Development of biochemical assays for immunotherapy drug discovery and development Andrew C. Newman, Aaron Snead, Stephen Edgcomb, and Henry Zhu BPS Bioscience, Inc. San Diego, CA 92121 Immunotherapy has become an important approach for the treatment of numerous diseases including cancer. A number of immunotherapies target one or more co-stimulatory or co-inhibitory pathways regulating immune activation such as cell surface receptors and enzymes like IDO1 and TDO. Reliable high throughput screening (HTS) methods are needed to successfully screen and identify small molecules, antibodies, or antibody fragments (Fab) that modulate these pathways. Here, we report the development of a toolbox of HTS biochemical assays to screen for inhibitors of immune receptor-ligand interaction and for screening inhibitors of IDO1 and TDO activity. These assays have been internally validated using known inhibitors. Future work will focus on establishing novel biochemical and cell-based assays for immunoregulatory pathways. INTRODUCTION Recognition and clearance of cancerous cells by the immune system requires a series of steps. Some of these steps involve signaling cascades that are initiated by proteins expressed on the surface of cancerous cells and/or immune cells that regulate immune activation. Many tumors have evolved specific mechanisms to evade the immune response. This often involves cancer cells expressing ligands that directly inhibit T cell activation by activating co-inhibitory pathways (Fig. 1). A number of therapies targeting T cell co-inhibitory receptors have demonstrated profound clinical benefit1,2,3. Indoleamine 2,3 dioxygenase 1 (IDO1) and tryptophan 2,3 dixoygenase (TDO) are enzymes that catalyze the rate-limiting step of tryptophan degradation and indirectly inhibits T cell activation through tryptophan depletion and generation of toxic metabolites. Importantly, both of these enzymes have been shown to be upregulated by subsets of cancers4 (Fig. 4) and IDO1 inhibitors are currently being pursued in the clinic5,6. Bioscience, Inc. San Diego, CA RESULTS A. Wash Strep HRP is added, followed by chemiluminescent or colorimetric substrate Biotin-labeled ligand is added with or without inhibitor Wash Figure 2. Immunotherapy biochemical assay design. BPS’ ELISA-based immunotherapy assays take advantage of our biotin-labeled proteins. One binding partner is allowed to adhere to a plate overnight and the next day the cognate binding partner along with test inhibitor is added. Binding is detected by addition of Strep-HRP along with chemiliuminescent or colorimetric HRP substrate. A. Figure 4. The role of IDO1 & TDO in the tumor microenvironement. (A) PD-1 inhibitors have shown clinical efficacy, but many tumors upregulate expression of IDO1 and/or TDO, resulting in Trp depletion in the tumor microenvironment and increased levels of the Trp metabolite, Kynunerinine (Kyn) which combine to induce T cell cycle arrest and apoptosis. (B) Inhibition of IDO1 and TDO has the potential to halt T cell inhibition caused by Trp catabolism. A. C. B. B. siWnt5a D. F. E. However, not all cancers have responded to current therapies, providing rationale for targeting other co-regulatory pathways. BPS has developed biochemical-based assays designed to screen for inhibitors of relevant immunotherapy targets in an effort to accelerate the discovery and development of novel immunotherapies. Figure 3. Validation of BPS’ Immunotherapy Receptor Inhibitor Screening Assay Kits. BPS Bioscience’s assay kits are validated by inhibiting receptor-ligand interaction with known inhibitors, when available, or with unlabeled ligand. Figure 3 depicts inhibition of (A) PD-1:PD-L1 (B) PD-1:PD-L2 (C) CD137:CD137L (D) CD28:B7-1 (E) CTLA4:B7-1 (F) CTLA4:B72, and (G) BTLA-HVEM interaction by PD-1 neutralizing antibody (BPS Cat. #71120), CTLA4 neutralizing antibody (BPS Cat. #71212), or unlabeled ligand as indicated. G. Figure 1. Immunotherapy targets. T cells are activated when the T cell receptor engages with peptides displayed by MHC class I or II by antigen presenting cells or cancer cells. A large repertoire of co-inhibitory and costimulatory receptors and ligands integrate a signaling network that regulates T cell activation, differentiation, survival, and effector function. B. Wells are coated with receptor Y Y Y Y 1 BPS Y Y Y Y ABSTRACT Catalog # Product Name Catalog # Product Name 72003 PD-1:PD-L1[Biotinylated] 72014 PD1:PDL1 Homogeneous Assay 72004 PD-1:PD-L2[Biotinylated] 72015 PD-1:PD-L2 Homogeneous Assay 72005 PD-1[Biotinylated]:PD-L1 72016 PD-1:PD-L1[Biotinylated] 72006 PD-1[Biotinylated]:PD-L2 72017 PD-1:PD-L2[Biotinylated] 72007 CD28:B7-1[Biotinylated] 72018 PD-1[Biotinylated]:PD-L1 72008 BTLA:HVEM[Biotinylated] 72019 PD-1[Biotinylated]:PD-L2 72009 CTLA4:B7-1[Biotinylated] 72024 CTLA4[Biotinylated]:B7-2 72012 PD-1:PD-L2 TR-FRET 72025 CD137[Biotinylated]:CD137L Table 1. BPS Bioscience’s Immunotherapy receptor portfolio. This table represents a complete list of BPS’ biochemical-based immunotherapy receptor assay kits as of April 2015. This portfolio is continually expanding and will eventually include all the receptor-ligand pairs in Figure 1 and more. Figure 5. Validation of BPS’ IDO1 and TDO inhibitor screening assay kit Both the IDO1 (BPS Cat. #72021) and TDO (BPS Cat. #72023) Inhibitor Screening Assay Kits measure enzyme activity by analyzing absorption of product (Kyn) at λ = 320-325 nm. (A) IDO1 activity in the presence of the IDO1-specific inhibitor INCB024360, the IC50 was determined to be 60 nM, demonstrating the ability of the kit to serve as a high throughput screening tool for IDO1 inhibitors (B) TDO activity in the presence of the TDO-specific inhibitor 680C91, the IC50 was determined to be 338 nM, demonstrating the ability of the kit to serve as a high throughput screening tool for TDO inhibitors. SUMMARY Immunotherapy is a rapidly growing field with a number of currently approved therapies that are showing excellent efficacy in the clinic, especially in cancer. However, these success stories also have their shortcomings as not all cancers have been responsive, emphasizing the need for novel therapeutics. In an effort to increase the rate of immunotherapy drug discovery and development, we have developed and validated biochemical high throughput screens for established drug targets as well as potential new targets. These assays are straightforward, easy-to-use, and come in different formats such as chemiluminescent, colorimetric, TR-FRET, and AlphaLISATM. In addition to these assay kits, BPS also provides screening services for its entire immunotherapy portfolio, allowing researchers to take advantage of our assay expertise. Future work will focus on developing assays for novel therapeutic targets in addition to cell-based assays that complement our portfolio of biochemical-based assays. REFERENCES/CONTACT INFO 1. 2. 3. 4. 5. 6. Robert, C., et al., N. Engl. J. Med. 2011; 364: 2517-2526 Wolchok, J.D., et al., N. Engl. J. Med. 2013; 369: 122-133. Sznol, M., et al., J. Clin. Oncol. 2014; 32: (Suppl.; abstr LBA9003). Platten, M., et al., Front. Immunol. 2015; 5(63): 1-7. Holmgaard, R.B. et al., J. Exp. Med. 2013; 210: 1389-1402 Gibney, G.T. et al., J. Clin. Oncol. 2014; 32: (Suppl.: abstr 301). For more information visit bpsbioscience.com/immunotherapy or contact Andrew Newman at [email protected] , for general information, email [email protected].
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