A Novel Human Her-2/neu Chimeric Molecule Expressed byListeria monocytogenesCan Elicit Potent HLA-A2 Restricted CD8-positive T cell Responses and Impact the Growth and Spread of Her-2/neu-positive Breast Tumors

Abstract

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...

Development of a live and highly attenuated Listeria monocytogenes-based vaccine for the treatment of Her2/neu-overexpressing cancers in human

“…3 In the first approaches to target the HER2/neu antigen using Listeria monocytogenes (Lm), Listeria-based vaccines expressing small fragments of the extra and intracellular domains of the rat and human HER2/neu protein were constructed. 4,5 We also generated a Listeria vaccine (Lm-LLO-ChHer2) using a chimeric protein by fusing two of the extracellular and one intracellular fragments of the human HER2/neu, including most of the known major histocompatibility complex (MHC) class I epitopes of the protein. 5 All of these vaccines were shown to be immunogenic and efficacious in regressing preestablished tumors in FVB/N mice and delay the onset of spontaneous mammary tumors in HER2/neu-expressing transgenic animals.…”

“…4,5 We also generated a Listeria vaccine (Lm-LLO-ChHer2) using a chimeric protein by fusing two of the extracellular and one intracellular fragments of the human HER2/neu, including most of the known major histocompatibility complex (MHC) class I epitopes of the protein. 5 All of these vaccines were shown to be immunogenic and efficacious in regressing preestablished tumors in FVB/N mice and delay the onset of spontaneous mammary tumors in HER2/neu-expressing transgenic animals. 5 Because of the high homology (B96%) between rat and human HER2/neu protein sequences and at least three known common H-2D q epitopes between the two proteins, these preliminary experiments suggested that Lm-LLO-ChHer2 could break the tolerance toward the HER2/neu self-antigen.…”

“…5 All of these vaccines were shown to be immunogenic and efficacious in regressing preestablished tumors in FVB/N mice and delay the onset of spontaneous mammary tumors in HER2/neu-expressing transgenic animals. 5 Because of the high homology (B96%) between rat and human HER2/neu protein sequences and at least three known common H-2D q epitopes between the two proteins, these preliminary experiments suggested that Lm-LLO-ChHer2 could break the tolerance toward the HER2/neu self-antigen. However, the Listeria-HER2/neu vaccines developed, thus far, have been based on an attenuated Listeria platform, which used the antibiotic marker (cat), for in vitro selection of the recombinant bacteria in the presence of chloramphenicol.…”

“…However, the Listeria-HER2/neu vaccines developed, thus far, have been based on an attenuated Listeria platform, which used the antibiotic marker (cat), for in vitro selection of the recombinant bacteria in the presence of chloramphenicol. 5 In order to advance this vaccine toward human use, we have generated another recombinant Listeria-Her2/neu vaccine (ADXS31-164) using the highly attenuated LmddA vaccine vector, which has a deletion of the virulence gene actA and is devoid of antibiotic selection markers. 6 ADXS31-164 retains the plasmid for Her2/neu expression in vivo and in vitro by auxotrophic complementation of the dal gene, which is absent in the LmddA strain and required for the synthesis of D-alanine.…”

“…This study compares the immunogenicity and efficacy of this optimized vaccine for human use, with our previous chloramphenicol resistant version. 5 …”