Brett Schrand

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78–83%), high retention of cell viability (71–74%), high tumour cell enrichment against leukocytes (1.7–2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4–0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Targeting 4-1BB Costimulation to the Tumor Stroma with Bispecific Aptamer Conjugates Enhances the Therapeutic Index of Tumor Immunotherapy

Schrand and colleagues report the efficacy in five murine tumor models of an immunotherapeutic approach whereby systemic administration of tumor stroma-targeted 4-1BB aptamer conjugates, which target disseminated tumor lesions, elicits potent antitumor immunity with minimal dose-limiting toxicity. Despite the recent successes of using immune modulatory Abs in patients with cancer, autoimmune pathologies resulting from the activation of self-reactive T cells preclude the dose escalations necessary to fully exploit their therapeutic potential. To reduce the observed and expected toxicities associated with immune modulation, here we describe a clinically feasible and broadly applicable approach to limit immune costimulation to the disseminated tumor lesions of the patient, whereby an agonistic 4-1BB oligonucleotide aptamer is targeted to the tumor stroma by conjugation to an aptamer that binds to a broadly expressed stromal product, VEGF. This approach was predicated on the premise that by targeting the costimulatory ligands to products secreted into the tumor stroma, the T cells will be costimulated before their engagement of the MHC–peptide complex on the tumor cell, thereby obviating the need to target the costimulatory ligands to noninternalizing cell surface products expressed on the tumor cells. Underscoring the potency of stroma-targeted costimulation and the broad spectrum of tumors secreting VEGF, in preclinical murine tumor models, systemic administration of the VEGF-targeted 4-1BB aptamer conjugates engendered potent antitumor immunity against multiple unrelated tumors in subcutaneous, postsurgical lung metastasis, methylcholantrene-induced fibrosarcoma, and oncogene-induced autochthonous glioma models, and exhibited a superior therapeutic index compared with nontargeted administration of an agonistic 4-1BB Ab or 4-1BB aptamer. Cancer Immunol Res; 2(9); 867–77. ©2014 AACR.

Reducing Toxicity of Immune Therapy Using Aptamer-Targeted Drug Delivery

Modulating the function of immune receptors with antibodies is ushering in a new era in cancer immunotherapy. With the notable exception of PD-1 blockade used as monotherapy, immune modulation can be associated with significant toxicities that are expected to escalate with the development of increasingly potent immune therapies. A general way to reduce toxicity is to target immune potentiating drugs to the tumor or immune cells of the patient. This Crossroads article discusses a new class of nucleic acid–based immune-modulatory drugs that are targeted to the tumor or to the immune system by conjugation to oligonucleotide aptamer ligands. Cell-free chemically synthesized short oligonucleotide aptamers represent a novel and emerging platform technology for generating ligands with desired specificity that offer exceptional versatility and feasibility in terms of development, manufacture, and conjugation to an oligonucleotide cargo. In proof-of-concept studies, aptamer ligands were used to target immune-modulatory siRNAs or aptamers to induce neoantigens in the tumor cells, limit costimulation to the tumor lesion, or enhance the persistence of vaccine-induced immunity. Using increasingly relevant murine models, the aptamer-targeted immune-modulatory drugs engendered protective antitumor immunity that was superior to that of current “gold-standard” therapies in terms of efficacy and lack of toxicity or reduced toxicity. To overcome immune exhaustion aptamer-targeted siRNA conjugates could be used to downregulate intracellular mediators of exhaustion that integrate signals from multiple inhibitory receptors. Recent advances in aptamer development and second-generation aptamer–drug conjugates suggest that we have only scratched the surface. Cancer Immunol Res; 3(11); 1195–200. ©2015 AACR.

Reducing toxicity of 4–1BB costimulation: targeting 4–1BB ligands to the tumor stroma with bi-specific aptamer conjugates

Systemic administration of immune modulatory antibodies to cancer patients is associated with autoimmune pathologies. We have developed a clinically feasible and broadly applicable approach to limit immune stimulation to disseminated tumor lesions using a bi-specific agonistic 4–1BB oligonucleotide aptamer targeted to a broadly expressed stromal product (e.g., VEGF or osteopontin). The stroma-targeted aptamer conjugates engendered potent antitumor immunity against unrelated tumors and exhibited a superior therapeutic index compared to non-targeted agonistic 4–1BB antibody.

Radiation-induced enhancement of antitumor T cell immunity by VEGF-targeted 4-1BB costimulation

Radiotherapy can elicit systemic immune control of local tumors and distant nonirradiated tumor lesions, known as the abscopal effect. Although this effect is enhanced using checkpoint blockade or costimulatory antibodies, objective responses remain suboptimal. As radiotherapy can induce secretion of VEGF and other stress products in the tumor microenvironment, we hypothesized that targeting immunomodulatory drugs to such products will not only reduce toxicity but also broaden the scope of tumor-targeted immunotherapy. Using an oligonucleotide aptamer platform, we show that radiation-induced VEGF-targeted 4-1BB costimulation potentiated both local tumor control and abscopal responses with equal or greater efficiency than 4-1BB, CTLA-4, or PD1 antibodies alone. Although 4-1BB and CTLA-4 antibodies elicited organ-wide inflammatory responses and tissue damage, VEGF-targeted 4-1BB costimulation produced no observable toxicity. These findings suggest that radiation-induced tumor-targeted immunotherapy can improve the therapeutic index and extend the reach of immunomodulatory agents. Cancer Res; 77(6); 1310-21. ©2017 AACR.

Tipping a favorable CNS intratumoral immune response using immune stimulation combined with inhibition of tumor-mediated immune suppression

ABSTRACT High-grade gliomas are notoriously heterogeneous regarding antigen expression, effector responses, and immunosuppressive mechanisms. Therefore, combinational immune therapeutic approaches are more likely to impact a greater number of patients and result in longer, durable responses. We have previously demonstrated the monotherapeutic effects of miR-124, which inhibits the signal transducer and activator of transcription 3 (STAT3) immune suppressive pathway, and immune stimulatory 4–1BB aptamers against a variety of malignancies, including genetically engineered immune competent high-grade gliomas. To evaluate potential synergy, we tested an immune stimulatory aptamer together with microRNA-124 (miRNA-124), which blocks tumor-mediated immune suppression, and found survival to be markedly enhanced, including beyond that produced by monotherapy. The synergistic activity appeared to be not only secondary to enhanced CD3+ cell numbers but also to reduced macrophage immune tumor trafficking, indicating that a greater therapeutic benefit can be achieved with approaches that both induce immune activation and inhibit tumor-mediated immune suppression within the central nervous system (CNS) tumors.

Potentiating Tumor Immunity Using Aptamer-Targeted RNAi to Render CD8+ T Cells Resistant to TGFβ Inhibition

ABSTRACT TGFβ secreted by tumor cells and/or tumor infiltrating stromal cells is a key mediator of tumor growth and immune suppression at the tumor site. Nonetheless, clinical trials in cancer patients targeting the TGFβ pathway exhibited at best a modest therapeutic benefit. A likely reason, a common limitation of many cancer drugs, is that the physiologic roles of TGFβ in tissue homeostasis, angiogenesis, and immune regulation precluded the dose escalation necessary to achieve a profound clinical response. Murine studies have suggested that countering immune suppressive effects of TGFβ may be sufficient to inhibit tumor growth. Here we describe an approach to render vaccine-activated CD8+ T cells transiently resistant to TGFβ inhibition using an siRNA against Smad4 to inhibit a key step in the canonical TGFβ signaling pathway. The siRNA was targeted to vaccine activated CD8+ T cells in the mouse by conjugation to a 4–1BB binding oligonucleotide (ODN) aptamer ligand (4–1BB-Smad4 conjugate). In vitro the 4–1BB-Smad4 conjugate rendered T cells partially resistant to TGFβ inhibition, and treatment of tumor bearing mice with systemically administered 4–1BB-Smad4 conjugate enhanced vaccine- and irradiation-induced antitumor immunity. Limiting the inhibitory effects of TGFβ to tumor-specific T cells will not interfere with its multiple physiologic roles and hence reduce the risk of toxicity.

Hapten-mediated recruitment of polyclonal antibodies to tumors engenders antitumor immunity

Uptake of tumor antigens by tumor-infiltrating dendritic cells is limiting step in the induction of tumor immunity, which can be mediated through Fc receptor (FcR) triggering by antibody-coated tumor cells. Here we describe an approach to potentiate tumor immunity whereby hapten-specific polyclonal antibodies are recruited to tumors by coating tumor cells with the hapten. Vaccination of mice against dinitrophenol (DNP) followed by systemic administration of DNP targeted to tumors by conjugation to a VEGF or osteopontin aptamer elicits potent FcR dependent, T cell mediated, antitumor immunity. Recruitment of αGal-specific antibodies, the most abundant naturally occurring antibodies in human serum, inhibits tumor growth in mice treated with a VEGF aptamer–αGal hapten conjugate, and recruits antibodies from human serum to human tumor biopsies of distinct origin. Thus, treatment with αGal hapten conjugated to broad-spectrum tumor targeting ligands could enhance the susceptibility of a broad range of tumors to immune elimination.Efficient tumor antigen uptake by dendritic cells is facilitated by antibody coating of tumor cells. Here, the authors develop an approach based on hapten vaccination and hapten/tumor targeting ligand conjugates that potentiate anti-tumor immunity by generating and redirecting serum antibodies to tumors.

Osteopontin mediates glioblastoma-associated macrophage infiltration and is a therapeutic target

Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene–deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin &agr;v&bgr;5 (ITG&agr;v&bgr;5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. Systemic administration in mice of 4-1BB–OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.

Abstract 2617: Inducing neoantigens in therapeutic and prophylactic cancer immunotherapy

Correlation between clonal neoantigen burden (neoantigens generated early in tumorigenesis and therefore represented at high frequency in all tumor lesions) and responsiveness to checkpoint blockade has underscored the relevance of neoantigens in promoting tumor immunogenicity. Yet, the most of patients do not express, or express low numbers of, clonal neoantigens, and consequently will be less likely to benefit from checkpoint blockade. We describe strategies to generate de novo neoantigens in the patient’ disseminated tumors and show that in mouse tumor models they potentiate checkpoint blockade. We previously showed that tumor inhibition of the Nonsense-mediated mRNA decay (NMD) results in the neoantigens’ induction and reduces tumor growth (Pastor et al., 2010). We now demonstrate that tumor-targeted NMD inhibition potentiates PD-1 blockade. A general concern and potential limitation of this approach is that a significant proportion of the induced neoepitopes come from mutated products and hence will not be shared by all tumor lesions. To that end, we are exploring alternative strategies by targeting key components of antigen presentation pathways, specifically the TAP transporter, ERAAP peptidase, and Invariant chain. Studies show that downregulation of these products, not only reduces the canonical antigenic presentation but also upregulates alternative pathways that present new, otherwise silent or subdominant, epitopes. Since such epitopes are not generated by random events they are more likely to represent clonal neoepitopes. We are developing approaches to inhibit the aforementioned mediators using corresponding siRNAs targeted to tumor cells by conjugation to a nucleolin-binding aptamer. Nucleolin, a nucleolar product, is translocated to the surface the majority of tumors and thereby serves as a broad target to deliver therapeutic cargo to the disseminated tumors. We show that nucleolin aptamer-targeted downregulation of TAP, ERAAP or Invariant chain inhibits tumor growth and potentiates PD-1 blockade. Recent studies suggest that tumor neoantigen-specific T cells are dysfunctional due to constant antigenic exposure. Transiently expressed siRNA inhibition-induced neoantigens are not expected to be defective. Ongoing studies explore the combinatorial use of neoantigen induction methods with others immune potentiating strategies. Prophylactic cancer vaccination obviates the limitations of therapeutic vaccination. A major barrier for developing this modality is the choice of antigens that will appear in the future cancer. The ability to induce tumor neoantigens can serve the basis for developing a new approach to prophylactic, though not preventative, cancer vaccination whereby neoantigens are induced in the healthy individual (at risk for cancer), and if or when a cancer develops induce the same antigens in the patient’ tumor by the methods described above. Preliminary studies in mice show that the approach has merit. Citation Format: Greta Garrido Hidalgo, Agata Levay, Alexey Berezhnoy, Brett Schrand, Eli Gilboa. Inducing neoantigens in therapeutic and prophylactic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2617. doi:10.1158/1538-7445.AM2017-2617