Joyce Reyes

Combining Human and Rat Sequences in Her-2 DNA Vaccines Blunts Immune Tolerance and Drives Antitumor Immunity

Immune tolerance to tumor-associated self-antigens poses a major challenge in the ability to mount an effective cancer vaccine response. To overcome immune tolerance to HER-2, we formulated DNA vaccines that express both human HER-2 and heterologous rat Neu sequences in separate plasmids or as single hybrid constructs that encode HER-2/Neu fusion proteins. Candidate vaccines were tested in Her-2 transgenic (Tg) mice of BALB/c (BALB), BALB/cxC57BL/6 F1 (F1), or C57BL/6 (B6) background, which exhibit decreasing immune responsiveness to HER-2. Analysis of various cocktails or hybrid vaccines defined a requirement for particular combination of HER/2/Neu sequences to effectively prime immune effector cells in HER-2 Tg mice. In B6 HER-2 Tg mice, rejection of HER-2-positive tumors protected mice from HER-2-negative tumors, providing evidence of epitope spreading. Our findings show that a strategy of combining heterologous antigen with self-antigens could produce a potent DNA vaccine that may be applicable to other tumor-associated antigens.

Cryotherapy with Concurrent CpG Oligonucleotide Treatment Controls Local Tumor Recurrence and Modulates HER2/neu Immunity

Percutaneous cryoablation is a minimally invasive procedure for tumor destruction, which can potentially initiate or amplify antitumor immunity through the release of tumor-associated antigens. However, clinically efficacious immunity is lacking and regional recurrences are a limiting factor relative to surgical excision. To understand the mechanism of immune activation by cryoablation, comprehensive analyses of innate immunity and HER2/neu humoral and cellular immunity following cryoablation with or without peritumoral CpG injection were conducted using two HER2/neu(+) tumor systems in wild-type (WT), neu-tolerant, and SCID mice. Cryoablation of neu(+) TUBO tumor in BALB/c mice resulted in systemic immune priming, but not in neu-tolerant BALB NeuT mice. Cryoablation of human HER2(+) D2F2/E2 tumor enabled the functionality of tumor-induced immunity, but secondary tumors were refractory to antitumor immunity if rechallenge occurred during the resolution phase of the cryoablated tumor. A step-wise increase in local recurrence was observed in WT, neu-tolerant, and SCID mice, indicating a role of adaptive immunity in controlling residual tumor foci. Importantly, local recurrences were eliminated or greatly reduced in WT, neu tolerant, and SCID mice when CpG was incorporated in the cryoablation regimen, showing significant local control by innate immunity. For long-term protection, however, adaptive immunity was required because most SCID mice eventually succumbed to local tumor recurrence even with combined cryoablation and CpG treatment. This improved understanding of the mechanisms by which cryoablation affects innate and adaptive immunity will help guide appropriate combination of therapeutic interventions to improve treatment outcomes.

Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination

Gibson and colleagues show that outbred domestic cats develop mammary tumors similar to those in humans. Electrovaccination of heterologous or point-mutated feline HER2 DNA overcomes T-cell immune tolerance in 40% of healthy cats and induces antibodies with distinct specificity. Domestic cats share human living environments and genetic traits. They develop spontaneous feline mammary carcinoma (FMC) with similar histopathology to human breast cancer. HER2 and AKT phosphorylation was demonstrated in primary FMC by immunoblot analysis, indicating HER2 as a therapeutic target. FMC lines K12 and K248 expressing HER1, HER2, and HER3 were sensitive to receptor tyrosine kinase (RTK) inhibitors gefitinib and lapatinib. To test HER2 vaccine response in cats, purpose-bred, healthy cats were electrovaccinated with heterologous (xenogeneic) or point-mutated feline HER2 DNA. T-cell reactivity to feline self-HER2 was detected in 4 of 10 cats that received bear HER2, human–rat fusion HER2 (E2Neu) or mutant feline HER2 (feHER2-K), which contains a single amino acid substitution. The variable T-cell responses may resemble that in the genetically heterogeneous human population. All immune sera to heterologous HER2 recognized feline HER2 expressed in 3T3 cells (3T3/HER2), but not that in FMC K12 or K248. Immune sera to mutant pfeHER2-K bound 3T3/HER2 cells weakly, but they showed better recognition of K12 and K248 cells that also express HER1 and HER3, suggesting distinct HER2 epitopes displayed by FMC that may be simulated by feHER2-K. In summary, HER2 DNA electroporation overcomes T-cell immune tolerance in approximately 40% of healthy cats and induces antibodies with distinct specificity. Vaccination studies in domestic cats can expedite vaccine iteration to guide human vaccine design and better predict outcome, with the added benefit of helping feline mammary tumor patients. Cancer Immunol Res; 3(7); 777–86. ©2015 AACR.

IFNγ PET Imaging as a Predictive Tool for Monitoring Response to Tumor Immunotherapy

IFNγ is an attractive target for imaging active antitumor immunity due to its function in the T-cell signaling axis. Here, we test an IFNγ immuno-PET (immunoPET) probe for its capacity to identify adaptive immunotherapy response after HER2/neu vaccination in both spontaneous salivary and orthotopic neu+ mouse mammary tumors. IFNγ immunoPET detected elevated cytokine levels in situ after vaccination, which inversely correlated with tumor growth rate, an indicator of response to therapy. In a model of induced T-cell anergy where CD8 T cells infiltrate the tumor, but upregulate PD-1, IFNγ tracer uptake was equivalent to isotype control, illustrating a lack of antitumor T-cell activity. The IFNγ immunoPET tracer detected IFNγ protein sequestered on the surface of tumor cells, likely in complex with the IFNγ receptor, which may explain imaging localization of this soluble factor in vivo Collectively, we find that the activation status of cytotoxic T cells is annotated by IFNγ immunoPET, with reduced off-target binding to secondary lymphoid tissues compared with imaging total CD3+ tumor-infiltrating lymphocytes. Targeting of soluble cytokines such as IFNγ by PET imaging may provide valuable noninvasive insight into the function of immune cells in situ Significance: This study presents a novel approach to monitor therapeutic outcomes via IFNγ-targeted positron emission tomography. Cancer Res; 78(19); 5706-17. ©2018 AACR.

Abstract 2361: Novel animal models for prediction of cancer vaccine response

Predicting cancer vaccine response is a challenge that must be addressed in immune competent animals that recapitulate human disease and human response to therapy. Furthermore, genetically heterogeneous humans respond differently to vaccination due, in part, to their varying MHC haplotypes and polymorphisms including those found in costimulatory or checkpoint genes. We previously showed that human HER2 transgenic (Tg) mice of different genetic backgrounds have distinct response to HER2 vaccines in the order of BALB > (BALBxB6)F1 > (B6xDR3)F1 > B6 (Radkevich-Brown 2010). We anticipate even greater variance among human populations, and seek genetically diverse animal models to project human cancer vaccine response. Domestic felines develop mammary tumors with similar pathology and etiology as human cancer, and expression of EGFR, HER2 and HER3 genes that are highly homologous to their human counterparts. Intramuscular electrovaccination of healthy outbred domestic shorthair cats with heterologous (xenogeneic) or single point-mutated self HER2 DNA induces HER2-specific T cells in ∼30% of vaccinated cats (Gibson 2015). Humoral immune responses were induced by both vaccine constructs, with point-mutated feline HER2 vaccine producing antibodies to unique epitopes on feline mammary carcinoma (FMC). The latter inhibits FMC growth in vitro and delays tumor growth in SCID mice. Vaccination with wild type feline HER2 DNA fails to induce specific immunity and even hinders feline HER2 immune activation by other vaccines. Studies in domestic cats provide novel insight on the range and nature of vaccine response in healthy cats which can also be tested in feline cancer patients. Strategic modification of minimal residues may be most effective in inducing reactivity to cancer associated self antigens. To address genetic regulation of vaccine response, diversity outbred (DO) mice offer a pragmatic option. These mice were generated by cross-breeding 8 inbred mouse strains to ensure broad genetic diversity among the mice (Svenson 2012). The human HER2 gene is introduced by breeding DO mice with our human HER2 transgenic (Tg) mice. F1 mice represent a genetically heterogeneous population that expresses human HER2 as a self antigen. Specific genes impacting vaccine response will be identified by genome-wide quantitative trait locus (QTL) scan to associate haplotype or single nucleotide variants with vaccine response. Findings in these mice may lead to the identification of critical genes that regulate cancer vaccine response. Candidate vaccines identified in DO mice can be further validated in cats and expanded to human trials. With these novel animal models, cancer vaccines can be designed and tested with greater precision, and patients with reactive genotypes may be identified before vaccination. (Supported by CA76340) Citation Format: Heather Gibson, Richard F. Jones, Joyce D. Reyes, Wei-Zen Wei. Novel animal models for prediction of cancer vaccine response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2361.

Abstract 470: Electrovaccination of domestic cats with a hybrid heterologous Her2 DNA vaccine overcomes immune tolerance to self Her2.

About 20% of non-castrated female domestic cats develop malignant mammary tumors with similar histopathology and disease progression as human breast cancer. Her2 over-expression is reported in 40-85% of feline mammary carcinomas, indicating this disease is particularly suited for testing Her2 DNA vaccination. To corroborate these findings, expression of Her2 was evaluated in 4 feline mammary tumors resected from three feline patients. Immunohistochemistry analysis with anti-human Her2 mAb showed regions of intense membrane staining in all four tumor samples, indicating high frequency of Her2 expression among feline mammary tumors and immune cross-reactivity of human and feline Her2. Feline mammary tumor lines K248 (Minke, et. al. 1991) and K12 (Modiano, et. al. 1991) both express Her2 as measured by flow cytometry, further demonstrating Her2 as a common antigen in feline mammary tumors. Feline Her2 cDNAs cloned from K248 and K12 (GenBank Accession JN990983) are identical to each other and show 93% sequence identity with human Her2. To test the feasibility of conducting DNA electrovaccination in cats, 3 pathogen-free domestic short hair cats between 1-2 years of age were injected i.m. in the biceps femoris of the hind legs or triceps of the front legs with vaccine DNA. Each cat received admix of 1.5mg of pE2Neu encoding a hybrid human Her2 and rat neu fusion protein and 1.5mg of feline pGM-CSF. Each injection site was subjected to electroporation with a NEPA21 electroporator, consisting of three 50msec 100V degenerating bipolar pulses. The vaccination was repeated at 3 week intervals and blood was drawn 2 weeks after each vaccination. Anti-human Her2 antibody was detected in the immune serum after 2 vaccinations, as demonstrated by binding to SKOV3 cells that over-express human Her2. After 4 vaccinations, anti-human Her2 antibody titers ranged from 50,000 to 200,000. Therefore, DNA electrovaccination is an effective means to induce immune response in cats. To test if immunization with heterologous pE2Neu induced cross-reactive antibody to self feline Her2, immune serum was incubated with 3T3/fHer2 cells that express feline Her2. Specific binding to 3T3/fHer2 was observed after 3 vaccinations at 1:100 serum dilutions. These results provide the first evidence that immune tolerance in cats to self Her2 is overcome by a hybrid DNA vaccine encoding a fusion protein of two heterologous genes: human Her2 and rat neu. This vaccine regimen is a candidate veterinary therapeutic agent and the insight gained from cat vaccination provides strong support for human vaccine trials. Citation Format: Heather M. Gibson, Richard Jones, Marie Piechocki, Joyce Reyes, Michele Sauerbrey, Ulka Vaishampayan, Wei-Zen Wei. Electrovaccination of domestic cats with a hybrid heterologous Her2 DNA vaccine overcomes immune tolerance to self Her2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 470. doi:10.1158/1538-7445.AM2013-470