Denise L. Cecil

A Multiantigen Vaccine Targeting Neu, IGFBP-2, and IGF-IR Prevents Tumor Progression in Mice with Preinvasive Breast Disease

A multiantigen multipeptide vaccine, targeting proteins expressed in preinvasive breast lesions, can stimulate type I CD4+ T cells which have been shown to be deficient in both patients with breast cancer and mice that develop mammary tumors. Transgenic mice (TgMMTV-neu) were immunized with a multiantigen peptide vaccine specific for neu, insulin—like growth factor-binding protein 2 and insulin-like growth factor receptor-I at a time when some of the animals already had preinvasive lesions (18 weeks of age). Although immunization with each individual antigen was partially effective in inhibiting tumor growth, immunization with the multiantigen vaccine was highly effective, blocking development of palpable lesions in 65% of mice and slowing tumor growth in the infrequent palpable tumors, which did arise. Protection was mediated by CD4+ T cells, and the few slow-growing tumors that did develop demonstrated a significant increase in intratumoral CD8+ T cells as compared with controls (P = 0.0007). We also combined the vaccine with agents that were, by themselves, partially effective inhibitors of tumor progression in this model; lapatinib and the RXR agonist bexarotene. Although the combination of lapatinib and vaccination performed similarly to vaccination alone (P = 0.735), bexarotene and vaccination significantly enhanced disease-free survival (P < 0.0001), and approximately 90% of the mice showed no pathologic evidence of carcinomas at one year. The vaccine also demonstrated significant clinical efficacy in an additional transgenic model of breast cancer (TgC3(I)-Tag). Chemoimmunoprevention combinations may be an effective approach to breast cancer prevention even when the vaccine is administered in the presence of subclinical disease. Cancer Prev Res; 6(12); 1273–82. ©2013 AACR.

Elimination of IL-10–Inducing T-Helper Epitopes from an IGFBP-2 Vaccine Ensures Potent Antitumor Activity

Immunization against self-tumor antigens can induce T-regulatory cells, which inhibit proliferation of type I CD4(+) T-helper (TH1) and CD8(+) cytotoxic T cells. Type I T cells are required for potent antitumor immunity. We questioned whether immunosuppressive epitopes could be identified and deleted from a cancer vaccine targeting insulin-like growth factor-binding protein (IGFBP-2) and enhance vaccine efficacy. Screening breast cancer patient lymphocytes with IFN-γ and interleukin (IL)-10 ELISPOT, we found epitopes in the N-terminus of IGFBP-2 that elicited predominantly TH1 whereas the C-terminus stimulated TH2 and mixed TH1/TH2 responses. Epitope-specific TH2 demonstrated a higher functional avidity for antigen than epitopes, which induced IFN-γ (P = 0.014). We immunized TgMMTV-neu mice with DNA constructs encoding IGFBP-2 N-and C-termini. T cell lines expanded from the C-terminus vaccinated animals secreted significantly more type II cytokines than those vaccinated with the N-terminus and could not control tumor growth when infused into tumor-bearing animals. In contrast, N-terminus epitope-specific T cells secreted TH1 cytokines and significantly inhibited tumor growth, as compared with naïve T cells, when adoptively transferred (P = 0.005). To determine whether removal of TH2-inducing epitopes had any effect on the vaccinated antitumor response, we immunized mice with the N-terminus, C-terminus, and a mix of equivalent concentrations of both vaccines. The N-terminus vaccine significantly inhibited tumor growth (P < 0.001) as compared with the C-terminus vaccine, which had no antitumor effect. Mixing the C-terminus with the N-terminus vaccine abrogated the antitumor response of the N-terminus vaccine alone. The clinical efficacy of cancer vaccines targeting self-tumor antigens may be greatly improved by identification and removal of immunosuppressive epitopes.

Th1 epitope selection for clinically effective cancer vaccines.

New cancer immunotherapies mark progress in our understanding of tumor biology and harnessing the immune systems management of self. However, protein- and peptide-based vaccines are not yet consistently efficacious. Recent work uncovers principles governing the genesis of T helper type-restrictive immunity to self-antigens elicited by vaccine epitopes, enabling vaccines to skew the balance from tolerogenic Type II (Th2) to inflammatory Type I (Th1) T cells, and invigorating this cancer immunotherapeutic approach.

Selection of epitopes from self-antigens for eliciting Th2 or Th1 activity in the treatment of autoimmune disease or cancer.

Vaccines have been valuable tools in the prevention of infectious diseases, and the rapid development of new vectors against constantly mutating foreign antigens in viruses such as influenza has become a regular, seasonal exercise. Harnessing the immune response against self-antigens is not necessarily analogous or as achievable by iterative processes, and since the desired outcome includes leaving the targeted organism intact, requires some precision engineering. In vaccine-based treatment of autoimmunity and cancer, the proper selection of antigens and generation of the desired antigen-specific therapeutic immunity has been challenging. Both cases involve a threshold of existing, undesired immunity that must be overcome, and despite considerable academic and industry efforts, this challenge has proven to be largely refractory to vaccine approaches leveraging enhanced vectors, adjuvants, and administration strategies. There are in silico approaches in development for predicting the immunogenicity of self-antigen epitopes, which are being validated slowly. One simple approach showing promise is the functional screening of self-antigen epitopes for selective Th1 antitumor immunogenicity, or inversely, selective Th2 immunogenicity for treatment of autoimmune inflammation. The approach reveals the importance of confirming both Th1 and Th2 components of a vaccine immunogen; the two can confound one another if not parsed but may be used individually to modulate antigen-specific inflammation in autoimmune disease or cancer.

Immunization against HIF-1α Inhibits the Growth of Basal Mammary Tumors and Targets Mammary Stem Cells In Vivo

Purpose: Triple-negative breast cancer (TNBC) represents a cancer stem cell–enriched phenotype. Hypoxia-inducible factor-1α (HIF-1α) induces the expression of proteins associated with stemness and is highly upregulated in TNBC. We questioned whether HIF-1α was immunogenic and whether vaccination targeting HIF-1α would impact the growth of basal-like mammary tumors in transgenic mice. Experimental Design: We evaluated HIF-1α–specific IgG in sera from controls and patients with breast cancer. Class II epitopes derived from the HIF-1α protein sequence were validated by ELISPOT. To assess therapeutic efficacy, we immunized Tg-MMTVneu and C3(1)Tag mice with HIF-1α Th1-inducing peptides. Stem cells were isolated via magnetic bead separation. Levels of HIF-1α and stem cells in the tumor were quantitated by Western blotting and flow cytometry. Results: The magnitude (P < 0.001) and incidence (P < 0.001) of HIF-1α–specific IgG were elevated in TNBC patients compared with controls. Both breast cancer patients and donors showed evidence of HIF-1α–specific Th1 and Th2 immunity. Three HIF-1α–specific Th1 class II restricted epitopes that were highly homologous between species elicited type I immunity in mice. After HIF-1α vaccination, mammary tumor growth was significantly inhibited in only C3(1)Tag (basal-like/stem cellhigh; P < 0.001) not TgMMTV-neu (luminal/neu/stem celllow; P = 0.859) murine models. Vaccination increased type I T cells in the tumor (P = 0.001) and decreased cells expressing the stem cell marker, Sca-1, compared with controls (P = 0.004). Conclusions: An HIF-1α vaccine may be uniquely effective in limiting tumor growth in TNBC. Inhibiting outgrowth of breast cancer stem cells via active immunization in the adjuvant setting may impact disease recurrence. Clin Cancer Res; 23(13); 3396–404. ©2016 AACR.

Clinical Application of Plasmid-Based Cancer Vaccines

Decades of work on DNA-based cancer vaccination has produced numerous clinical candidates, most targeting single tumor-associated antigens, a few targeting multiple antigens or epitopes, and virtually all predominantly aimed at the induction of CD8+ cytotoxic T cells. Clinical trials in melanoma, prostate, breast, colorectal, and cervical carcinomas reporting results in the past few years are herein reviewed. Clinical and immunological responses were generally small and detected in only a minority of patients. High doses, extended immunization periods, and multifaceted platforms have modestly improved immunogenicity. Appreciating the important role polyfunctional CD4+ helper T cells play in tumor rejection, and using recent advances in epitope mapping and informatics, a new generation of DNA-based vaccines targeting CD4+ epitopes that are both tumor reactive and bind multiple MHC class II haplotypes are now entering clinical trials. With such an empirical approach, DNA-based vaccines may be poised to become potent weapons in the armamentarium against cancer.

Abstract NTOC-097: VACCINATION TARGETING INSULIN–LIKE GROWTH FACTOR BINDING PROTEIN–2 (IGFBP–2) IN ADVANCED OVARIAN CANCER: SAFETY, IMMUNOGENICITY, AND SURVEILLANCE, EPIDEMIOLOGY, AND END RESULTS (SEER) COMPARISON

BACKGROUND: Immunization against self-antigens can induce regulatory responses that inhibit desirable Type 1 antitumor immune responses. Deletion of epitopes that favor a regulatory phenotype may improve the efficacy of therapeutic vaccines. We have developed a novel IGFBP-2 targeting DNA plasmid vaccine that selectively induces Type 1 immunity. IGFBP-2 regulates invasiveness and metastases in ovarian cancer. Eradication of ovarian cancer cells expressing IGFBP-2 through effective immunization could prevent disease relapse or metastasis. METHODS: Twenty-five patients with advanced stage or recurrent ovarian cancer treated to complete remission after primary or salvage therapy received 3 monthly doses of an IGFBP-2 DNA vaccine in a single-arm, non-randomized study. ELISPOT and flow cytometry were used to characterize antigen specific T-cell responses. Serum antibodies were measured using ELISA and Western blot. The SEER database was reviewed to identify women diagnosed between 2006 and 2012 matched for age, year of diagnosis and stage of diagnosis. The difference between dates of diagnosis and enrollment (lead time) was calculated for each patient receiving vaccine. Only SEER patients who survived at least as long as the lead time of their matches plus an additional 6 months were kept for analysis. In cases where this resulted in no SEER matched patients, unmatched vaccine patients were excluded. Overall survival (OS) was analyzed using Cox models and the Kaplan-Meier method. RESULTS: 206 adverse events (AE) were recorded. Fatigue (12%) and injection site reactions (12%) were the most common. 97% of AE were grades 1-2, 3% grade 3, and no grades 4 or 5. In preliminary immune analysis (16 patients), IGFBP-2 specific T-cell precursor frequencies are significantly elevated over baseline levels at 4 (p CONCLUSIONS: IGFBP-2 Th1 selective immunization is well tolerated, generates significant Type I immunity, and may demonstrate clinical efficacy. Citation Format: John B. Liao, Denise L. Cecil, Yushe Dang, Kelsey K. Baker, Kelsie J. Ovenell, Jessica Reichow, Stephanie Parker, Doreen M. Higgins, Jennifer S. Childs, Elizabeth K. Broussard, Andrew L. Coveler, Lupe G. Salazar, Barbara A. Goff, Mary W. Redman, Mary L. Disis. VACCINATION TARGETING INSULIN–LIKE GROWTH FACTOR BINDING PROTEIN–2 (IGFBP–2) IN ADVANCED OVARIAN CANCER: SAFETY, IMMUNOGENICITY, AND SURVEILLANCE, EPIDEMIOLOGY, AND END RESULTS (SEER) COMPARISON [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-097.

Vaccine targeting HIF1A in triple negative breast cancer

The high rates of relapse in triple negative breast cancer (TNBC) are thought to be due to the presence of increased levels of cancer stem cells (CSC), which have been shown to be resistant to standard therapies. It has been demonstrated that hypoxia-inducible factor 1 (HIF1A) can induce the expression of numerous gene products associated with stem-ness and epithelial-mesenchymal transition in breast cancer cells and has been shown to be hyperactivated in TNBC. In this study, we aimed to target HIF1A with a therapeutic immune response through active immunization. HIF1A is a tumor-associated antigen. We have determined that both the magnitude and incidence of HIF1A-specific IgG is significantly elevated in TNBC compared to volunteer donors. We identified epitopes derived from HIF1A that selectively elicited IFN-gamma secretion with little to no IL-10 secretion in human peripheral blood mononuclear cells and T cell lines generated with these epitopes responded to recombinant HIF1A protein. Furthermore, these epitopes are highly homologous between mouse and man. To evaluate therapeutic efficacy, we immunized MMTV-neu (HER2+ model) and C3(1)Tag (TNBC model) mice with a plasmid-based vaccine containing an extended sequence of the identified epitopes. Tumor growth was inhibited over 80% (p < 0.0001) in the TNBC model; however, growth was inhibited only by 40% (p < 0.01) in the HER2+ model. We determined the majority of the tumor cells from the TNBC model expressed the mouse stem cell marker, Sca-1, whereas only a minority of the cells derived from the HER2+ model expressed the marker. Finally, we detected a 52% decrease in tumor Sca-1 expression after HIF1A-specific vaccination in the TNBC model (p = 0.004). Targeting HIF1A via active immunization may be an effective way to prevent disease relapse in patients with TNBC.

Abstract 4392: Vaccination targeting IGF-IR sensitizes tumors to tamoxifen therapy in an anti-estrogen resistant mouse model

Tamoxifen is a standard treatment for estrogen receptor (ER)-positive breast cancer patients. However, acquired or de novo resistance to therapy is a major clinical problem. In hormone-resistant tumors, there is increased activation of insulin-like growth factor-1 receptor (IGF-IR) and subsequent downstream signaling molecules, such as AKT. We have determined that IGF-IR is immunogenic in breast cancer and is a potential target for active immunization. It has been demonstrated that natural immunogenic human epitopes can be predicted by high binding affinity across multiple class II alleles, thus, we used a combined scoring system from five algorithms for predicting class II binding to determine Th epitopes of IGF-IR. Of the 20 potentially immunogenic peptides identified, five peptides (p1166-1181, p1212-1227, p1301-1316, 1307-1322 and p1311-1326) in the C-terminal domain were determined to elicit a predominantly inflammatory Th1 response compared to an immunosuppressive Th2 response in human PBMC. Overexpression of AKT induces tamoxifen resistance, while inhibition restores tamoxifen sensitivity. The tumor suppressor, PTEN, actively inhibits AKT. Data we have generated has demonstrated that vaccination restored PTEN activity in tumor cells. We questioned if modulation of PTEN could render tumors in the anti-estrogen-resistant MMTV-neu mouse model sensitive to tamoxifen therapy. Vaccination demonstrated a robust Th1 response (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4392. doi:1538-7445.AM2012-4392

Abstract 1571: Developing antigen-specific Th1 epitope based vaccines by excluding immunosuppressive peptide sequences

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We have determined that IGFBP-2 is immunogenic in ovarian cancer. Our aim is to develop a multi-epitope vaccine that will elicit Th1 immunity to IGFBP-2. Antigen specific Th1 can modulate the tumor microenvironment to enhance cross priming, supporting the proliferation of cytotoxic T cells which are capable of eradicating ovarian cancer cells. Although some clinical benefit has been demonstrated with this strategy, recent ex vivo analyses of human PBMC have described the presence of antigen-specific CD4+ T regulatory cell (Tregs) in cancer patients that were not detected in healthy individuals. Thus, we questioned if we could optimize our vaccine to include only Th1-stimulating epitopes. Using a combined scoring system from five algorithms for predicting class II binding to determine Th epitopes, we identified 14 IGFBP-2 peptides. Th1 immunogenicity (IFN-gamma) and potential immunosuppression (IL-10) was evaluated by ELISPOT for 40 different donors. Twenty-two percent of the donors only responded with IL-10 secretion to any peptide, 22% only responded with IFN-gamma and 53% of the patients had a mixed IFN-gamma and IL-10 response. To determine which peptides would induce a predominantly Th1 response in the greatest number of people, we used a ratio of IFN-gamma to IL-10 and analyzed both the magnitude and frequency of ELISPOT responses for each of peptides using the following algorithm: (corrected mean SPW) x (percent of responding donors). The peptides were then ranked from highest IL-10 response to highest IFN-gamma response. Interestingly, 6 of the 14 peptides demonstrated a preference to secrete IFN-gamma over IL-10 and are only located in the N-terminus (amino acids 1-163) of IGFBP-2; the remaining potentially immunosuppressive peptides are located in the C-terminus (amino acids 164-328). Vaccination with p1-163 in MMTV-neu mice demonstrated a robust Th1 response (p=0.03) and concomitant inhibition of tumor growth by 70% compared to adjuvant only control animals, p164-328-vaccinated or full length protein-vaccinated mice (p<0.001 for all). Vaccination with p164-328 or full length protein did not inhibit tumor growth. These data suggest that more effective vaccines can be designed when both Th1 epitopes and immunosuppressive epitopes are screened simultaneously and epitopes that are most likely to induce robust Th1 responses in the majority of individuals can be identified and included as vaccine components. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1571. doi:1538-7445.AM2012-1571