Browsing by Author "Mitchell, Duane A"

Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. The heterogeneity of MB molecular subtypes as well as the failure of standard therapies to treat metastatic or recurrent disease necessitates more potent targeted approaches that minimize collateral toxicity. Immunotherapy presents a promising strategy by specifically targeting cancer cells and to date, there have been few successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting the proliferation and differentiation of progenitor cell populations within the developing cerebellum. To test the developing cerebellum as a source of tumor rejection antigens, we adapted two animal models of MB recapitulating human Sonic Hedgehog (SHH) and Group 3 tumors for immunotherapeutic evaluation. Immunologic characterization of these murine models revealed subtype-specific differences in the tumor microenvironment and a differential response to immune checkpoint blockade. We used total embryonic RNA from the developing mouse cerebellum (P5) to generate antigen-specific T cells and confirmed the immunogenicity of targeting developmentally regulated antigens in vitro. Developmental antigen-specific T cells produced high levels of Th1-type cytokines in response to two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells did not show any cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens conferred a significant survival benefit and long term cures in intracranial treatment models of SHH and Group 3 tumor bearing animals. We additionally tested whether the enrichment of select developmental antigens through the exclusion of normal brain transcripts would potentiate antitumor responses in both animal models. Finally, we evaluated the relevance of targeting fetal antigens across human MB subtypes. Our studies demonstrate that developmental antigens can safely target multiple MB subtypes and can be further refined to preferentially target individual subgroups. Further studies targeting immunogenic developmental antigens and leveraging this strategy with specific immune modulatory interventions represent a novel approach at utilizing patient molecular classification information to mediate safe and effective immunotherapy.

Glioblastoma (GBM) is a grade IV astrocytoma in which the median overall survival is approximately 15 months at time of diagnosis. Even with the current multi- modal therapeutic approach of surgery, chemotherapy with the DNA alkylating agent temozolomide, and radiation therapy, GBM remains uniformly lethal. Immunotherapeutic interventions are a burgeoning field in many different cancer treatments. They offer the exquisite specificity endowed by the immune system with minimal toxicities and new methods are being developed to enhance the endogenous immune responses.

With the recent identification of human cytomegalovirus (CMV) present within glioblastoma tissue but void in the surrounding normal healthy parenchyma there have been significant efforts aimed at understanding the biologic implications of the presence of the virus within GBM tissues with preliminary work demonstrating several capabilities of the virus to enhance the oncogenic process.

Likewise, a key area of importance in the development and design of effective immunotherapeutic platforms is the identification and targeting of tumor-specific antigens. The success of any immunotherapy platform relies heavily on the ability to selectively target antigens present within tumors but absent on healthy tissue, regardless of its role in tumorigenesis, as well as having robust immunogenic properties.

CMV offers a plethora of possible targets, as it is the largest known DNA virus that infects humans, yet very little is known about its biological significance in glioblastoma pathogenesis as well as the most efficacious and immunogenic targets for immunotherapeutic development.

We have been able to elucidate more thoroughly the feasibility and potency of an immunologic platform targeting CMV within glioblastoma utilizing a multi-antigen multi-component peptide based strategy that demonstrated significant immunogenicity and anti-tumor activity in pre-clinical models utilizing various assays. We have also developed several sensitive and specific detection methodologies including: 1) custom gene expression microarrays, 2) multiplex real time quantitative polymerase chain reaction (RT-qPCR) assays, 3) a massively parallel RNA deep sequencing platform, and 4) immunological assays. We have also successfully determined the capacity for endogenous CMV gene expression to be maintained in primary glioblastoma cell lines as well as examining the preponderance of CMV gene expression in a subpopulation of glioma stem cell-like cells, the slow cycling GBM cells established from primary tumor tissues, in an attempt to illuminate some of the biologic underpinnings of CMV with respect to GBM pathogenesis.

Taken together, these data lay the groundwork for the development of a more efficacious vaccination strategy targeting CMV in GBM. The screening strategies employed throughout this work will allow for an accurate antigenic profile of CMV in GBM which will subsequently permit the design of a more robust peptide vaccine for the next generation of cancer vaccine. We have also begun to describe some of the interesting biologic phenomena associated with CMV in GBM, as our results demonstrate continued viral gene expression in glioma stem cell-like cell populations indicating viral tropism for certain cell types.

Despite aggressive surgical resection, cytotoxic chemotherapy, and external beam radiotherapy, most cases of glioblastoma (GBM) remain recalcitrant. These outcomes necessitate novel developmental therapeutics that spare normal tissue. Immunotherapy is a promising novel adjuvant treatment that can harness the cytotoxic capacity of the immune system against tumor-associated antigens with exquisite specificity. To circumvent the challenges associated with the advancement of adoptive cellular immunotherapy, we developed a novel treatment platform, which leverages the use of commercially available and clinically translatable nanoparticles (NPs) that can be combined with tumor derived RNA to peripherally activate T cells against GBM antigens. Although cancer vaccines have suffered from weak immunogenicity, we have advanced a NP vaccine formulation that can reshape a host’s immune profile through combinatorial delivery of RNAs encoding for tumor antigens and RNAs encoding for immunomodulatory molecules to mediate long-lived T cell persistence.

We sought to assess if vaccination with amplified tumor derived RNA encapsulated in lipophilic NPs could be assembled to transfect antigen presenting cells (APCs) in vivo and induce therapeutic anti-tumor immunity in pre-clinical murine tumor models. We hypothesized that RNA encapsulated nanoliposomes would localize to reticuloendothelial organs such as the spleen and liver, transfect APCs therein and induce peripheral antigen specific T cell immunity against GBM. Since activated T cells can cross the blood brain barrier and exert their effector functions against GBM antigens, peripheral transfection of APCs by RNA-NPs represents an attractive vaccination approach for priming endogenous immunity against refractory brain tumors.

We screened several translatable NP formulations for their ability to transfect dendritic cells (DCs) in vitro with GFP mRNA. We demonstrated that the NP DOTAP was the most promising translatable formulation compared to alternative cationic liposomal preparations and linear polyethylenimine NPs with and without DC targeting mannose receptors. RNA-NP vaccines formulated in DOTAP were shown to induce in vivo gene expression and preserve RNA stability over time. We determined that intravenous (IV) injection of RNA-NPs was requisite for inducing functional antigen specific immunity, which was superior to standard peptide vaccines formulated in complete Freund’s adjuvant (CFA). IV administered RNA-NPs localized to splenic and hepatic white blood cells (WBCs); these cells expanded antigen specific T cells when transferred to naïve immunocompetent mice. RNA-NPs induced increased percentages of B7 co-stimulatory molecules, but also elicited compensatory PD-L1 expression. We enhanced the immunogenicity and anti-tumor efficacy of RNA-NP vaccines by combining RNA-NPs with immune checkpoint blockade against PD-L1. We also enhanced the immunogenicity and efficacy of this platform by simply combining mRNAs encoding for immunomodulatory cytokines (i.e. GM-CSF). Finally, we demonstrated that RNA-NP vaccines mediate anti-tumor efficacy against intracranial and subcutaneous melanomas and engender therapeutic anti-tumor efficacy in a cellular immunotherapy model against a radiation/temozolomide resistant invasive murine high-grade glioma.

GBM remains invariably associated with poor patient outcomes thus necessitating development of more targeted therapeutics. Clinically translatable RNA-NPs form stable complexes making them amenable to overnight shipping. They induce potent immune responses when administered systemically and mediate robust anti-tumor efficacy that can be enhanced through co-delivery of immunomodulatory RNAs.

This technology can simultaneously bypass the complexity of cellular therapeutics while cutting down the time to generation of personalized vaccines. Since RNA-NP vaccines can be made within days from a tumor biopsy, providing near immediate immune induction against GBM, these formulations can provide a more feasible and effective therapy with a wide range of applicability for all malignancies that can be targeted using RNA obtained from surgical resection of solid tumors.