Adam Swartz

Rindopepimut: a promising immunotherapeutic for the treatment of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive glial cell-derived primary tumor. Current standard of care for patients with GBM includes maximal tumor resection plus adjuvant radiotherapy and temozolomide chemotherapy, increasing median overall survival to a mere 15 months from diagnosis. Because these therapies are inherently nonspecific, there is an increased likelihood of off-target and incomplete effects; therefore, targeted modalities are required for enhanced safety and efficacy. Rindopepimut is emerging as a safe and potentially effective drug for the treatment of GBM. Rindopepimut consists of a 14-mer peptide that spans the length of EGF receptor variant III, a mutant variant of EGF receptor found on approximately 30% of primary GBM, conjugated to the carrier protein keyhole limpet hemocyanin. Vaccination with rindopepimut has been shown to specifically eliminate cells expressing EGF receptor variant III. Phase II clinical trials have suggested that vaccination of newly diagnosed GBM patients with rindopepimut plus adjuvant granulocyte-macrophage colony-stimulating factor results in prolonged progression-free and overall survival with minimal toxicity. This review will outline the development of rindopepimut, as well as the current status of this vaccine.

Peptide vaccines for the treatment of glioblastoma

Glioblastoma multiforme (GBM) is an extremely malignant brain tumor for which current therapies do little to remedy. Despite aggressive treatment with surgery, radiation therapy, and chemotherapy, tumors inevitably recur as a direct consequence of the infiltrative nature of GBM. The poor prognosis of patients with GBM underscores the clear and urgent need for more precise and potent therapies. Immunotherapy is emerging as a promising means to treat GBM based on the immune system’s capacity to mediate tumor-specific cytotoxicity. In this review, we will discuss the use of peptide vaccines for the treatment of GBM. The simplicity of peptide vaccines and their ability to elicit tumor antigen-specific immune responses make them an invaluable tool for the study of brain tumor immunotherapy.

Enhancing dendritic cell-based vaccination for highly aggressive glioblastoma

Introduction: Patients with primary glioblastoma (GBM) have a dismal prognosis despite standard therapy, which can induce potentially deleterious side effects. Arming the immune system is an alternative therapeutic approach, as its cellular effectors and inherent capacity for memory can be utilized to specifically target invasive tumor cells, while sparing collateral damage to otherwise healthy brain parenchyma. Areas covered: Active immunotherapy is aimed at eliciting a specific immune response against tumor antigens. Dendritic cells (DCs) are one of the most potent activators of de novo and recall immune responses and are thus a vehicle for successful immunotherapy. Currently, investigators are optimizing DC vaccines by enhancing maturation status and migratory potential to induce more potent antitumor responses. An update on the most recent DC immunotherapy trials is provided. Expert opinion: Targeting of unique antigens restricted to the tumor itself is the most important parameter in advancing DC vaccines. In order to overcome intrinsic mechanisms of immune evasion observed in GBM, the future of DC-based therapy lies in a multi-antigenic vaccine approach. Successful targeting of multiple antigens will require a comprehensive understanding of all immunologically relevant oncological epitopes present in each tumor, thereby permitting a rational vaccine design.

Architectural and biochemical expressions of mustard gas keratopathy: preclinical indicators and pathogenic mechanisms.

A subset of victims of ocular sulfur mustard (SM) exposure develops an irreversible, idiotypic keratitis with associated secondary pathologies, collectively referred to as mustard gas keratopathy (MGK). MGK involves a progressive corneal degeneration resulting in chronic ocular discomfort and impaired vision for which clinical interventions have typically had poor outcomes. Using a rabbit corneal vapor exposure model, we previously demonstrated a clinical progression with acute and chronic sequelae similar to that observed in human casualties. However, a better understanding of the temporal changes that occur during the biphasic SM injury is crucial to mechanistic understanding and therapeutic development. Here we evaluate the histopathologic, biochemical and ultrastructural expressions of pathogenesis of the chronic SM injury over eight weeks. We confirm that MGK onset exhibits a biphasic trajectory involving corneal surface regeneration over the first two weeks, followed by the rapid development and progressive degeneration of corneal structure. Preclinical markers of corneal dysfunction were identified, including destabilization of the basal corneal epithelium, basement membrane zone abnormalities and stromal deformation. Clinical sequelae of MGK appeared abruptly three weeks after exposure, and included profound anterior edema, recurring corneal erosions, basement membrane disorganization, basal cell necrosis and stromal degeneration. Unlike resolved corneas, MGK corneas exhibited frustrated corneal wound repair, with significantly elevated histopathology scores. Increased lacrimation, disruption of the basement membrane and accumulation of pro-inflammatory mediators in the aqueous humor provide several mechanisms for corneal degeneration. These data suggest that the chronic injury is fundamentally distinct from the acute lesion, involving injury mechanisms that operate on different time scales and in different corneal tissues. Corneal edema appears to be the principal pathology of MGK, in part resulting from persistent necrosis of the basal corneal epithelium and deterioration of the basement membrane. The findings also provide a potential explanation as to why administration of anti-inflammatories transiently delays, but does not prevent, the development of MGK sequelae.

Dendritic cells enhance polyfunctionality of adoptively transferred T cells which target cytomegalovirus in glioblastoma

Median survival for glioblastoma (GBM) remains <15 months. Human cytomegalovirus (CMV) antigens have been identified in GBM but not normal brain, providing an unparalleled opportunity to subvert CMV antigens as tumor-specific immunotherapy targets. A recent trial in recurrent GBM patients demonstrated the potential clinical benefit of adoptive T-cell therapy (ATCT) of CMV phosphoprotein 65 (pp65)-specific T cells. However, ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8+ T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8+ T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ+, TNFα+, and CCL3+ polyfunctional, CMV-specific CD8+ T cells. These increases in polyfunctional CMV-specific CD8+ T cells correlated (R = 0.7371, P = 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256-64. ©2017 AACR.

Preconditioning Vaccine Sites for mRNA-Transfected Dendritic Cell Therapy and Antitumor Efficacy

Messenger RNA (mRNA)-transfected dendritic cell (DC) vaccines have been shown to be a powerful modality for eliciting antitumor immune responses in mice and humans; however, their application has not been fully optimized since many of the factors that contribute to their efficacy remain poorly understood. Work stemming from our laboratory has recently demonstrated that preconditioning the vaccine site with a recall antigen prior to the administration of a dendritic cell vaccine creates systemic recall responses and resultantly enhances dendritic cell migration to the lymph nodes with improved antitumor efficacy. This chapter describes the generation of murine mRNA-transfected DC vaccines, as well as a method for vaccine site preconditioning with protein antigen formulations that create potent recall responses.

369 Chimeric Antigen Receptors Deficient in Lck Signaling Require 4-1BB Costimulation to Expand in Vivo, Resist Regulatory T-Cell Suppression, and Treat Solid Tumors in Immune-Intact Hosts.

INTRODUCTION Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is an effective immunotherapy for hematological cancers but requires a rethinking for clinical efficacy against solid tumors, where CARs have largely failed. Lymphodepletive preconditioning regimens can enhance CAR activity in vivo by promoting T-cell expansion and depleting immunoinhibitory cells that counteract cellular immunity. These nonspecific regimens, however, can be exceedingly toxic and contribute to poor quality of life. Novel strategies are needed to bypass the intratumoral inhibition of CARs. CD4+FoxP3+ regulatory T cells (Tregs) play a critical role in treatment failure, and, importantly, CARs have been shown to inadvertently potentiate Tregs by providing a local source of interleukin-2 (IL-2) for Treg consumption. We explored whether specific disruption of this axis would confer efficacy against solid tumors. METHODS We developed second (CD28z) and third (CD28-4-1BBz) generation CARs targeting the tumor-specific mutation, EGFRvIII. To eliminate secretion of IL-2, 2 amino acid substitutions were introduced in the PYAP Lck binding motif of the CD28 domain (xCD28) of CAR transgenes. We evaluated these modified second- and third-generation CARs against established B16 melanomas expressing EGFRvIII. RESULTS Second-generation CD28z CARs fail to expand in vivo. Addition of 4-1BB in third-generation CARs improves expansion, but this modification alone was insufficient for CD28-4-1BBz CARs to treat tumors without prior host lymphodepletion. CARs deficient in Lck signaling, however, significantly retarded tumor growth in immune-intact mice without prior lymphodepletion, and this was dependent on inclusion of 4-1BB in CAR design. To determine if deficient Lck signaling altered CAR vulnerability to Tregs, we lymphodepleted mice and transferred CARs ± Tregs. Cotransfer was sufficient to abrogate the efficacy of CD28-4-1BBz CARs, whereas the efficacy of xCD28-4-1BBz CARs remained unperturbed. CONCLUSION xCD28-4-1BBz CARs may be an effective immunotherapy for solid tumors infiltrated with Treg and may mitigate the need for toxic lymphodepletive preconditioning.INTRODUCTION: Von Hippel-Lindau (VHL) disease is an autosomal dominant neoplastic disorder leading to formation of multiple central nervous system hemangioblastomas (HBs). (1) Although most VHL-associatedHBs remain quiescent (Q-HB), some ( 50%) progress to symptomatic tumor growth or cyst formation (S-HB). (2) We recently reported increased erythropoietin (EPO) transcription, focal activation of EPO receptors, and resultant JAK-STAT signaling cascade activation in S-HB (when compared with paired Q-HB). Although, polycythemia is rarely reported in VHL, an autocrine/paracrine EPO-EPOR loop in HB may be similar to ones reported in malignant tumors. (3) Here, we investigated the locality (distant vs autocrine/apocrine) of EPO in VHL and report focal production of EPO that supports an autocrine/paracrine mechanism for activation of EPO-R cascade.

CD27 stimulation unveils the efficacy of linked class I/II peptide vaccines in poorly immunogenic tumors by orchestrating a coordinated CD4/CD8 T cell response

ABSTRACT Despite their promise, tumor-specific peptide vaccines have limited efficacy. CD27 is a costimulatory molecule expressed on CD4+ and CD8+ T cells that is important in immune activation. Here we determine if a novel CD27 agonist antibody (αhCD27) can enhance the antitumor T cell response and efficacy of peptide vaccines. We evaluated the effects of αhCD27 on the immunogenicity and antitumor efficacy of whole protein, class I-restricted, and class II-restricted peptide vaccines using a transgenic mouse expressing human CD27. We found that αhCD27 preferentially enhances the CD8+ T cell response in the setting of vaccines comprised of linked class I and II ovalbumin epitopes (SIINFEKL and TEWTSSNVMEERKIKV, respectively) compared to a peptide vaccine comprised solely of SIINFEKL, resulting in the antitumor efficacy of adjuvant αhCD27 against intracranial B16.OVA tumors when combined with vaccines containing linked class I/II ovalbumin epitopes. Indeed, we demonstrate that this efficacy is both CD8- and CD4-dependent and αhCD27 activity on ovalbumin-specific CD4+ T cells is necessary for its adjuvant effect. Importantly for clinical translation, a linked universal CD4+ helper epitope (tetanus P30) was sufficient to instill the efficacy of SIINFEKL peptide combined with αhCD27, eliminating the need for a tumor-specific class II-restricted peptide. This approach unveiled the efficacy of a class I-restricted peptide vaccine derived from the tumor-associated Trp2 antigen in mice bearing intracranial B16 tumors. CD27 agonist antibodies combined with peptide vaccines containing linked tumor-specific CD8+ epitopes and tumor-specific or universal CD4+ epitopes enhance the efficacy of active cancer immunotherapy.

A simple and enzyme-free method for processing infiltrating lymphocytes from small mouse tumors for ELISpot analysis

The ELISpot assay prevails as one of the most sensitive and meaningful assays for the detection of antigen-specific, effector immune responses. Acquisition of cellular analyte for ELISpot analysis is typically not problematic when derived from tissues enriched in lymphocytes (e.g., lymphoid organs and blood); however, cell processing becomes more difficult when lymphocytes represent only a very minor population relative to the source tissue, especially when the source tissue is in limited supply (e.g., small mouse tumors). Traditional enzymatic-based methods for dissociating tumors often result in poor yields, inconsistent lymphocyte enrichment, and can have deleterious effects on lymphocyte phenotype and function. To address these limitations, we have developed an enzyme-free protocol for processing tumor infiltrating lymphocytes (TILs) from small mouse tumors, which enables the enumeration of antigen-specific effector lymphocytes using ELISpot analysis. This procedure is predicated on the dissociation of tumor tissue using gentle agitation with a paddle blender followed by a brief in vitro culture period to remove adherent cells, as well as to revive lymphocytes from a non-responsive state. Although this method is demonstrated with mouse intracerebral tumors, we have found that this protocol is applicable to peripheral tumors and may likely extend to alternative tissue sources wherein lymphocytes exist in low numbers.

Promising vaccines for treating glioblastoma

ABSTRACT Introduction: Conventional therapies for glioblastoma (GBM) typically fail to provide lasting antitumor benefits, owing to their inability to specifically eliminate all malignant cells. Cancer vaccines are currently being evaluated as a means to direct the adaptive immune system to target residual GBM cells that remain following standard-of-care treatment. Areas covered: In this review, we provide an overview of the more noteworthy cancer vaccines that are under investigation for the treatment of GBM, as well as potential future directions that may enhance GBM-vaccine effectiveness. Expert opinion: To date, no cancer vaccines have been proven effective against GBM; however, only a few have reached phase III clinical testing. Clinical immunological monitoring data suggest that GBM vaccines are capable of stimulating immune responses reactive to GBM antigens, but whether these responses have an appreciable antitumor effect on GBM is still uncertain. Nevertheless, there have been several promising outcomes in early phase clinical trials, which lend encouragement to this area of study. Further studies with GBM vaccines are, therefore, warranted.