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 × 10^3), 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

Despite the recent successes of using immune modulatory antibodies in cancer patients, autoimmune pathologies resulting from the activation of self reactive T cells preclude the dose escalations necessary to fully exploit their therapeutic potential. 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, vascular endothelial growth factor (VEGF). The 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 prior to their engagement of the MHC/peptide complex on the tumor cell, thereby obviating the need to target the costimulatory ligands to non-internalizing cell surface products expressed on the tumor cells. Underscoring the potency of stroma targeted costimulation and the broad spectrum of tumors secreting VEGF, systemic administration of the VEGF targeted 4-1BB aptamer conjugates engendered potent antitumor immunity against multiple unrelated tumors in subcutaneous, post surgical metastasis, and carcinogen-induced tumor models, and exhibited a superior therapeutic index compared to non-targeted administration of an agonistic 4-1BB antibody.</p

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

Abstract A25: Targeting costimulation to the tumor stroma with bispecific oligonucleotide aptamers

Abstract The paucity of costimulation at the tumor site compromises the ability of tumor-specific T cells to eliminate the tumor. The recent FDA approval of Ipilumimab, an antibody that blocks the inhibitory action of CTLA-4, has underscored the therapeutic potential of using monoclonal antibodies as ligands to stimulate protective immunity in human patients. Nonetheless, systemic administration of immunostimulatory ligands carries the risk of activating low avidity autoreactive T cells that could result in immune related pathologies. Indeed, treatment with Ipilimumab is associated with significant dose-limiting autoimmune toxicities. Similarly, treatment with an agonistic antibody that binds to 4-1BB, an immune stimulatory receptor expressed on activated CD8+ T cells, while demonstrating antitumor efficacy in cancer patients was associated with severe hepatic toxicity that led to the suspension of the clinical trial. Arguably, targeting costimulatory ligands to the disseminated tumor lesions of the patient would reduce drug associated toxicities. We have previously shown that systemic delivery of a bispecific aptamer composed of an agonistic 4-1BB binding aptamer, the therapeutic aptamer, conjugated to a an aptamer that bound to a product expressed on the surface of a tumor cell, the targeting aptamer, led to inhibition of tumor growth, was more effective than, and synergized with, vaccination, and exhibited a superior therapeutic index compared to nontargeted costimulation with 4-1BB antibodies or 4-1BB aptamers. The chemically synthesized oligonucleotide aptamers offer significant advantages over antibodies in terms of synthesis, cost, and conjugation chemistry to generate bispecific dimeric ligands. Nonetheless, the main current limitation of tumor targeted costimulation stems from the fact that the therapeutic cargo, the costimulatory (aptamer) ligand, has to be displayed on the surface of the targeted tumor cells. Consequently one is limited to target receptors that do not internalize upon ligand binding. While such receptor-ligand interactions exist, case in point is Rituximab, an anti-CD19 antibody that kills their targets via antibody-dependent cell cytotoxicity, most receptor-ligand complexes are internalized, and consequently the targeting choices are severely limited. Here we describe an approach that addresses this limitation and broadens the scope of tumor-targeted costimulation whereby the therapeutic aptamer ligand is targeted not to a product expressed on the surface of the tumor cells, but rather to a product that is secreted into the tumor stroma. Thus, the tumor infiltrating T cells will encounter the ligand and receive immune stimulation in advance to engaging their cognate MHC-restricted antigen on the tumor cells. In support of this approach we have shown that agonistic 4-1BB aptamers conjugated to aptamers which bind to either of two secreted products, VEGF or osteopontin, potentiated vaccine-induced protective antitumor immunity in mice that was superior to that of the non-targeted 4-1BB aptamers. Targeting secreted products will also significantly broaden the scope of tumor-targeted costimulation because secreted products like VEGF or osteopontin are broadly expressed in many tumors of disparate origin, unlike cell surface expressed products like PSMA or Her2 that exhibit a much more restricted expression pattern. Citation Format: Brett Schrand, Randall Brenneman, Alexey Berezhnoy, Eli Gilboa. Targeting costimulation to the tumor stroma with bispecific oligonucleotide aptamers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A25.</jats:p

Abstract A023: Inducing neoantigens in disseminated tumor lesions to enhance their susceptibility to PD-1 blockade therapy

Abstract Correlation between “clonal” neoantigen burden (neoantigens generated early during cancer development and therefore represented at high frequency in all tumor lesions) and responsiveness to checkpoint blockade therapy has underscored the importance of neoantigens in promoting tumor immunogenicity, and has spurred a broad effort to develop methods to identify neoantigens for use in vaccination protocols. Yet, the majority of patients do not express, or express low numbers of, clonal neoantigens, and consequently will be less likely to benefit from checkpoint blockade and other forms of immune potentiating therapies. Here we describe several strategies to generate de novo neoantigens in the patient' disseminated tumor lesions and show that in murine tumor models they potentiate checkpoint blockade therapy. We have previously shown that oligonucleotide aptamer-targeted siRNA inhibition of the Nonsense-mediated mRNA (NMD) process in tumor cells in situ results in the induction of neoatigens and inhibition of tumor growth (Pastor et al., Nature, 2010, 465:227). We now demonstrate that tumor-targeted NMD inhibition potentiates PD-1 blockade therapy. A general concern and potential limitation of the NMD approach is that a significant proportion of NMD inhibition-induced neoantigens are generated as a result of random mutations or aberrant splicing and hence will not be represented at high frequency in all the tumor lesions of the patient. To that end, we are exploring alternative strategies to generate clonal neoantigens by targeting key components of antigen presentation pathways, specifically the TAP transporter, ERAAP peptidase, and Invariant chain, the latter to generate class II-restricted neoantigens. Studies have shown that downregulation of these products, using genetic means or antisense RNA, not only inhibits the canonical antigen presentation pathway but also upregulates alternative pathways that present new, otherwise silent or subdominant, epitopes. Since such epitopes are not generated as a result of random mutations they are more likely to represent “clonal” neoepitopes generated in every cell in which the target was downregulated. Here we are developing clinically feasible broadly applicable approaches to inhibit the aforementioned mediators of antigen presentation using corresponding siRNAs targeted to tumor cells in situ by conjugation to a nucleolin-binding aptamer. Nucleolin, a nucleolar product, is translocated to the surface the majority of tumor cells of both human and murine origin and thereby serves as a broad, if not universal, target to deliver therapeutic cargo to the disseminated tumor cells of the patient. We show that nucleolin aptamer-targeted downregulation of ERAAP or Invariant chain in tumor-bearing mice inhibits tumor growth and potentiates PD-1 blockade therapy. Ongoing studies explore the combinatorial use of neoantigen induction methods and combination of neoantigen induction with immune potentiating strategies including but not limited to checkpoint blockade. Citation Format: Greta Garrido Hidalgo, Agata Levay, Alexey Berezhnoy, Brett Schrand, Eli Gilboa. Inducing neoantigens in disseminated tumor lesions to enhance their susceptibility to PD-1 blockade therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A023.</jats:p

Aptamer-Targeted Attenuation of IL-2 Signaling in CD8 + T Cells Enhances Antitumor Immunity

Immune responses elicited against cancer using existing therapies such as vaccines or immune stimulatory antibodies are often not curative. One way to potentiate antitumor immunity is to enhance the long-term persistence of anti-tumor CD8 T cells. Studies have shown that the persistence of activated CD8 T cells is negatively impacted by the strength of interleukin 2 (IL-2) signaling. Here, we used small interfering RNAs (siRNAs) against CD25 (IL-2Rα) to attenuate IL-2 signaling in CD8 T cells. The siRNAs were targeted to 4-1BB-expressing CD8 T cells by conjugation to a 4-1BB-binding oligonucleotide aptamer. Systemic administration of the 4-1BB aptamer-CD25 siRNA conjugate downregulated CD25 mRNA only in 4-1BB-expressing CD8 T cells promoting their differentiation into memory cells. Treatment with the 4-1BB aptamer-CD25 siRNA conjugates enhanced the antitumor response of a cellular vaccine or local radiation therapy. Indicative of the generality of this approach, 4-1BB aptamer-targeted delivery of an Axin-1 siRNA, a rate-limiting component of the β-catenin destruction complex, enhanced CD8 T cell memory development and antitumor activity. These findings show that aptamer-targeted siRNA therapeutics can be used to modulate the function of circulating CD8 T cells, skewing their development into long-lasting memory CD8 T cells, and thereby potentiating antitumor immunity

Abstract 2617: Inducing neoantigens in therapeutic and prophylactic cancer immunotherapy

Abstract 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</jats:p

Abstract 4992: Development of a novel radiation-induced targeted immunotherapy strategy through oligonucleotide aptamer conjugation

Abstract Monoclonal antibodies (mAbs) produce dramatic anti-tumor responses in multiple solid tumors. Unfortunately, systemic administration can result in end organ retention and dose-limiting toxicities. Radiotherapy (RT) is a standard-of-care treatment modality that induces direct tumor cell kill through DNA damage. While studies assessing the synergy of RT-induced tumor cell kill with mAbs are ongoing, an additional unexploited effect of RT is the induction of stress response products in the tumor microenvironment, such as vascular endothelial growth factor (VEGF). Oligonucleotide aptamers are short pieces of nucleic acid that exhibit little to no immunogenicity compared to mAbs, making them ideal delivery vehicles for therapeutic mAbs. The purpose of this study was to assess the feasibility of a novel strategy for targeted tumor delivery of existing immunotherapeutic mAbs through conjugation to ligands that bind to radiation induced products. An antibody recognizing the costimulatory molecule 41BB was covalently modified with a 21-mer ssDNA oligonucleotide linker sequence that serves as an anchor site for the VEGF aptamer synthesized with a 3’ extension. Annealing of complementary strands formed a 41BB mAb-VEGF aptamer conjugate. First, VEGF aptamer conjugation to modified 41BB mAb was verified by polyacrylamide gel electrophoresis analysis which revealed the expected size shift of the 41BB mAb-VEGF aptamer conjugate versus the unannealed controls. Conjugation was then verified using flow cytometry. Recombinant mouse 41BB-Fc was immobilized on Protein A Dynabeads and 41BB mAb conjugated with 5’ biotinylated VEGF aptamer. 41BB mAb-VEGF aptamer conjugate retention on Dynabeads was confirmed by using streptavidin-PE 5’ biotin of aptamer attached to mAb in conjugate binding epitope target. Functionality of 41BB mAb-VEGF aptamer conjugate was confirmed in vitro using a CD8+ T cell costimulation assay. CD8+ T cells were isolated from 8 week old female C57BL6/J splenocytes, and exposed to sub-optimal levels of CD3 mAb. 41BB mAb-VEGF aptamer conjugate, unconjugated 41BB mAb and isotype control were added to activated cells. Elevated levels of interferon gamma were observed and equivalent in conjugated and unconjugated 41BB mAbs. These results indicate that conjugation of 41BB mAb-VEGF aptamer is feasible and that conjugation does not affect 41BB mAb functionality. Use of this novel targeting approach to improve the therapeutic index of immunotherapeutic mAbs requires validation in murine tumor models. Citation Format: Ana Paula Benaduce, Randall Brenneman, Diana Cardero, Brett Schrand, Eli Gilboa, Adrian Ishkanian. Development of a novel radiation-induced targeted immunotherapy strategy through oligonucleotide aptamer conjugation. [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 4992.</jats:p

Potentiating tumor immunity using aptamer-targeted RNAi to render CD8 +

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