumor antigen (Ag) (GA733-2/epithelial cell adhesion molecule), using a viral vector encoding a monoclonal antibody (mAb) specific for said tumor Ag (CO17-1A). To this end, we have constructed recombinant vaccinia viruses expressing the sequences corresponding to mAb CO17-1A, which recognizes a specific Ag (GA733-2) that is present on the surface of most gastrointestinal carcinomas. The recombinant vectors encoding either a secreted or membrane-anchored form of CO17-1A mAb were used to infect effector cells, which were subsequently assessed for their cytotoxic activity. The recombinant viruses were able to infect both granulocyte-macrophage colony-stimulating factor-activated human macrophages and Ag-stimulated murine cytotoxic T lymphocytes. Infected granulocyte-macrophage colony-stimulating factor-activated macrophages were found to be able to kill GA733-2-expressing tumor cells. Likewise, infected cytotoxic T lymphocytes, although conserving their original alloreactivity, gained the capability of killing GA733-2-expressing cancer cells.

MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines.

Analyses of MUC1-specific cytotoxic T cell precursor (CTLp) frequencies were performed in mice immunized with three different MUC1 vaccine immunotherapeutic agents. Mice were immunized with either a fusion protein comprising MUC1 and glutathione S-transferase (MUC1-GST), MUC1-GST fusion protein coupled to mannan (MFP) or with a recombinant vaccinia virus expressing both MUC1 and interleukin-2. Mouse strain variations in immune responsiveness have been observed with these vaccines. We have constructed mice transgenic for the human MUC1 gene to study MUC1-specific immune responses and the risk of auto-immunity following MUC1 immunization. Transgenic mice immunized with MUC1 were observed to be partially tolerant in that the MUC1-specific antibody response is lower than that observed in syngeneic but non-transgenic mice. However, a significant MUC1-specific CTLp response to all three vaccines was observed, indicating the ability to overcome T cell, but to a lesser extent B cell, tolerance to MUC1 in these mice. Histological analysis indicates no evidence of auto-immunity to the cells expressing the human MUC1 molecule. These results suggest that it is possible to generate an immune response to a cancer-related antigen without damage to normal tissues expressing the antigen.

Lack of evidence for an immunosuppressive role for MUC1.

The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1-and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells.

Selective induction of peripheral and mucosal endothelial cell addressins with peripheral lymph nodes and Peyer's patch cell-conditioned media.

Vascular endothelial cell addressins play an important role in lymphocyte homing in secondary lymphoid organs and in chronic inflammatory areas. A SV40 large T antigen-immortalized cell line from peripheral lymph nodes, HECa1O [Bizouarne et al., 1993a], was used to characterize the location of addressins with regard to environmental factors and cytokines. For this purpose, two monoclonal antibodies, MECA 79 and MECA 367, specific for peripheral lymph node vascular addressin and for mucosal addressin (Peyer's patches), respectively, were bound to unstimulated HECa1O cells. Both mucosal and peripheral addressins were detected inside the cells and in cellular extracts of the resting cells. On the cell surface, both addressins could be evidenced on the same cells at a moderate level of expression. They partly mediate the EL4/EL4IL2 lymphoma cells' adhesion to HECa1O cells. Supernatants of cultured peripheral lymph node or Peyers' patch cells induced expression of MECA 79 or MECA 367 antigens, respectively, on the surface of HECa1O cells. Interleukins, IL-7, IL-3, and IL-8, induced the cell-surface appearance of MECA 79 but not of MECA 367 antigen. Therefore, the same cell type synthesizes both antigens, but the expression of these antigens on the cell surface is independently regulated, thus uncovering a characteristic tissue type-specific as well as environment-sensitive properties of microvascular endothelial cells.

Characterization of membrane sugar-specific receptors in cultured high endothelial cells from mouse peripheral lymph nodes.

The culture of specialized high endothelial cells (HEC) from lymphoid organs (peripheral lymph nodes (PLN) and Peyer's patches (PP)) was undertaken in order to study and characterize the cell surface molecules which are involved in lymphocyte recognition and allow homing. Cells were stimulated in vivo by a graft versus host (GVH) type of reaction before isolation and culture. The resulting adherent and growing cells were characterized as endothelial cells because of their typical aspect and their ability to produce angiotensin-converting enzyme and factor VIII-related antigen. They possess tissue-specific endothelial addressins. MECA 79 antigen is present on cells isolated from PLN while MECA 367 antigen is detected on cells from PP. Surface receptors for glycans were studied cytochemically using neoglycoproteins and fluorescence microscopy and quantified by flow cytometry experiments which showed that the specificity of sugar receptors depends upon endothelial cell origin. Indeed, sugar receptors for alpha-L-fucosyl residues were specifically expressed by endothelial cells from PLN. These receptors were inducible upon action of activated lymphocyte-conditioned medium. Further characterization of endothelial cells from peripheral lymph nodes indicates that they indeed mediate adhesion of lymphocytes in vitro. The role of protein-sugar interactions in this process was assessed by inhibition experiments performed with the help of neoglycoproteins. Best inhibitory effects were obtained when endothelial cells had been preincubated with alpha-L-fucosyl-BSA and when lymphoid cells were preincubated with beta-D-galactosyl-BSA. Concomitant inhibition assays indicate the participation of sugar specific receptors--endogenous lectins--on the surface of both endothelial and lymphoid cells to achieve recognition and adhesion.

A SV-40 immortalized murine endothelial cell line from peripheral lymph node high endothelium expresses a new alpha-L-fucose binding protein.

Endothelial cells from mouse peripheral lymph nodes were immortalized by cationic liposome-mediated transfection using a plasmid construct containing both the gene coding for the large T antigen of simian virus 40 and a geneticin resistance gene suitable for selection. A cell line (HECa10) was isolated on the basis of its capacity to specifically bind fucoside carrying glycoconjugates; these cells present the main characteristics of endothelial cells: production of angiotensin converting enzyme and of factor VIII-related antigen. Upon stimulation, they express E-selectin which binds oligosaccharides containing the Lewisx determinant (Fuc alpha 3[Gal beta 4 GlcNAc beta 3Gal beta) and the MECA 79 addressin which is characteristic for the peripheral lymph node high endothelium and is a L-selectin ligand. HECa10 cells, as well as peripheral lymph node high endothelial cells in primary culture, express a second fucoside binding protein which differs from E-selectin. Indeed, this new fucoside-binding protein is constitutively expressed on unstimulated cells while E-selectin is not. Furthermore, HECa10 cells mediate selective lymphoid cell adhesion in a selectin/addressin-dependent mechanism, mainly inhibited by MECA 79 antibody and, in a fucose-binding lectin-dependent manner, mainly inhibited by the specific neoglycoprotein.