Matthew M. Seavey

Cancer Immunotherapy Targeting the High Molecular Weight Melanoma-Associated Antigen Protein Results in a Broad Antitumor Response and Reduction of Pericytes in the Tumor Vasculature

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-HMW-MAA-C) that expresses and secretes a fragment of HMW-MAA (residues 2,160-2,258) fused to the first 441 residues of the listeriolysin O (LLO) protein. Immunization with Lm-LLO-HMW-MAA-C was able to impede the tumor growth of early established B16F10-HMW-MAA tumors in mice and both CD4(+) and CD8(+) T cells were required for therapeutic efficacy. Immune responses to a known HLA-A2 epitope present in the HMW-MAA(2160-2258) fragment was detected in the HLA-A2/K(b) transgenic mice immunized with Lm-LLO-HMW-MAA-C. Surprisingly, this vaccine also significantly impaired the in vivo growth of other tumorigenic cell lines, such as melanoma, renal carcinoma, and breast tumors, which were not engineered to express HMW-MAA. One hypothesis is that the vaccine could be targeting pericytes, which are important for tumor angiogenesis. In a breast tumor model, immunization with Lm-LLO-HMW-MAA-C caused CD8(+) T-cell infiltration in the tumor stroma and a significant decrease in the number of pericytes in the tumor blood vessels. In conclusion, a Lm-based vaccine against HMW-MAA can trigger cell-mediated immune responses to this antigen that can target not only tumor cells but also pericytes in the tumor vasculature.

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

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.

An Anti-Vascular Endothelial Growth Factor Receptor 2/Fetal Liver Kinase-1 Listeria monocytogenes Anti-Angiogenesis Cancer Vaccine for the Treatment of Primary and Metastatic Her-2/neu+ Breast Tumors in a Mouse Model

Thirty years after angiogenesis was shown to play an enabling role in cancer, modern medicine is still trying to develop novel compounds and therapeutics to target the tumor vasculature. However, most therapeutics require multiple rounds of administration and can have toxic side effects. In this study, we use anti-angiogenesis immunotherapy to target cells actively involved in forming new blood vessels that support the growth and spread of breast cancer. Targeting a central cell type involved in angiogenesis, endothelial cells, we immunized against host vascular endothelial growth factor receptor 2 to fight the growth of Her-2/neu+ breast tumors. Using the bacterial vector, Listeria monocytogenes (Lm), we fused polypeptides from the mouse vascular endothelial growth factor receptor 2 molecule (fetal liver kinase-1) to the microbial adjuvant, listeriolysin-O, and used Lm to deliver the Ags and elicit potent antitumor CTL responses. Lm-listeriolysin-O-fetal liver kinase-1 was able to eradicate some established breast tumors, reduce microvascular density in the remaining tumors, protect against tumor rechallenge and experimental metastases, and induce epitope spreading to various regions of the tumor-associated Ag Her-2/neu. Tumor eradication was found to be dependent on epitope spreading to HER-2/neu and was not solely due to the reduction of tumor vasculature. However, vaccine efficacy did not affect normal wound healing nor have toxic side effects on pregnancy. We show that an anti-angiogenesis vaccine can overcome tolerance to the host vasculature driving epitope spreading to an endogenous tumor protein and drive active tumor regression.

Targeting tumor vasculature with novel Listeria-based vaccines directed against CD105

The FDA approval of bevacizumab (Avastin®, Genentech/Roche), a monoclonal antibody raised against human VEGF-A, as second-line therapy for colon and lung carcinoma validated the approach of targeting human tumors with angiogenesis inhibitors. While the VEGF/VEGFR pathway is a viable target for anti-angiogenesis tumor therapy, additional targets involved in tumor neovascularization have been identified. One promising target present specifically on tumor vasculature is endoglin (CD105), a member of the TGF-β receptor complex expressed on vascular endothelium and believed to play a role in angiogenesis. Monoclonal antibody therapy and preventive vaccination against CD105 has met with some success in controlling tumor growth. This report describes the in vivo proof-of-concept studies for two novel therapeutic vaccines, Lm-LLO-CD105A and Lm-LLO-CD105B, directed against CD105 as a strategy to target neovascularization of established tumors. Listeria-based vaccines directed against CD105 lead to therapeutic responses against primary and metastatic tumors in the 4T1-Luc and NT-2 mouse models of breast cancer. In a mouse model for autochthonous Her-2/neu-driven breast cancer, Lm-LLO-CD105A vaccination prevented tumor incidence in 20% of mice by week 58 after birth while all control mice developed tumors by week 40. In comparison with previous Listeria-based vaccines targeting tumor vasculature, Lm-LLO-CD105A and Lm-LLO-CD105B demonstrated equivalent or superior efficacy against two transplantable mouse models of breast cancer. Support is provided for epitope spreading to endogenous tumor antigens and reduction in tumor vascularity after vaccination with Listeria-based CD105 vaccines. Reported here, these CD105 therapeutic vaccines are highly effective in stimulating anti-angiogenesis and anti-tumor immune responses leading to therapeutic efficacy against primary and metastatic breast cancer.

Therapeutic Efficacy of CEP-33779, a Novel Selective JAK2 Inhibitor, in a Mouse Model of Colitis-Induced Colorectal Cancer

Constitutively activated STAT3 and STAT5 are expressed in a wide variety of human malignancies including solid and hematopoietic cancers and often correlate with a poor prognosis and resistance to multiple therapies. Given the well established role of STAT3 in tumorigenesis, inhibition of Janus-activated kinase 2 (JAK2) activity might represent an attractive therapeutic approach. Using a mouse model of colitis-induced colorectal cancer, we show that a novel, orally active, selective JAK2 inhibitor, CEP-33779, induced regression of established colorectal tumors, reduced angiogenesis, and reduced proliferation of tumor cells. Histopathologic analysis confirmed reduced incidence of histologic-grade neoplasia by CEP-33779. Tumor regression correlated with inhibition of STAT3 and NF-κB (RelA/p65) activation in a CEP-33779 dose–dependent manner. In addition, the expression of proinflammatory, tumor-promoting cytokines interleukin (IL)-6 and IL-1β was strongly reduced upon JAK2 inhibition. The ability of CEP-33779 to suppress growth of colorectal tumors by inhibiting the IL-6/JAK2/STAT3 signaling suggests a potential therapeutic utility of JAK2 inhibitors in multiple tumors types, particularly those with a strong inflammatory component. Mol Cancer Ther; 11(4); 984–93. ©2012 AACR.

Immunoregulation of fetal and anti-paternal immune responses

Immunological tolerance to the fetus is essential for fetal survival during pregnancy. The semi-allogeneic fetus expresses genes foreign to the mother that can be recognized by maternal T cells. Under times of stress or infection, deleterious immune responses can result in fetal destruction and/or maternal death. Exposure to non-maternal antigens begins as early as insemination and some of the mechanisms required to prevent maternal priming against these antigens are in place before sexual encounter. Continuous and overlapping regulatory mechanisms must cooperate to allow the best chances for fertilization, implantation, and healthy gestation, simultaneously protecting the fetus from maternal immune attack yet making minimal compromises in resistance to infection. Several types of immune cell from both the innate and adaptive arms of the immune system help protect both the mother and fetus during pregnancy. It’s the intricate communication and interplay between the immune system and the endocrine system that will ultimately decide the success or fate of the developing fetus.

Paternal Antigen-Bearing Cells Transferred during Insemination Do Not Stimulate Anti-Paternal CD8+ T Cells: Role of Estradiol in Locally Inhibiting CD8+ T Cell Responses

Maternal immunological tolerance of the semiallogeneic fetus involves several overlapping mechanisms to balance maternal immunity and fetal development. Anti-paternal CD8+ T cells are suppressed during pregnancy in some but not all mouse models. Since semen has been shown to mediate immune modulation, we tested whether exposure to paternal Ag during insemination activated or tolerized anti-paternal CD8+ T cells. The uterine lumen of mated female mice contained male MHC I+ cells that stimulated effector, but not naive, CD8+ T cells ex vivo. Maternal MHC class I+ myeloid cells fluxed into the uterine lumen in response to mating and cross-presented male H-Y Ag to effector, but not naive, CD8+ T cells ex vivo. However, neither unprimed nor previously primed TCR-transgenic CD8+ T cells specific for either paternal MHC I or H-Y Ag proliferated in vivo after mating. These T cells subsequently responded normally to i.p. challenge, implicating ignorance rather than anergy as the main reason for the lack of response. CD8+ T cells responded to either peptide Ag or male cells delivered intravaginally in ovariectomized mice, but this response was inhibited by systemic estradiol (inducing an estrus-like state). Subcutaneous Ag induced responses in both cases. Allogeneic dendritic cells did not induce responses intravaginally even in ovariectomized mice in the absence of estradiol. These results suggest that inhibition of antiallogeneic responses is restricted both locally to the reproductive tract and temporally to the estrous phase of the menstrual cycle, potentially decreasing the risk of maternal immunization against paternal Ags during insemination.

The ubiquitin-like protein, ISG15, is a novel tumor-associated antigen for cancer immunotherapy

The recent announcement of the first FDA-approved therapeutic vaccine for prostate cancer, Sipuleucel-T, is a watershed moment for the field of tumor immunotherapy. However, while Sipuleucel-T provides a powerful tool to clinicians for the most prevalent form of cancer in men, there remains an unmet need for a similar therapeutic strategy against breast cancer, the most prevalent cancer in women. While current breast cancer vaccines in development target several antigens, the most prevalent is the tumor-associated antigen, HER2. Initial results with HER2 vaccines appear promising in terms of efficacy; however, the lack of HER2 overexpression by a majority of breast tumors and the safety concerns associated with current HER2-targeted immunotherapy suggest that additional therapeutic strategies would be beneficial. Recently, several studies have identified ISG15 as a molecule highly expressed in numerous malignancies. ISG15 is a small ubiquitin-like protein regulated by type-I interferon and classically associated with viral defense. Elevated ISG15 expression in breast cancer is especially well documented and is independent of HER2, progesterone receptor, and estrogen receptor status. Additionally, high ISG15 expression in breast cancer correlates with an unfavorable prognosis and poor responses to traditional treatment strategies such as chemotherapy and radiation. To overcome these challenges, we employ a novel strategy to specifically target tumor-associated ISG15 expression with immunotherapy. We demonstrate that vaccination against ISG15 results in significant CD8-mediated reductions in both primary and metastatic mammary tumor burden. These results validate ISG15 as a tumor-associated antigen for cancer immunotherapy.

Estradiol-induced vaginal mucus inhibits antigen penetration and CD8+ T cell priming in response to intravaginal immunization

Although vaginal immunization has been explored as a strategy to induce mucosal immunity in the female reproductive tract, this site displays unique immunological features that probably evolved to inhibit anti-paternal T cell responses after insemination to allow successful pregnancy. We previously demonstrated that estradiol, which induces an estrus-like state, prevented CD8(+) T cell priming during intravaginal immunization of mice. We now show that estradiol prevented antigen loading of vaginal antigen presenting cells (APCs) after intravaginal immunization. Histological examination confirmed that estradiol prevented penetration of peptide antigen into the vaginal wall. Removal of the estradiol-induced mucus barrier by mucinase partially restored antigen loading of vaginal APC and CD8(+) T cell proliferation in vivo. The estradiol-induced mucus barrier may thus prevent exposure to antigens delivered intravaginally, supplementing additional estradiol-dependent mechanism(s) that inhibit CD8(+) T cell priming after insemination or vaginal vaccination.

Antiangiogenesis immunotherapy induces epitope spreading to her-2/neu resulting in breast tumor immunoediting

Targeting tumors using cancer vaccine therapeutics has several advantages including the induction of long-term immunity, prime boost strategies for additional treatments and reduced side effects compared to conventional chemotherapeutics. However, one problem in targeting tumor antigens directly is that this can lead to antigen loss or immunoediting. We hypothesized that directing the immune response to a normal cell type required for tumor growth and survival could provide a more stable immunotherapeutic target. We thus examined the ability of an antiangiogenesis, Listeria monocytogenes (Lm)-based vector to deliver extracellular and intracellular fragments of the mouse vascular endothelial growth factor receptor-2/Flk-1 molecule, Lm-LLO-Flk-E1, and Lm-LLO-Flk-11 respectively, in an autochthonous model for Her-2/neu(+) breast cancer. We found that these vaccines could cause epitope spreading to the endogenous tumor protein Her-2/neu and significantly delay tumor onset. However, tumors that grew out overtime accumulated mutations in the Her-2/neu molecule near or within cytotoxic T lymphocytes epitopes. We show here for the first time how an antiangiogenesis immunotherapy can be used to delay the onset of a spontaneous tumor through epitope spreading and determine a possible mechanism of how immunoediting of an endogenous tumor protein can allow for tumor escape and outgrowth in an autochthonous mouse model for Her-2/neu(+) breast cancer.