OX40 Agonist
OX40 MARKET LANDSCAPE OX40 (a.k.a. CD134) is a costimulatory molecule belonging to the TNF receptor family expressed primarily on activated effector T (T eff) cells and naive regulatory T cells. Ligation of OX40, primarily on CD4 + T cells, activates NF-κB pathway and up-regulates antiapoptotic molecules of the Bcl-2 family, leading to T cell clonal expansion, activation, memory, and cytokine production. OX40 engagement on CD4 + FoxP3 + Treg cells leads to expansion, deactivation, or cell death depending on the local milieu. Given that OX40 triggering can potently stimulate T cells and potentially block/eliminate regulatory T cells, OX40 agonists have been investigated in multiple preclinical tumor models and anti-human OX40 monoclonal antibodies are currently being evaluated in clinical trials. ¹PLOS l ONE; http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089350 CURRENT MARKET PARTICIPANTS Agent Target Structure Phase Company Tavolixizumab (MEDI0562*) Pogalizumab (MOXR0916/ RG7888) OX40 Humanized anti-ox40 mab Phase 1 AstraZeneca OX40 Humanized anti-ox40 mab Phase 1 Genentech/Roche PF-04518600 OX40 Fully Human anti-ox40 mab Phase 1 Pfizer GSK3174998 OX40 Humanized anti-ox40 mab Phase I GSK *MEDI0562 replaced murine MEDI6469 whose trials were recently halted MEDI6383 is an OX40L-Fc fusion protein also in trials
OX40 mab LEAD DISCOVERY PROJECT PLAN 1 Immunization and Serum Assay 2 B-Cell Harvest and IgM Depletion B-cell repertoire harvested from AbeoMouse 3 High-Speed, High-Content Single B-Cell Sorting Purify B-cells surface expressing via FACS (For identification of putative agonists) 4 Antibody Amplification & Expression PCR amplify and clone heavy and light variable regions into human IgG expression vectors 5 High-Throughput Screening Transfection and screening of 1000 s of recombinant antibodies 6 Scale-Up and Cellular Assay Validation Scale-up highest affinity mabs to 100ug scale and screen for mabs which function in OX40 Activation Assay
1 Immunization and Serum Assay OX40 mab LEAD DISCOVERY PROJECT STATUS Mouse 359 Mouse 360 Pre Post1 Post2 Pre Post1 Post2 1:500 1:2K 1:8K 10 mice immunized subcutaneously with the extracellular domain of human OX40 in a proprietary adjuvant Mouse serum is assayed for its ability to bind human OX40 extracellular domain at varying dilutions 1:32K 1:128K 1:512K 1:2.048M
OX40 mab LEAD DISCOVERY PROJECT STATUS 2 B-Cell Harvest 8 Cervical 2 Axillary 2 Brachial 3 Pyloric Spleen= 1.01 g 3 Renal 3 Mesenteric 2 Inguinal 2x10 8 lymphocytes, spleen mass 1.01g harvested from a single AbeoMouse 3 Lumbar 2 Popliteal 5.6x10 7 cells remained after IgM depletion Spleen Bone Marrow Lymph Nodes
OX40 mab LEAD DISCOVERY PROJECT STATUS 3 High-Speed, High-Content Single B-Cell Sorting 0.016% of B cells from the first AbeoMouse immunized with OX40 surface-express an antibody binding to OX40 1632 Ig+OX40+ cells were single-cell deposited for amplification and expression
OX40 mab LEAD DISCOVERY PROJECT STATUS 4 Antibody Amplification & Expression 96-Well Gel of Variable Region PCR Products Fully Synthetic (Plasmid-Free) Gene Expression Cassettes Proprietary Single-Cell RT-PCR Process Proprietary Expression Vector Systems
OX40 mab LEAD DISCOVERY PROJECT STATUS 5 High-throughput Screening Rapid Expression And Assay Systems Test 1000 s of mabs for binding strength per day
OX40 mab LEAD DISCOVERY ASSAY VALIDATION 6 Scale-up and Cellular Assay Validation Abeome has an inhouse human Jurkat cell line engineered with a NFkappaB luciferase reporter which is activated and detected upon OX40 activation Abeome has synthesized, expressed and purified a reference antibody PF-04518600 (Pfizer clinical control)
R F U R F U Functional Screen: Activation of OX40 Reporter Line O X 4 0 A s s a y - P la te 1 (7 /1 5 /1 6 ) O X 4 0 A s s a y - P la te 2 (7 /1 5 /1 6 ) 6 0 0 6 0 0 4 0 0 1.0 u g /m L 40 0.1 0 u g /m L 1.0 u g /m L 0.1 u g /m L 2 0 0 2 0 0 0 0 U T C A B M 2 1 2 A B M 2 1 3 A B M 2 1 4 A B M 2 1 5 A B M 2 1 6 A B M 2 1 7 A B M 2 1 8 C o n tr o l U T C A B M 2 1 9 A B M 2 2 0 A B M 2 2 1 A B M 2 2 2 A B M 2 2 3 A B M 2 2 4 A B M 2 2 5 A B M 1 9 3 C o n tr o l *note: All treatments are in presence of anti-human F(ab )2 to cross-link Analysis of 40+ antibodies in cellular assay revealed 3 functional agonists
N o r m a liz e d R L U Titration of mabs in OX40 Reporter Jurkat Cell Line ABM193, Clinical Control and Isotype Control 1 0 0 0 O X 4 0 A s s a y - A B M 1 9 3 (9 /2 3 /1 6 ) 9 0 0 8 0 0 7 0 0 6 0 0 5 0 0 4 0 0 A B M 1 9 3 C lin ic a l Is o ty p e C o n tro l EC50: 0.15 ug/ml 0.26 ug/ml 3 0 0 2 0 0 1 0 0 0-4 - 3-2 - 1 0 1 m A b lo g (u g /m L ) ABM193 demonstrates more total activation of T cells AND Better potency than Pfizer s PF-04518600
Activation of Primary T Cells ABM193, Clinical Control and Isotype Control ABM193 Activates Primary T Cells
N o r m a liz e d R L U Titration of Humanized Antibodies habm193.2, habm193.3 and habm193.4 1 5 0 0 O X 4 0 A s s a y - H u m a n iz e d A B M 1 9 3 (1 0 /1 9 /1 6 ) 1 0 0 0 5 0 0 A B M 1 9 3 h A B M 1 9 3.2 h A B M 1 9 3.3 h A B M 1 9 3.4 EC50 (ug/ml): 0.081 0.091 0.097 n/a 0-5 0 0-4 - 3-2 - 1 0 1 m A b lo g (u g /m L ) *anti-human Fab cross-linker added to top dose at 2.5ug/mL habm193.2 and habm193.3 demonstrate equivalent potency to chimeric parent ABM193
Protein G Dynabead Antibody Capture 5mg 1mg 0.1mg 0.01mg ABM2 (control) Beads only acd3 ABM193 Visual Clustering of T Cells with Bead-Bound anti-ox40 mab ABM193
OX40 Agonist mab Summary: ABM193 shows superior in vitro activity compared to Pfizer s PF- 04518600 in 2-cell assay and equivalent in primary T cells ABM193 has been successfully humanized without significant loss of potency Mouse model of human allogeneic tumors in combination with PD-1 or PDL-1 would be a logical next step on the development path NSG and NSG -SGM3 mice Abeome is seeking a partner to move this lead forward and possibly to generate further leads