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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.

Radiotherapy (RT)-potentiated immunomodulation is an emerging field in solid tumor malignancies. RT induces peripheral antitumor immunity that can lead to the reduction of distant, non-irradiated tumor lesions (abscopal response). This has provided rationale for current early phase trials combining existing checkpoint blockade and co-stimulatory mAbs with stereotactic RT. Objective responses remain suboptimal with checkpoint blockade monotherapy, and dose limiting toxicity is widely observed with these modalities. We hypothesize that targeting immunotherapy to the tumor using oligonucleotide aptamers that bind to RT induced tumor stress products will significantly enhance the therapeutic index. We used the aptamer platform to generate a bi-specific construct containing a costimulatory aptamer ligand specific to murine 4-1BB which was conjugated to an aptamer specific to products secreted into the tumor stroma (VEGF or osteopontin). Tumor targeting, local control, abscopal responses and toxicity were compared to unconjugated 4-1BB-VEGF constructs, or mAB based therapies, when used in combination with RT in murine subcutaneous and autochthonous MCA tumor models. RT induced VEGF upregulation was demonstrated in null/low VEGF secreting tumors that led to preferential accumulation of VEGF-41BB aptamers in the irradiated tumor.12Gy x1 elicited optimal tumor targeting of bi-specific VEGF-4-1BB conjugate. Significant anti-tumor immune responses were observed in s.c. murine tumor models. This conjugate showed dramatic reduction in systemic toxicity compared to the equivalent gold standard 4-1BB mAb. RT-VEGF-4-1BB treatment significantly improved local control, overall survival and induced potent abscopal responses compared to unconjugated and non-irradiated controls in the Balb/c 4T1 and MCA murine tumor models. Furthermore, RT-VEGF-4-1BB treatment showed similar anti-tumor effects as CTLA-4 mAB with significantly less systemic toxicity. This approach increased intra-tumoral CD4+ and CD8+ T cells, the CD8+ to Treg ratio, and induced significantly more tumor cell death. In conclusion, this strategy has been shown to induce potent anti-tumor immune responses in relevant murine tumor models and significantly improves the therapeutic index over non-targeted mABs. Furthermore, radiotherapy can expand the scope of tumor targeted immune modulation to virtually all solid malignancies and induce potent abscopal responses. These results provide the rationale for developing clinical grade bispecific aptamers for future testing. Citation Format: Brett Schrand, Bhavna Verma, Agata Levay, Shradha Patel, Iris Castro, Ana Paula Benaduce, Randall Brenneman, Oliver Umland, Hideo Yagita, Eli Gilboa, Adrian Ishkanian. Radiation-induced vegf-targeted 4-1bb costimulation enhances immune control of tumor growth [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 1700. doi:10.1158/1538-7445.AM2017-1700

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.

Circulating tumor cells (CTCs) play a critical role in metastasis. Functional characterization of CTCs is limited by a lack of available methods to enrich viable CTCs. We have developed a next-generation (next-gen) platform, modifying our current size-based CTC enrichment technology, for viable CTC capture. Here, we employed our next-gen microfilter device for viable CTC capture in a syngeneic breast cancer mouse model system to show that such cells can be cultured with high efficiency for further characterization. Balb/c mice received subcutaneous injection of 4T1 (highly metastatic) or 4T07 (poorly metastatic) mouse mammary tumor cell lines originally derived from the same spontaneously arising tumor. Primary tumors were excised on day 21, digested and placed in culture, and viable CTCs were isolated using the next-gen microfilter device from 0.5ml whole blood draws for culture directly on-chip. Successfully established cell cultures were characterized by morphology, IHC for markers associated with epithelial-mesenchymal transition (EMT), and anchorage-independent growth to compare abilities to form tumorispheres. Tumors developed in 100% (6/6) of mice injected; cultures were obtained from 100% dissociated primary tumors (n=3 4T1, n=3 4T07), and isolated CTCs yielded cultures from 33% of mice injected with both cell lines (1/3 each). While 4T07-CTC cultures demonstrate homogenous morphology similar to the corresponding primary tumor, 4T1-CTC cultures demonstrate two morphologically distinct subclones: “A," spheroid-like, different from the primary tumor, and “B," spindle-like, similar to the primary tumor. “A” cells demonstrate the ability to produce progeny with spherical as well as spindle-like morphology in vitro. EMT profiling revealed that both 4T1-CTC subclones demonstrate epithelial and mesenchymal expression characteristics, while 4T07-CTCs predominately show mesenchymal character. CTCs had a greater ability to form tumorispheres in anchorage-independent growth conditions than corresponding primary tumors, with 4T1 “A” having greater tumorisphere-forming ability than 4T1 “B”. These studies demonstrate our ability to capture and culture viable CTCs from mice injected with tumor cells of varying metastatic capability. The characterization of CTCs may help identify functionally important subsets of CTCs. Molecular and functional analyses of CTCs are ongoing, and include epigenomic, transcriptomic and proteomic expression profiling, assessment of stem cell-like properties, and their mechanistic role in metastatic disease. Our work could be the basis to establish CTC cultures in human patients, and provide a transformative approach to the functional characterization of CTCs, leading to an exciting new technology for improved cancer research and individualized patient management. Citation Format: Anthony Williams, Brett Schrand, Siddarth Rawal, Zheng Ao, Randall Brenneman, Eli Gilboa, Siyang Zheng, Ram Datar, Richard Cote. Capture, culture and characterization of viable circulating tumor cells in a syngeneic breast cancer mouse model system. [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 1447. doi:10.1158/1538-7445.AM2013-1447

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.