Discovery and Development of Therapeutic Antibodies (Maastricht University)

expert session course Gen2305/2024 ‘Engineering the Immune System Meet the Expert Session Discovery and development of Therapeutic Antibodies March 12, 2024 Rene Hoet Chief Innovation Officer, FairJourney Biologics, Porto & Lecturer Position, Biopharmaceutics, University Maastricht, Dept. Pathology,...

expert session course Gen2305/2024 ‘Engineering the Immune System Meet the Expert Session Discovery and development of Therapeutic Antibodies March 12, 2024 Rene Hoet Chief Innovation Officer, FairJourney Biologics, Porto & Lecturer Position, Biopharmaceutics, University Maastricht, Dept. Pathology, [email protected] 1 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Introduction René azM Make the best antibody products for patients Building Bridges Team effort Clear goals Synergy Transparency GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 2 Key Learnings Outline Introduction Antibody Therapeutics Human Antibody Lead Discovery Case Studies Case study Arzerra/Ofatumumab (Genmab, transgenic mice) Case study FXIa Ab (Bayer, phage display) Case study Optimizing Abs (FairJourney Biol. , Mammalian Display Technology) Future Challenges and Opportunities for Therapeutic Antibodies „the high hanging fruit“ Case Studies CGRP (Migraine) Antibodies Next gen. Immuno-oncology : Anti-BTN3A Ab activating gdT cells (Imcheck) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 3 Learning goals Structure of human antibodies Difference between chimeric, human and humanized antibodies Technologies how to make human therapeutic antibodies Most important mechanisms of action how therapeutic antibodies work as drugs in humans Advised Literature: Saeed et al. (2017) Front. Microbiol. 8:495. Case study: Therapeutic Antibody Discovery by transgenic mice Ofatumumab (CD20) Advised Literature:Bagacean et al. Immunotherapy (2016) 8(5), 569–581 Therapeutic Antibody Discovery and Optimization by phage display ex. Library Dyax, Hoet et al. Nature Biotech. 2005, Mammalian Display Technology see: Parthiban et al (2019), Mabs: 11(5), 884-898., Dyson M.R. et al. (2020) Mabs, 12 (1), 18 Approved Antibody Therapeutics See Link Anibody Scociety: www.antibodysociety.org/antibody-therapeutics-productdata/ FDA approves 100th monoclonal antibody product, Nature Reviews Drug Discovery, 2021, 40, 491-495 Future Developments therapeutic antibodies Antibody Drug Conjugates Bispecific antibodies: Review Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI., Nat Rev Drug Discov. 2019 Aug;18(8):585-608. -new potential antibody therapeutics, High hanging fruit Carter, P.J. and G.A. Lazar, Nature Rev. Drug Discovery (2018), 17, 197-223 - Mammalian Display: Beyond affinity: selection of antibody variants with optimal biophysical properties and reduced immunogenicity from mammalian display libraries. Dyson MR, et al. MAbs. 2020 Jan-Dec;12(1):1829335 Case studies novel mAbs -CGRP (anti-migraine) antibodies Edvinsson L, et al. Nat Rev Neurol. 2018 Jun;14(6):338-350. doi: 10.1038/s41582-018-0003-1. -BTN3A antibody targeting gdT cells (Imcheck) A. De Gassart et al., Sci Transl Med. 2021 Oct 20;13(616):eabj0835. GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 4 Outline Key Learnings Introduction Antibody Therapeutics Human Antibody Lead Discovery Case Studies Case study Arzerra/Ofatumumab (Genmab, transgenic mice) Case study FXIa Ab (Bayer, phage display) Case study Optimizing Abs (FairJourney Biol. , Mammalian Display Technology) Future Challenges and Opportunities for Therapeutic Antibodies „the high hanging fruit“ Case Studies CGRP (Migraine) Antibodies Next gen. Immuno-oncology : Anti-BTN3A Ab activating gdT cells (Imcheck) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 5 100 approved therapeutic Antibody milestone reached in 2021 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 6 Strengths and limitations of therapeutic antibodies Strengths The availability of several well-established methods for antibody generation and optimization. A high success rate compared to other drugs: 18% for humanized antibodies from the first human trial to regulatory approval (SMOL only 9%). Well-established and broadly applicable production technologies. Mostly well tolerated by patients. Limitations Expensive, reflecting high production costs and commonly large doses, potentially limiting patient access or clinical applications. Clinical applications currently limited to cell surface or extracellular targets. Cannot be orally administered. The large size, particularly of the IgG format (~150 kDa), may limit tissue penetration. There is limited penetration of the central nervous system by IgG owing to inefficient penetration of the blood–brain barrier. GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 7 New Therapeutic Development Drug Approvals by different modalities About 50% in 2023 are Biologics From Antibody Discovery till Therapeutic Approval, Rene Hoet AIM Lisboa Sept 28, 2023 Page 8 Antibodies first approvals in EU,US and ROW From Antibody Discovery till Therapeutic Approval, Rene Hoet AIM Lisboa Sept 28, 2023 9 Antibody Approvals in 2023 for Cancer and non-cancer indications Cancer Non-Cancer Data from Antibody Society (Jan 2024) A searchable table of the figure data is available at www.antibodysociety.org/antibody-therapeutics-product-data/ From Antibody Discovery till Therapeutic Approval, Rene Hoet AIM Lisboa Sept 28, 2023 10 From Antibody Discovery till Therapeutic Approval, Rene Hoet AIM Lisboa Sept 28, 2023 11 How Is Antibody Diversity Created ? Combinatorial diversity V(VH,VL), (D) and J segments Combination Heavy and Light Chain Diversity, Somatic Mutations GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 12 Antibody Format & Specificity Human IgG1 V-regions Specificity & affinity CDR loops Fc–region Antibody format GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 13 Glycosylation of Human IgG Improved therapeutic efficacy Scanlan CN; PNAS 2008 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 14 Chimeric, Humanized and Human antibodies Expected Immunogenicity Ruuls et al., Biotechnology Journal, Aug 2008 Only CDRs Murine GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 15 Human Therapeutic Antibody Technologies In vitro or in vivo technology? In vivo In vitro Mammalian Display §It is all about antibody diversity to your target!!! GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 16 Outline Key Learnings Introduction Antibody Therapeutics Human Antibody Lead Discovery Case Studies Case study Arzerra/Ofatumumab (Genmab, transgenic mice) Case study FXIa Ab (Bayer, phage display) Case study Optimizing Abs (FairJourney Biol. , Mammalian Display Technology) Future Challenges and Opportunities for Therapeutic Antibodies „the high hanging fruit“ Case Studies CGRP (Migraine) Antibodies Next gen. Immuno-oncology : Anti-BTN3A Ab activating gdT cells (Imcheck) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 17 CD20 Characteristics Non-glycosylated, 33-37 kDa protein Expression – From pre-B cell stage, throughout B-cell maturation until plasmacytoid immunoblast (not lymphoid stem cells or plasma cells) Normal function – putative role in B cell activation, B cell growth & calcium flux Immunotherapy: – Clinically well-validated target through rituximab rituximab binding site ofatumumab binding site Opportunity: – Generate novel human antibody with improved effector function GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 18 Human Antibodies UltiMab platform (Genmab) Antibodies by immunization High affinities & specificities Complete IgG, immediately select for inhibition of function GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 19 ms IgH ms Igk hu IgH HuMAbMouse™ hu Igk Four distinct genetic modifications. Functionally replace the mouse immunoglobulin loci with human immunoglobulin transgenes Page 20 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Hybridoma Technology to produce Antibodies GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 21 Ofatumumab Kills resistant tumor cells by ADCC and CDC Specific lysis (%) PMN 40 ** MNC Plasma Primary B-CLL Whole blood * 20 * (n>12) 0 no antibody ofatumumab rituximab Teeling et al. (2004) Blood 104, 1793-1800 22 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Ofatumumab triggers efficient complement-dependent cytotoxicity Raji Daudi SU-DHL-4 % lysed cells 100 80 60 Ofatumumab rituximab 40 20 0 0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100 mAb (µg/ml) Complement inhibitors CD20 expression low high high low Teeling et al. (2004) Blood 104, 1793-1800 Teeling et al. (2006) J. Immunol. 177; 362-371 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 23 Ofatumumab: kills effectively at low CD20 expression ofatumumab Teeling et al. (2006) J. Immunol. 177; 362-371 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 24 ADCC and CDC cooperate in cell killing Inflammatory mediators attract effector cells and induce FcgR upregulation ADCC Antibody Dependent Cellular Cytotoxicity Granzymes cytokines FcgR Apoptosis CDC C1q MAC Complement Dependent Cytotoxicity Ofatumumab Kills Tumors More Effectively Control Ofatumumab Daudi-Luc Xenografts in SCID mice 26 Ofatumumab: small loop epitope rituximab ofatumumab Binding to the small loop epitope generates high avidity C1q binding and efficient C3b deposition. GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 27 Ofatumumab (ArzerraTM ) Rapid development RA Phase I/II March 21 2002 Parent clone ofatumumab FL Phase I/II B-CLL Phase II combi FL Phase III MS Phase II RA Phase III B-CLL, FDA approved Oct.2009 GSK alliance B-CLL Phase III in vivo POC B-CLL Phase I/II DLBCL Phase II FL phase II combi B-CLL, EMA approved April 2010 28 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Ofatumumab (ArzerraTM) Characteristics Unique human CD20 mAb Epitope Binds novel small loop epitope Function More effective in vitro CDC than rituximab Relatively insensitive to complement regulatory factors Effective CDC of cells with low CD20 expression including CLL More effective depletion of B cells and tumor cell in in vivo models Clinical: Well tolerated Positive phase III data: Clinical benefit demonstrated in refractory CLL population with no standard treatment options FDA/EMA approved rituximab binding site ofatumumab binding site GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 29 Approved CD20 mabs International nonproprietary name, US product identifier Brand name Tositumomab-I131 Bexxar MabThera, Rituximab Rituxan Ibritumomab Zevalin tiuxetan Ofatumumab Arzerra Gazyva, Obinutuzumab Gazyvaro Ocrelizumab OCREVUS Target; Format CD20; Murine IgG2a Indication first approved or reviewed Non-Hodgkin lymphoma First EU approval year NA First US approval year 2003# CD20; Chimeric IgG1 Non-Hodgkin lymphoma 1998 1997 CD20; Murine IgG1 CD20; Human IgG1 CD20; Humanized IgG1 Glycoengineered CD20; Humanized IgG1 Non-Hodgkin lymphoma Chronic lymphocytic leukemia 2004 2010# 2002 2009 Chronic lymphocytic leukemia Multiple sclerosis 2014 2018 2013 2017 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 CD20 Therapeutic Antibodies What’s next? Bispecific Antibodies From Antibody Discovery till Therapeutic Approval, Rene Hoet AIM Lisboa Sept 28, 2023 The Technology: What are Bispecific Antibodies? Monospecific vs. Bispecific Monospecific antibodies Binds to a single target, A Binds A Binds A How do Bispecifics disruptive SoC? Greater efficacy on a single patho-mechanism Address multiple patho-mechanisms simultaneously Novel options to modulate or kill cells (Target, recruit, activate or destroy specific cell types) Bispecific antibodies Bind to two targets (A and B) Tunable stoichiometry (1A:1B, 2A:1B, 2A:2B) Binds A Binds A Fc ü Enables previously undruggable approaches ü Will disrupt SoC in many indication areas From Antibody Discovery till Therapeutic Approval, Rene Hoet AIM Lisboa Sept 28, 2023 Binds B Binds B Page 32 CD20 xCD3 Bispecific T cell engagers recently approved in the clinic CD From Antibody Discovery till Therapeutic Approval, Rene Hoet AIM Lisboa Sept 28, 2023 20 Page 33 From Antibody Discovery till Therapeutic Approval, Rene Hoet AIM Lisboa Sept 28, 2023 34 Bispecifics can enable first-in-class (bispecific >> combination of IgGs 1 +1 >2) Activate two co-receptors/ co-factors Enhance receptor inhibition 2014 Fitzgerald et al. (Merrimack) 2015 Igawa (Chugai) Enhance ADC internalization (biparatopic) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Increase therapeutic window (cell-specific targeting) 2016 Tang et al. Page 35 Outline Key Learnings Introduction Antibody Therapeutics Human Antibody Lead Discovery Case Studies Case study Arzerra/Ofatumumab (Genmab, transgenic mice) Case study FXIa Ab (Bayer, phage display) Case study Optimizing Abs (FairJourney Biol. , Mammalian Display Technology) Future Challenges and Opportunities for Therapeutic Antibodies „the high hanging fruit“ Case Studies CGRP (Migraine) Antibodies Next gen. Immuno-oncology : Anti-BTN3A Ab activating gdT cells (Imcheck) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 36 3 7 Human antibodies made in vitro GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Antibody Discovery using in vitro display selection platforms (Hoogenboom, Nat. Biotech, 2005) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 38 Antibody Engineering GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 39 How Does Phage Display work? Bacteriophage Antibody genes are linked to bacteriophage (M13)-protein (gene III) displayed on surface of bacteriophage Antibody gene bacteriophage Sir Greg Winter Nobel Prize 2018 “displayed” antibody (Fab) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 40 What is an antibody library Many different antibody genes and corresponding antibody proteins that create enormous diversity Over 10 billion Fabs in antibody library GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 41 Constructing Antibody Libraries in Phage VH (or VHCH1) g onin l c andning y l b sem ise clo s a e pw Gen or ste P CR VL (or VLCL) Synthetic germline Vgene diversity VH VL V-genes from Human B cells from naïve or immune donors Dis ve play cto r Intro d into E uce.coli Rescue GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Immune, naïve or synthetic antibody library of human Fab or ScFv fragments Page 42 Powerful screening process GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 43 In vitro human antibody technology Antibody Phage Display Library Dyax (now part of Shire/Takeda) Many Therapeutic Antibodies from 1 Library l: k: 1.1x1010 1.2x1010 RACE VL CL VH-CDR3: 2x109 Clone Prepare mRNA VH-CDR1&2: 4.2x107 Isolate lymphocytes Collect blood Synthetic DNA § VL/CL and VH-CDR3 diversity from human B cell repertoire § VH-CDR1,2: synthetic targeted diversity § Single 3-23 framework for VH Displayed antibody Fab fragments Source: Hoet et al, Nature Biotech, 2005 4 First in class Antibodies approved (Cyramza, Lilly, 2014, Necitumumab; Lilly, 2015, Avelumab EMD Serono/Pfizer 2017, Lanadelumab, Shire) Many additional Antibodies from this library in clinical trial (Lilly, Merck Serono, Amgen, Biogen-Idec, Merrimack, Baxter, Athera, Bayer) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 44 Therapeutic Ab Discovery example Bayer: FXIa Antibody Discovery by functional Fab screening Currently in Phase II clinical trial Phage Selections of Dyax Fab library (2.1010) on FXI/FXIa 50,000 clones screened in ELISA on rabbit/human FXI/FXIa 1100 hits identified Sequencing identified 198 unique clones FXIa assay (RFU) 4800 hits screened in Biochemical Assay on FXI/FXIa In vitro dose response for 198 candidates In detail in vitro and in vivo characterization top candidates Lead Antibody Candidate BAY 1213790 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 45 Outline Key Learnings Introduction Antibody Therapeutics Human Antibody Lead Discovery Case Studies Case study Arzerra/Ofatumumab (Genmab, transgenic mice) Case study FXIa Ab (Bayer, phage display) Case study Optimizing Abs (FairJourney Biol. , Mammalian Display Technology) Future Challenges and Opportunities for Therapeutic Antibodies „the high hanging fruit“ Case Studies CGRP (Migraine) Antibodies Bispecific Ab to FIXa and FX (mimicing FVII (Hemophilia) Next gen. Immuno-oncology : Anti-BTN3A Ab activating gdT cells (Imcheck) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 46 47 ANTIBODY TECHNOLOGIES AVAILABLE AT FAIRJOURNEY BIOLOGICS FJB INTRODUCTION TO DEVELOP DRUG CANDIDATES SHORT TIMELINES HIGH AFFINITY / POTENCY IMPROVED DIVERSITY + HUMAN DONORS TYPICAL SIZE: >1010 CFU LLAMAS HUMAN DONORS TYPICAL SIZE: 109 CFU TYPICAL SIZE: 5x108 CFU NON-IMMUNE Fab, scFv, VHH POTENTIAL DIVERSITY: 1027 CFU SEMI-SYNTHETIC “EXPLORER” IMMUNE VHH-hFc, scFv-hFc SYNTHETIC DESIGN DIVERSE REPERTOIRES PHAGE MAMMALIAN ALTERNATE DISPLAY METHODS IMPROVED DEVELOPABILITY Mammalian Display to Select Antibodies for Developability GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 48 MAMMALIAN DISPLAY PLATFORM 49 INTRODUCTION TO MAMMALIAN DISPLAY Target intron in mammalian genome Exon 1 (0) Exon 2 (0) Left HA cleavage Right HA Blasticidin (0) pEF light chain CMV heavy chain Left HA (750bp) Right HA (830bp) Parthiban et al (2019), MAbs: 11(5): 884-898. CHALLENGE: Large library with 1 gene/cell SOLUTION: Nuclease-directed integration Screen 10’s millions of clones Ability to work in IgG or another final format (including multispecific antibodies) Screen directly in production cell type 50 MAMMALIAN AND DEVELOPABILITY MAMMALIAN DISPLAY ALLOWS SELECTION OF CLONES WITHDISPLAY OPTIMAL BIOPHYSICAL PROPERTIES BOCOCIZUMAB CASE STUDY Mouse hybridoma à humanized (5a10i) Affinity matured by Phage Display (300pM à 5pM) Maturation led to multiple developability “red flags” according to Jain et al (2017), including evidence of selfinteraction and low specificity Significant immunogenicity found in phase 3 clinical trial Compromised developability properties again reflected in poorer surface display Can we “fix” Bococizumab by Mammalian Display? IgG display (a-Fc) Dyson et al (2020), MAbs: 12(1), 884-898. 51 MAMMALIAN DISPLAY ALLOWS SELECTION OF CLONES WITH OPTIMAL BIOPHYSICAL PROPERTIES BOCOCIZUMAB CASE STUDY Identification hydrophobic and positive charged residues HEK293 cells displaying library of bococizumab variants Mutagenesis library & Mammalian Display selection Sequence analysis & IgG screening Biophysical Characterization: bococizumab variant mAbs Characterisation & immunogenicity assessment Proliferation of CD4 + T cells by selected bococizumab variants A unique platform to fast-track clinical candidates - Screen 10’s millions of clones Ability to select or engineer in IgG or other final formats - Allows fixing of “broken” antibodies Dyson et al (2020), MAbs: 12(1), 884-898. Comparison phage and mammalian display systems for mAb libraries Display System Max. Library size Phage Mammalian Cells 10 +10 -10 +11 10+6-10+7 Selection Scope Main Strength/Limitations Suitable mAb formats Versatile Incl. Cell Selections Large mAb panels, Technically robust, easy to use, Functional screening difficult, no screenig for developability scFv, VHH, Fab, Diabody Limitations Cell Selections hIgG with immune effector, possible to select for functional mAbs, selection for final product format, limited library size, slower system, FACS sorting expertise & equipment needed, select for developability, technically more challenging system. IgG, scFv, Fab, VHH GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 52 FUTURE ANTIBODY DISCOVERY PLATFORMS AT FJB 53 “EXPLORER” MODULAR ANTIBODY PLATFORM: COMBINING PHAGE & MAMMALIAN DISPLAY POTENTIAL TO EXPLORE 1027 DIVERSITY Select Heavy Chain (VH) and Light Chain (VL) Scaffolds Most Frequently used in Humans Well Expressed Validated in Clinically Approved Therapeutics Select Antibody Diversity Mimicking Human Diversity Synthetic CDR1 & CDR2 Heavy and Light Chains mimicking Diversity in humans CDR3-H3 and CDR3-L3 Diversity from Human Donors Reduction Biophysical Liabilities in Design Large Modular Human Library in Phage Display of Highly Developable Antibodies Total Diversity individual Modules 1027 Phage Library > 2x1010 Simple Transfer to Mammalian Display & Expression to Optimize Ab Repertoires by “shuffling” modules (e.g. HCDR1&2 and VL) in Final Ab Format Select for binding & developability Opportunity to Optimize Any Ab repertoire (including bispecifics) Incorporation Microfluidics Technologies for soluble functional screening Outline Key Learnings Introduction Antibody Therapeutics Human Antibody Lead Discovery Case Studies Case study Arzerra/Ofatumumab (Genmab, transgenic mice) Case study FXIa Ab (Bayer, phage display) Case study Optimizing Abs (FairJourney Biol. , Mammalian Display Technology) Future Challenges and Opportunities for Therapeutic Antibodies „the high hanging fruit“ Case Studies CGRP (Migraine) Antibodies Next gen. Immuno-oncology : Anti-BTN3A Ab activating gdT cells (Imcheck) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 54 Pursuit of the “high-hanging fruit“ New targets (Biology) and Improve efficacy of Antibodies Carter, P.J. and G.A. Lazar, Nature Rev. Drug Discovery, 2018, 17, 197-223 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 55 Case Study 1 Migrane Antibodies International nonproprietary name Brand name Target; Format Erenumab Aimovig CGRP receptor; Human IgG2 2018 2018 Fremanezumab Ajovy CGRP; Humanized IgG2 2019 2018 Galcanezumab Emgality CGRP; Humanized IgG4 2018 2018 NA 2020 Eptinezumab VYEPTI First EU First US approval year approval year CGRP; Humanized IgG1 CGRP=Calcitonin Gene-Related Peptide GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 56 Antibodies to CGRP (receptor) for Migraine treatment show Prophylactic Treatment Migraine is a neurological disorder Migraine pathophysiology involves, amongst others, high plasma concentrations of calcitonin gene-related peptide (CGRP). CGRP plasma levels are elevated in migraine attacks. CGRP infusion induces migraine-like pain, and therapy that lowers CGRP levels improves migraine symptoms. Human(ized) monoclonal antibodies against CGRP or its receptor have been developed as antimigraine drugs. Human and humanized CGRP function blocking mAbs have demonstrated promising migraine prophylaxis efficacy, with favorable side effect profiles in several phase II and III studies. 4 antibodies have now been FDA approved GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 57 An overview of mechanisms/actions of CGRP (receptor) antimigraine drugs Abimael González-Hernández, (2018), Expert Opinion on Drug Metabolism & Toxicology,14:1, 25-41 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 58 Fremanezumab Phase III trial results Primary and Secondary End Points Silberstein SD et al. N Engl J Med 2017;377:2113-2122. Summary GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 60 Outline Key Learnings Introduction importance of Biologics Introduction Antibody Therapeutics Human Antibody Lead Discovery Case Studies Case study Arzerra/Ofatumumab (Genmab, transgenic mice) Case study FXIa Ab (Bayer, phage display) Case study Optimizing Abs (FairJourney Biol. , Mammalian Display Technology) Future Challenges and Opportunities for Therapeutic Antibodies „the high hanging fruit“ Case Studies CGRP (Migraine) Antibodies Next gen. Immuno-oncology : Anti-BTN3A Ab activating gdT cells (Imcheck) GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 61 Emerging and Established Ways of Antibody Targeting in Oncology Carter, P.J. and G.A. Lazar, Nature Rev. Drug Discovery (2018), 17, 197-223 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Success of Immune modulator Therapeutic Antibodies in Oncology International non- Brand name proprietary name Target; Format Indication first approved or reviewed First EU First US approval year approval year Ipilimumab Blinatumomab Yervoy Blincyto CTLA-4; Human IgG1 CD19, CD3; Murine bispecific tandem scFv Metastatic melanoma Acute lymphoblastic leukemia 2011 2015 2011 2014 Nivolumab Pembrolizumab Atezolizumab Avelumab Durvalumab Cemiplimab Opdivo Keytruda Tecentriq Bavencio IMFINZI Libtayo PD1; Human IgG4 PD1; Humanized IgG4 PD-L1; Humanized IgG1 PD-L1; Human IgG1 PD-L1; Human IgG1 PD-1; Human mAb Melanoma, non-small cell lung cancer Melanoma Bladder cancer Merkel cell carcinoma Bladder cancer Cutaneous squamous cell carcinoma 2015 2015 2017 2017 2018 2019 2014 2014 2016 2017 2017 2018 Madorsky Rowdo et al, Front Immunol. 2015; 6: 127 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 Page 63 Current limitations Immunotherapy How to address “Cold Tumors” Bonaventura et al., Frontiers in Immunol. 2019 Tumors escape from immune surveillance by downregulation of the immune system Approved Antibodies as inhibitors of immune checkpoint regulators: aCTLA-4, aPD1 and aPDL1 Current focus on new combinations with aCTLA-4 and aPD-1(L) therapy for additional cancer indications (over 2000 clinical trials) However so far only subset of cancer and subsets of patients respond (average about 20-30%) Need for new approaches to induce immune response GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 64 gd T-cells: a promising yet untapped immune cell population Innate immunity Adaptive immunity gd T-cells unique features o A dual effect: direct tumor lysis & immune responses modulation o High frequency & tumor infiltrating capabilities in a large number of cancers o Highly correlated with favorable clinical outcomes Prognostic impact of genes & infiltrating immune cells across cancers1 o No MHC restriction i.e., no barrier imposed by patients’ MHC alleles & no escape for advanced tumors which often lose MHC expression o No correlation between αβ TILs and TCR Vγ9Vδ2+ γδ TILs opens synergy with approved checkpoint inhibitors antiPD1/PD-L1/CTLA-4 A. Gentles et al. Nat Med. 2015 Aug;21(8):938-945 E. Lo Presti et al. Frontiers in Immunol. Review, 2014, 1-8; 3 G. Chitadze et al. 2017, Trends in Immunol 2017, Tosolini et al, Oncoimmunology 2017 1 2 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 6 6 BTN3A is over-expressed on many different tumors Transcriptomic data (GEPIA) BTN3A1 8 6 4 BTN3A2 8 6 4 2 10 HNSC KIRP BLGG SARC CHOL ESCA PAAD DLBC STAD SCM GBM KIRC 2 AML 6 BTN3A3 log2(TPM + 1) 2 Expression profile BTN3A is differentially expressed on immune cells (gd T-cells only target cells under stress) & across a broad range of solid tumors1 (PDAC, breast cancer, ovarian cancer…) & blood cancers (AML) 1,2 Protein (IHC data) Ctrl Pancreatic tissues Pancreatic ductal adenocarcinoma Normal and multi-tumor TMAs currently screened with several BTN3A mABs Benyamine A et al., OncoImmunol, 2017 1 Compte 2 Rhodes E, et al., Eur. J. Immunol.2004; Le Page C, et al., Plos one, 2012; Cubillos Ruiz et al,Oncotarget 2010; DA, et al., J. Immunol. 2015; Benyamine A et al., OncoImmunol, 2017 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 ICT01: Activating Anti-BTN3A mAb for IO & Infectious Diseases ICT01-mediated Indirect Activation Endogenous gd T Cell Activation Stress è phosphoantigen prod. è BTN3A1 active conformation ICT01 binding è BTN3A1/A2/A3 active conformation (stress mimicking mechanism bypasses pAgs) Cytokine Production Cytokine Production IFNg & TNFa release Proliferation Granzyme & Perforin release gd T Cells IFNg & TNFa release Proliferation Granzyme & Perforin release gd T Cells Vg9Vδ2 TCR Vg9Vδ2 TCR Lysis 3A2 3A1 B30.2 3A3 B30.2 Phosphoantigens Malignant Cancer cell/ transformation Infected Cell Pathogens Zoledronate (Mevalonate pathway dependent effect; can be mutated in CA as immune escape) Lysis 3A2 3A1 3A3 B30.2 B30.2 Cancer cell/ Infected Cell § ex-vivo ~20% ICT01 target occupancy achieves ~90% g9d2 T cell activation § Activated g9d2 T cells migrate from blood to tumors & sites of infections § Leads to activation of other immune cells, including ab T Cells, dendritic cells/APCs & B cells GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 67 BTN3A Ab is a strong activator of g9d2 T-cells against a wide range of tumors γ9δ2 T-cells + IgG or anti-BTN3A + Daudi γδ T-cells alone TCR Panγδ 6 8 γδ T-cells + Control Isotype +DAUDI γδ T-cells γδ T-cells + agonist CD277 + antagonist CD277 (mAb 103.2) +DAUDI (mAb 20.1) +DAUDI functional activity Anti-BTN3A activates the cytotoxicity and cytokine responses of g9δ2 T-cells in vitro1 0.78% CD107a/b γδ T-cells Anti-BTN3A induces a strong γ9δ2 T-cell response in a wide range of tumor cells 1 Christelle Harly et al. Blood 2012;120:2269-2279 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 69 ICT01 Safely Induces Selective Activation and Migration of g9d2 T Cells from the Circulation of Cancer Patients Preliminary results EVICTION Cohort 1: Dosing Patient (µg) In one Melanoma patient post ICT01 increase of gd T cells and CD3+ T cells infiltration 1. Good safety profile: no DLTs, related SAEs or cytokine release syndrome Safety Review Committee has approved dose escalation to Cohort 3 2. Specific activation of g9d2 T Cells after each dose 3. Preliminary evaluation of tumor biopsies suggest an increase in tumor infiltrating gd T cells, CD3, CD4 and CD8 T cells GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024 70 New (Antibody) Drug Discovery Unit at the University of Maastricht Bridge the gap…… New targets Concepts New Biologics Drugs for patients 71 GEN3505/2024 ‘Engineering the Immune System, Discovery and Optimization of mAbs, Rene Hoet March 12, 2024