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Blockade of programmed cell death protein 1 (PD-1) is approved for treatment of multiple human cancers and is the focus of multiple studies due to its key role in T cell function. However, only patients with ‘'hot'' tumors (i.e. those with increased numbers of tumor-infiltrating CD8+ T cells) respond well to the blockade. This suggests that new strategies to increase infiltration of CD8+ T cells into tumors will increase the number of tumor types and patients benefiting from anti-PD-1 therapy. CD5, a member of the scavenger receptor cysteine-rich (SRCR) superfamily, is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signaling and impair cytotoxic T lymphocyte (CTL) activation. CD5 knockout mice have increased anti-tumor immunity and reduced homograft tumor growth: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumor response and increase tumor-infiltrating CD8+ T cells. We report that in vivo administration of anti-CD5 blocking MAb treatment increased primary T cell activation in response to 4T1 tumor cell homografts and ex vivo activation as measured by increased CD69, Fas, Fas ligand, IFNγ, PD-1, and apoptosis. Further, anti-CD5 treatment enhanced the capacity of primary T cells to kill 4T1 tumor cells ex vivo. Mice receiving anti-CD5 and anti-PD-1 in combination exhibited increased overall survival compared to mice treated with either agent alone. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumor immunity and PD-1 blockade treatment. Citation Format: Faizah Alotaibi, Mateusz Rytelewski, Rene Figueredo, Ronak Zareardalan1, Saman Maleki Vareki, Xiufen Zheng, Meng Zhang, Peter Ferguson, Mikal El-Hajjar, Yousef Najajreh, Wei-ping Min, James Koropatnick. Functional blocking of CD5 on T cells to enhance the efficacy of therapeutic PD-1 blockade in treatment of mouse 4T1 breast tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 917.

Tumor cells frequently evade macrophage-mediated destruction by increasing cell surface expression of CD47, which delivers an anti-phagocytic (“do-not-eat”) signal by binding the inhibitory signal regulatory protein α (SIRPα) receptor on macrophages. Previous studies have shown that blockade of the CD47-SIRPα pathway using TTI-621, a soluble SIRPα-IgG1 Fc fusion protein, triggers macrophage phagocytosis of tumor cells in vitro, and potently inhibits tumor growth in vivo. In the current study, the in vitro and in vivo efficacy of TTI-622, a soluble SIRPα-Fc variant protein containing an IgG4 Fc tail, was evaluated in multiple model systems. Unlike CD47-blocking antibodies, TTI-622 binds minimally to human erythrocytes, and does not induce hemagglutination in vitro. Therefore, it avoids a large circulating antigen sink, and is less likely to cause anemia in patients. Additionally, TTI-622 potently induces phagocytosis of a broad panel of tumor cells derived from patients with both hematological and solid tumors. Although in vitro phagocytosis of human platelets is also observed, TTI-622 preferentially induces phagocytosis of tumor cells over platelets in a competitive phagocytosis assay. The in vivo efficacy of TTI-622 monotherapy and/or combination therapy was evaluated in different tumor models. In a DLBCL (Toledo) xenograft tumor model, both early and delayed treatments resulted in statistically significant decreases in tumor growth, and improved survival relative to treatment with control Fc. In the Burkitt lymphoma (Daudi) and multiple myeloma (MM.1S) xenograft tumor models, the potential of combining TTI-622 with daratumumab (anti-CD38 antibody) was also explored. In both models, TTI-622 monotherapy demonstrated partial tumor growth inhibition. However, the therapeutic efficacy was further enhanced when TTI-622 was combined with daratumumab. Intriguingly, a TTI-622 non-responsive head and neck cancer (FaDu) xenograft tumor model became responsive by combining TTI-622 with suboptimal doses of cetuximab (anti-EGFR antibody). The combination of TTI-622 with cetuximab resulted in a statistically significant decrease in tumor growth, and improved survival relative to monotherapy treatments. Collectively, these results demonstrate that TTI-622 induces potent, tumor-specific macrophage phagocytosis across a range of hematological and solid tumors, and is efficacious as a monotherapy agent in a DLBCL xenograft tumor model. Furthermore, TTI-622 potentiates the efficacy of daratumumab and cetuximab in hematological and solid xenograft tumor models, respectively. These data support the clinical evaluation of TTI-622 in combination with anti-tumor antibodies in cancer patients with both solid and hematological malignancies. Citation Format: Gloria H. Y. Lin, Natasja N. Viller, Marilyse Chabonneau, Laura Brinen, Tapfuma Mutukura, Karen Dodge, Simone Helke, Vien Chai, Violetta House, Vivian Lee, Hui Chen, Alison O'Connor, Debbie Jin, Rene Figueredo, Saman Maleki Vareki, Mark Wong, Emma Linderoth, Lisa D. Johnson, Xinli Pang, James Koropatnick, Jeff Winston, Penka S. Petrova, Robert A. Uger. TTI-622 (SIRPα-IgG4 Fc), a CD47-blocking innate immune checkpoint inhibitor, suppresses tumor growth and demonstrates enhanced efficacy in combination with antitumor antibodies in both hematologic and solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2709.

Indoleamine 2,3-dioxygenase-1 (IDO) is an immunosuppressive molecule expressed by most human tumors. IDO levels correlate with poor prognosis in cancer patients and IDO inhibitors are under investigation to enhance endogenous anticancer immunosurveillance. Little is known of immune-independent functions of IDO relevant to cancer therapy. We show, for the first time, that IDO mediates human tumor cell resistance to a PARP inhibitor (olaparib), γ radiation, cisplatin, and combined treatment with olaparib and radiation, in the absence of immune cells. Antisense-mediated reduction of IDO, alone and (in a synthetic lethal approach) in combination with antisense to the DNA repair protein BRCA2 sensitizes human lung cancer cells to olaparib and cisplatin. Antisense reduction of IDO decreased NAD+ in human tumor cells. NAD+ is essential for PARP activity and these data suggest that IDO mediates treatment resistance independent of immunity and at least partially due to a previously unrecognized role for IDO in DNA repair. Furthermore, IDO levels correlated with accumulation of tumor cells in G1 and depletion of cells in G2/M of the cell cycle, suggesting that IDO effects on cell cycle may also modulate sensitivity to radiation and chemotherapeutic agents. IDO is a potentially valuable therapeutic target in cancer treatment, independent of immune function and in combination with other therapies. Supported by grants to JK and WM from the Canadian Institutes of Health Research (CIHR). Citation Format: Saman Maleki Vareki, Mateusz Rytelewski, Rene Figueredo, Di Chen, Peter J. Ferguson, Mark Vincent, Weiping Min, Xiufen Zheng, James Koropatnick. Indoleamine 2,3-dioxygenase mediates immune-independent human tumor cell resistance to olaparib, γ radiation, and cisplatin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3765. doi:10.1158/1538-7445.AM2014-3765

BRCA2 is involved in homologous recombination repair of double-stranded DNA breaks in human cells. Inactivating mutations in BRCA2 predispose to early onset cancer of the breast, ovary and other tissues. However, patients with tumors that harbor BRCA2 mutations respond better to cancer therapy. Therefore, reducing BRCA2 in cancer cells capable of homologous recombination repair may sensitize otherwise resistant tumors to DNA-damaging anti-cancer treatment. We developed a second generation antisense oligodeoxynucleotide (BR-1) that specifically targets BRCA2. BR-1 decreased BRCA2 mRNA and protein and inhibited BRCA2-mediated RAD51 repair focus formation. BR-1 potently sensitized A549 (lung), SKOV-3 (ovarian), and MDA-MB-231 (breast) cells to cisplatin as evidenced by decreased cell proliferation. However, non-cancerous HK-2 kidney cells were not sensitized to cisplatin by BR-1. In A549 cells BR-1 enhanced cisplatin or ionizing radiation-induced inhibition of in vitro colony formation. Cisplatin-resistant head and neck squamous cancer cells (HN-15a) were rendered as sensitive to cisplatin following BR-1 treatment as the parent population. In addition, BR-1 plus cisplatin treatment decreased A549 cell metastatic frequency in an in vivo chicken chorio-allantoic membrane (CAM) model by over 70% when compared with drug treatment alone. Treatment with BR-1 and cisplatin decreased cellular respiration (decreased oxygen consumption) compared to control oligonucleotide plus cisplatin treatment, suggesting that downregulation of BRCA2 in the context of platinating drug treatment alters cellular metabolism. This change in respiration occurred independently of changes in mitochondrial number. We are continuing to develop and test BR-1 with the aim of applying it in an adjuvant setting with cisplatin in human clinical trials in the future. Supported by grants from the Ontario Centres of Excellence (OCE) Citation Format: Mateusz Rytelewski, Jessica Tong, Adrian Buensuceso, Hon Leong, Peter Ferguson, Saman Maleki Vareki, Christine Di Cresce, Larissa Romanow, Trevor Shepherd, Bonnie Deroo, Ann Chambers, Mark Vincent, James Koropatnick. A BRCA2-targeting antisense oligodeoxynucleotide enhances cisplatin effectiveness by decreasing human tumor cell proliferation, metastatic frequency, and metabolic response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1687. doi:10.1158/1538-7445.AM2014-1687

Chemotherapy plays a major role in treatment of many common cancers. However, it is limited by intrinsic and/or acquired tumor cell resistance to anticancer drugs, often mediated by increased expression of genes mediating that resistance. An approach to increase chemotherapy effectiveness is to reduce expression of such genes using small molecules or RNAi to silence specific targets that cancer cells rely on for escape from death due to exogenous treatment or immune cell attack. We have previously shown that siRNA downregulation of thymidylate synthase (TS), the enzyme responsible for the de novo synthesis of thymidylate and a common target for some anti-cancer drugs (5FUdR, pemetrexed, and others) enhances the activity of these drugs in vitro. Using lower concentrations of anticancer drugs has the potential to decrease deleterious side effects, including therapy-induced toxicity to T cells capable of recognizing and killing tumor cells. In addition, certain tumor proteins including indoleamine 2,3-dioxygenase [IDO] can regulate immunosurveillance by inhibiting antitumor T and NK cell activity. IDO can also mediate tumor cell resistance to some anticancer drugs, such as imatinib. To assess the capacity of combined siRNA knockdown of TS and IDO in human tumor cells to increase sensitivity to anticancer drugs, we report that: a) TS siRNA downregulates TS by 90% in HCT-15 colon carcinoma cells, over 90% percent in A549 lung carcinoma cells, and by 80% in H441 lung carcinoma cells, b) shRNA expression vectors stably expressing IDO siRNA in multiple clonally-derived populations of A549 reduces IFNγ-induced IDO protein expression by over 95%. Transiently-transfected TS siRNA is being used, alone and in combination with stable shRNA-mediated reduction in IDO, to assess the utility of targeting both mRNAs to enhance sensitivity to TS-targeting drugs (5-FU, 5-FUdR, pemetrexed), DNA-damaging anticancer drugs (cisplatin, melphalan) and both, both in vitro and against human A549-derived tumour xenografts in immunocompromised mice without T cells but competent with respect to NK cell activity. Combined antisense targeting of both TS and IDO in human tumor cells has the potential to increase the effectiveness of anti-TS and other drugs, both by increasing tumor cell drug sensitivity and by combined enhancement of sensitivity to drug treatment and reduced inhibition of NK cell activity. Citation Format: Saman Maleki Vareki, James Koropatnick. Enhancing chemotherapy efficacy and antitumor immune activity by combined antisense downregulation of TS and IDO. [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 A24.