Cell-based Assays for Immunotherapy Drug Development
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Learn about novel cell-based assays that enable improved immunotherapy drug development. See case studies utilizing checkpoint receptors such as PD-1, VISTA, and NIK.
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Cell-based Assays for Immunotherapy Drug Development
- 1. 1 Novel, Improved Cell-Based Assays to Enable Immunotherapy Drug Development for Checkpoint Receptors Jane E. Lamerdin, PhD Director of R&D, DiscoverX
- 2. 2 Target & Phenotypic Platforms Enabling Cancer Immunotherapy Drugs – From Screening to Clinics Target-Based Platform Phenotypic Platform PathHunter Cellular Assays BioMAP Human Primary Cell Assays APPLICATIONS • Screening & lead optimization • Bioassays for potency & stability testing • NAb assays for immunogenicity testing APPLICATIONS • Efficacy & biomarker selection • Pre-clinical safety studies • Combination studies
- 3. 3 Target & Phenotypic Platforms Enabling Cancer Immunotherapy Drugs – From Screening to Clinics Target ID and Validation Screening & Hit Identification Lead Optimization & Selection Efficacy and Biomarker Selection Safety & Pre- clinical Studies Clinical Combination Studies PathHunter® Checkpoint Assays PathHunter® Bioassays for QC Lot Release Testing BioMAP® Oncology Systems BioMAP® Diversity PLUS™ BioMAP® Combo ELECT
- 4. 4 • Harnesses the immune system to battle tumors • Selectively activate or inhibit T cells • Based on clinical success of molecules targeting two inhibitory receptors, CTLA4 and PD-1 • Combination therapy, personalized medicine • Customize treatment depending on patient’s tumor type Cancer Immunotherapy “Scientific Breakthrough of the Year” – 2013, Science Magazine
- 5. 5 Targeting T Cell Activation and Inhibitory Checkpoints Tools Are Needed to Screen for and Develop New Therapeutics Figure from: Nature 480 480–489 (22 December 2011) doi:10.1038/nature10673
- 6. 6 Challenges with Checkpoint Receptors • Difficult to create cell-based assays for checkpoint receptors • Often needs human blood tissue • Difficult to handle human samples • Donor variability • Long, complicated protocols • Assay not specific for target receptor Pembrolizumab
- 7. 7 PathHunter® Assay Technology Enzyme Fragment Complementation (EFC) Split β-galactosidase Enzyme 7 ProLink™ (PK) Enzyme Acceptor (EA) Inactive Fragments Active Enzyme ~40 aa peptide Large fragment Complemented Enzyme
- 8. 8 • PD-1 contains inhibitory motifs • Phosphorylation by Src kinases • SHP proteins recruited to phosphorylated motifs • SHP-2 attenuates TCR activation PD-1 Signaling Biology Assay based on native receptor biology PathHunter PD-1 assay quantifies this early activation event
- 9. 9 PathHunter® PD-1 Signaling Assay Target early step in PD-1 receptor activation Plate Jurkat PD-1 cells; add anti-PD-1 antibody Add U2OS PD-L1 or PD-L2-presenting cells Incubate 1hr Add Detection Reagents Incubate 1hr Read on Benchtop Luminometer Incubate 1-2hr Simple Add and Read Protocol
- 10. 10 PathHunter® PD-1 Signaling Assay Responds to Cell-presented Ligand (Co-culture with PD-L1 or PD-L2) Co-culture with PD-L2
- 11. 11 PathHunter® PD-1 Signaling Assay Rapid and Robust Response to Anti-ligand or Anti-PD-1 Antibodies Robust inhibition in < 2hr Inhibition with anti-PD-1 or anti-PD-L1 antibodies
- 12. 12 PathHunter® PD-1 Signaling Assay Performance Highly Specific and Reproducible Response RSD <4% Highly Specific Response Excellent Reproducibility
- 13. 13 PathHunter® Assay Applications Supports development of biologics and small molecules SMALL MOLECULES • Screening & lead optimization BIOLOGICS • Screening & lead optimization • Characterization Assays • Development of QC Lot Release Assays
- 14. 14 PathHunter® vs. Reporter Gene Assay 15X More Sensitive and 4X Faster PathHunter Assay Competitor Assay Data generated with same commercial anti-PD-1 antibody (BioLegend Cat # 329709) Total Assay Time <5 hrs >22 hrs
- 15. 15 PathHunter® Checkpoint Assay Advantages • Biologically relevant response • Without handling difficult and donor-variable human tissue • Easy Protocol With Fast Results • Simple add and read protocol and results in less than 5 hours • Multiple Applications • Supports development of biologics and small molecules • Highly Sensitive Response • 15X more sensitive than other assays
- 16. 16 Targeting T-cell Activation and Inhibitory Checkpoints Modulate immune response to destroy cancer cells Activating Receptors = TNFR superfamily members Signal through canonical and non- canonical NF-kB pathways Nature 480 480–489 (22 December 2011) doi:10.1038/nature10673
- 17. 17 • A number of co-stimulatory receptors have been reported to signal through both canonical and non-canonical NF-kB pathways: • BAFF • 4-1BB • OX40 • GITR • Interrogate non-canonical signaling by quantifying NIK stabilization TNFR Superfamily Receptor Signaling Signaling Through the Non-canonical NF-κB Pathway Immunol Rev. 2012 Mar; 246(1): 125–140
- 18. 18 NIK is Stabilized in Response to Ligand Engagement of Endogenous TWEAK Receptors in U-2 OS Cells • TWEAKR (Fn14) is a TNFR family member that is up- regulated in response to tissue damage • TWEAKR is a therapeutic target in multiple inflammatory diseases (e.g. RA, MS, atherosclerosis) and cancer (melanoma, glioma, etc.) • TWEAKR is endogenously expressed in U-2 OS cells
- 19. 19 NIK is Stabilized in Response to Ligand Engagement of Endogenous HVEM Receptors in U-2 OS Cells • HVEM is a TNFR family member that elicits either a co- stimulatory or inhibitory signal depending on the ligand • HVEM is endogenously expressed in U-2 OS cells • LIGHT delivers a co-stimulatory signal to HVEM+ cells, resulting in stabilization of NIK APC (naiive T cells) T cell
- 20. 20 NIK is Stabilized in Response to Activation of Endogenous CD40 in U-2 OS Cells by Soluble CD40 Ligand Bremer, E., ISRN Oncology 2013, Article ID 371854
- 21. 21 NIK is Stabilized in Response to Activation of Endogenous CD40 in U-2 OS Cells by Soluble and Oligomerized CD40 Ligand Soluble CD40L Oligomerized CD40L Bremer, E., ISRN Oncology 2013, Article ID 371854
- 22. 22 NIK is Stabilized in Response to Activation of Endogenous CD40 in U-2 OS Cells by CD40 Ligand and Agonistic Antibodies Soluble CD40L Oligomerized CD40L Agonistic CD40 Ab Bremer, E., ISRN Oncology 2013, Article ID 371854
- 23. 23 NIK is Stabilized in Response to OX40 Ligand Engagement • Exogenously expressed OX40 in U-2 OS NIK Signaling Cell Line • OX40 assay responds to soluble ligand and agonistic antibodies • Amenable to testing in 96-well or 384-well format (to conserve antibodies) 96-well 384-well
- 24. 24 • Simple and biologically relevant assay • Gain of signal assay • Robust response over broad incubation time period (4-6hr) • Compatible with biologics and small molecules • Applicable to diverse TNFR family members PathHunter® NIK Assay Benefits Measures Activation of Endogenous or Exogenously Expressed TNFR Superfamily Receptors with Soluble Ligand and Agonistic Antibodies
- 25. 25 • Blockade of PD-1 and other inhibitory receptors • CTLA-4, TIM-3, LAG3, TIGIT, CD244, CD160 • PD-1 blockade in combination with immunostimulators • Anti-OX40, anti-CD137, ICOS, TLR ligands, IL-2 • PD-1 blockade in combination with small molecules or other targeted inhibitors • e.g. angiogenesis inhibitors, HDAC or PARP inhibitors • PD-1 blockade in combination with vaccines, CAR-T or oncolytic viruses Immunotherapy’s Next Wave: Combination Therapy Monospecific vs Bi-specific antibodies
- 26. 26 Assay Concept for Bi-specific Assays Bi-specific Antibody
- 27. 27 Bi-specific Assays Developed for Immune Checkpoints Immune Checkpoint Bi-specific Assay • PD-1 / TIM3 • TIM3 / CEACAM • PD-1 / LAG3 • PD-1 / TIGIT • PD-1 / CTLA4 • PD-1 / 4-1BB Examples of Available Bi-specific Pools / Clones : 1 0 -1 1 1 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4 0 1 0 0 0 0 0 2 0 0 0 0 0 3 0 0 0 0 0 4 0 0 0 0 0 B is p e c ific A n tib o d y [g /m L ] DimerizationSignal(RLU) A n tib o d y 1 A n tib o d y 2
- 28. 28 VISTA Dimerization Assay • VISTA is a negative checkpoint regulator that suppresses T- cell activation and is highly expressed within the tumor micro- environment. • VISTA is expressed primarily on hematopoietic cells • VISTA blockade may offer an immunotherapeutic strategy for human cancer, especially in combination • VISTA is related to PD-L1; currently the receptor for VISTA is unknown • Dimer assay provides tool to rank order antibodies Figure from Cancer Immunol Res (2014); 2(6): 510-517 Highly Specific Response to anti-VISTA antibodies
- 29. 29 TIM3 Dimerization Assay • TIM3 is a negative checkpoint regulator expressed on multiple hematological cells • Recognizes ligands highly expressed on apoptotic cells, leading to phagocytosis of dying cells • Dimer assay provides tool to rank order antibodies Figure from Science Webinar Series, part 5: Gordon J. Freeman, Ph.D. APC T cell Highly Specific Response to anti-TIM3 antibodies
- 30. 30 Assays for CSF1 (M-CSF) and GM-CSF • Cell line responds robustly to M-CSF (CSF1) and IL-34 • Anti-M-CSF antibodies will lead to inhibition of ligand-induced dimerization Cytokine immunotherapy with GM-CSF: induces potent tumor-specific systemic immune responses ~30 assays available for cytokines and interleukins
- 31. 31 • SH2 recruitment (signaling) assays for inhibitory receptors • Clone available for PD-1 • Assays for other targets coming soon • Custom projects possible for additional novel checkpoint targets • Assays for co-stimulatory receptors • Assays for OX40, TWEAKR, HVEM and CD40 are available • Additional co-stimulatory receptors assays in progress • Assays for Bi-specific molecules Summary Multiple Assay Formats for Immune Checkpoint Receptors
- 32. 32 Thank you for your attention! Learn more at www.discoverx.com/checkpoint Contact info@discoverx.com for additional information