Two recombinant Listeria monocytogenes (rLm) strains were produced that secrete the human papilloma virus-16 (HPV-16) E7 protein expressed in HPV-16-associated cervical cancer cells. One, Lm-E7, expresses and secretes E7 protein, whereas a second, Lm-LLO-E7, secretes E7 as a fusion protein joined to a nonhemolytic listeriolysin O (LLO). Lm-LLO-E7, but not Lm-E7, induces the regression of the E7-expressing tumor, TC-1, established in syngeneic C57BL/6 mice. Both recombinant E7-expressing rLm vaccines induce measurable anti-E7 CTL responses that stain positively for H-2Db E7 tetramers. Depletion of the CD8+ T cell subset before treatment abrogates the ability of Lm-LLO-E7 to impact on tumor growth. In addition, the rLm strains induce markedly different CD4+ T cell subsets. Depletion of the CD4+ T cell subset considerably reduces the ability of Lm-LLO-E7 to eliminate established TC-1 tumors. Surprisingly, the reverse is the case for Lm-E7, which becomes an effective anti-tumor immunotherapeutic in mice lacking this T cell subset. Ab-mediated depletion of TGF-β and CD25+ cells improves the effectiveness of Lm-E7 treatment, suggesting that TGF-β and CD25+ cells are in part responsible for this suppressive response. CD4+ T cells from mice immunized with Lm-E7 are capable of suppressing the ability of Lm-LLO-E7 to induce the regression of TC-1 when transferred to tumor-bearing mice. These studies demonstrate the complexity of L. monocytogenes-mediated tumor immunotherapy targeting the human tumor Ag, HPV-16 E7.

The high molecular weight melanoma-associated antigen (HMW-MAA), also known as melanoma chondroitin sulfate proteoglycan, has been used as a target for the immunotherapy of melanoma. This antigen is expressed on the cell surface and has a restricted distribution in normal tissues. Besides its expression in a broad range of transformed cells, this antigen is also found in pericytes, which are important for tumor angiogenesis. We generated a recombinant Listeria monocytogenes (Lm-LLO-

Previous work in our laboratory has established that the fusion of tumor-associated antigens to a truncated form of the Listeria monocytogenes virulence factor listeriolysin O (LLO) enhances the immunogenicity and antitumor efficacy of the tumor antigen when delivered by Listeria or by vaccinia. LLO contains a PEST sequence at the NH 2 terminus. These sequences, which are found in eukaryotic proteins with a short cellular half-life, target proteins for degradation in the ubiquitin-proteosome pathway. To investigate whether the enhanced immunogenicity conferred by LLO is due to the PEST sequence, we constructed new Listeria recombinants that expressed the HPV-16 E7 antigen fused to LLO, which either contained or had been deleted of this sequence. We then compared the antitumor efficacy of this set of vectors and found that Listeria expressing the fusion protein LLO-E7 or PEST-E7 were effective at regressing established macroscopic HPV-16 immortalized tumors in syngeneic mice. In contrast, Listeria recombinants expressing E7 alone or E7 fused to LLO from which the PEST sequence had been genetically removed could only slow tumor growth. Because CD8 ؉ T cell epitopes are generated in the ubiquitin-proteosome pathway, we also investigated the ability of the vaccines to induce E7-specific CD8 ؉ T cells in the spleen and to generate E7-specific tumor-infiltrating lymphocytes. A strong correlation was observed between CD8 ؉ T-cell induction and tumor homing and the antitumor efficacy of the Listeria-E7 vaccines. These findings suggest a strategy for the augmentation of tumor antigen-based immunotherapeutic strategies that may be broadly applicable.

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Purpose: The aim of this study was to efficiently design a novel vaccine for human Her-2/ neu-positive (hHer-2/neu) breast cancer using the live, attenuated bacterial vector Listeria monocytogenes. Experimental Design:Three recombinant L. monocytogenes^based vaccines were generated that could express and secrete extracellular and intracellular fragments of the hHer-2/neu protein.In addition, we generated a fourth construct fusing selected portions of each individual fragment that contained most of the human leukocyte antigen (HLA) epitopes as a combination vaccine (L. monocytogenes^hHer-2/neu chimera). Results: Each individual vaccine was able to either fully regress or slow tumor growth in a mouse model for Her-2/neu-positive tumors. All three vaccines could elicit immune responses directed toward human leukocyte antigen-A2 epitopes of hHer-2/neu. The L. monocytogenes^hHer-2/ neu chimera was able to mimic responses generated by the three separate vaccines and prevent spontaneous outgrowth of tumors in an autochthonous model for Her-2/neu-positive breast cancer, induce tumor regression in transplantable models, and prevent seeding of experimental lung metastases in a murine model for metastatic breast cancer. Conclusion: This novel L. monocytogenes^hHer-2/neu chimera vaccine proves to be just as effective as the individual vaccines but combines the strength of all three in a single vaccination. These encouraging results support future clinical trials using this chimera vaccine and may be applicable to other cancer types expressing the Her-2/neu molecule such as colorectal and pancreatic cancer.The Her-2/neu receptor is considered to be an oncogene, because its overexpression (1, 2), point mutations (3), or deletions (4) have been associated with several types of cancers. Neoplasms that are linked to dysregulation of Her-2/neu include cancers of the breast, lung, colon, stomach, and pancreas (5 -7). Her-2/neu overexpression by human tumors is an independent predictor of poor prognosis (2,8,9) and is associated with aggressive disease and resistance to high-dose chemotherapies such as tamoxifen, cytoxan, methotrexate, and 5-fluorouracil (9). Her-2/neu is a member of the epithelial growth factor receptor family with tyrosine kinase activity (10). It is a large (185 KD) protein comprising three domains: an extracellular, a transmembrane, and an intracellular domain.Listeria monocytogenes is an intracellular facultative bacterium that has been investigated as a vaccine vector for cancer for over a decade (11). Once it enters the host, Listeria is rapidly taken up by immune cells such as dendritic cells and macrophages through phagocytosis. Due to the pore-forming action of a listerial cytotoxin called ref. 12), a fraction of the bacteria can escape from the phagosomes and multiply in the host cytosol. Because of this particular dual life cycle, which provides the bacterium with access to both phagosomal and intracellular compartments, antigens delivered by L. monocytogenes -based vaccines are presented on b...