ALTHEA Gold Libraries™ Novel A ntibody L ibraries for The rapeutic A ntibody Discovery Abstract Philippe Valadon 1 , Renae Nelson 1 1 Antibody Design Labs, San Diego, CA 92121 Philippe Valadon 1 , Sonia M. Pérez-Tapia 2 , Renae S. Nelson 1 , Omar U. Guzmán-Bringas 2 , Hugo I. Arrieta-Oliva 2 , Keyla M. Gómez-Castellano 2 , Mary Ann Pohl 3 , Juan C. Almagro 4 1 Antibody Design Labs, San Diego, CA 92121 3 Tri-Institutional Therapeutics Discovery Institute, New York, NY 10021 2 UDIBI, ENCB, Instituto Politécnico Nacional, México 2009-2013 4 GlobalBio Inc., Cambridge, MA 02138 Multi-stage Antibody Library Construction ALTHEA Gold libraries™ ALTHEA Gold Libraries™ are highly diversified scFv libraries (> 10 10 cfu each) enriched for developable antibodies resulting from the combination of in vitro selected scaffolds with in vivo validated CDR-H3 diversity. Flat scaffold for protein targets Grooved scaffold for peptide targets 3-23/3-20 PDB: 5I1i 3-23/4-1 PDB: 5I1d Alternative Topographies to Bind Protein and Peptide Targets The shape of the antigen-binding site determines the type of the antigen the antibody interacts with. Finlay & Almagro. Front Immunol. 3:342, 2012 ALTHEA Gold Libraries™ scaffolds provide two alternative antigen- binding site topographies: One flat suitable for protein antigens and the other grooved biased towards the recognition of peptides. Antigen-binding site Protein-A The V H scaffolds binds Protein-A and enables filtration of well-folded antibodies. One of the V L binds Protein-L and enables assay development and identification of variants coming out of the anti- peptide libraries when used combined with the anti-protein library Also… Biopanning with Protein Models Quality Control of ALTHEA Gold Libraries™ Summary Easy to analyze Highly Diverse Developable antibodies >10 10 members Two alternative topographies + CDR-H3 with a Gaussian distribution of lengths from 1 to 20 Highly stable (Tm >70 0 C) One universal V H scaffold Two alternative V L scaffolds Known structures Protein-A/L binding ~70% (>7 x 10 9 ) unique & functional antibody fragments Philippe Valadon, M.D., Ph.D. [email protected] Tel: +1.858.480.6213 www.abdesignlabs.com Juan C. Almagro, Ph.D. [email protected] Tel: +1.617.710.4487 www.globalbioinc.com For Information, please contact: Each set of ALTHEA Gold Libraries™ is unique, combining carefully designed human scaffolds of improved developability with natural CDR-H3 diversity. Please direct your inquiry for ownership to Antibody Design Labs or GlobalBio, Inc. Protein-A : scFv complex PDB: 1dee PDB: 1mhh Deep Sequencing of Over One Million of H3J fragments 0 2 4 6 8 10 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Anti-Protein Anti-Peptide The potential of ALTHEA Gold Libraries™ to generate specific and diverse antibodies has successfully been validated with several undisclosed targets, including a soluble target, a peptide/protein complex and a membrane protein After reshuffling natural H3J diversity from the pool of 200 donors into selected scFvs for high stability in the primary libraries, all the clones are unique, with ~70% being highly stable Frequency (%) CDR-H3 length (Kabat’s definition) 0 10 20 30 40 Anti-Protein Anti-Peptide Human IGHJ germline gene usage Frequency (%) We present here, the design, implementation and validation of ALTHEA (from the Greek “to heal ”) Gold Libraries™. ALTHEA Gold Libraries™ are semisynthetic scFv libraries built on synthetic human well-known V H and V L germline genes combined with natural H3J fragments obtained from a pool of 200 human donors. The three germline genes provide a universal V H scaffold paired with two distinct V L scaffolds. The V L scaffolds offer two alternative antigen-binding site topographies to bind protein or peptide targets. The antigen-binding site (except the CDR-H3) diversity is designed in positions identified as in contact with protein and peptide antigens in the known antigen- antibody complex structures. The diversification regimes are designed based on the amino acid frequencies of the repertoire of human germline genes and known human antibody sequences. Through a proprietary process comprising the selection for thermostable scFv variants, followed by shuffling of the natural H3J fragments, ALTHEA Gold Libraries™ provide a highly diverse, functional and developable repertoire of human antibody fragments suitable for antibody therapeutic discovery and development. The potential of ALTHEA Gold Libraries™ to generate specific antibody fragments has been assessed with multiple targets including protein models (Hen Egg White Lysozyme; HEL, Human Serum Albumin; HSA), several therapeutic targets including TNF, a protein/peptide complex, and membrane proteins. In all case studies, diverse and specific antibodies were obtained with Kd in the low nM range. Carefully designed scaffolds with neutral CDR-H3 fragments , displayed as scFvs Heat shock Highly stable scFv library Rescue with Protein A 200 donors Age < 40 years 100 males & 100 females RT-PCR with specific primers 10 min @ 70 0 C Diverse H3J fragments Primary Libraries Natural H3J fragments Sfi I V H (3-23) CDR-H3 + Joining Linker GS19 Vk (3-20/4-1) PelB Sfi I Neutral/Natural Diversity ~ 100 bp Synthetic ~ 800 bp Tags Designed Diversity Designed Diversity Diversity: 10 6 Diversity: 10 6 Protein-L Antigen-binding site HEL-coated Immunotubes 100 ug/mL TDI libraries Washed away non-bound Ф bound Ф Eluted with Trypsin Rescued with helper Ф Anti-Protein or Anti-Peptide (10 12 Ф) ~100x coverage Plated in 2xYT/Carb Human Serum Albumin ALTHEA Gold Libraries™ SL2 by Direct Capture Yield specific scFvs against multiple targets using diverse biopanning strategies Highly functional Phage output (UFC/ml ) x 10 5 0 100 200 300 400 500 600 700 800 Round 1 Round 2 Round 3 Round 4 0 200 400 600 800 1000 1200 1400 1600 Round 1 Round 2 Round 3 Round 4 Lysozyme Parallel Biopanning of the Anti-protein and Anti-peptide Libraries and Trypsin Elution Protein-A binding after 72 0 C (10 min) Carefully Designed Diversity Identified positions in contact with protein and peptide targets and diversified based on human germline genes and known antibody sequences amino acid frequencies 0% 15% 30% 45% 1.20 - 1.49 1.50 - 1.99 2.00 - 2.49 2.50 - 3.00 RESOLUTION > 50% below 3.0 Å Macaca mulatta Rattus norvegicus Humanized Chimeric Mus musculus Homo sapiens SPECIES Anti-Protein Anti- Peptide GENERAL SPECIFITIES Studied over 2,500 antibodies known structure to design ALTHEA Gold Libraries™ CDR-H3 V L 90 0 V H 0% 20% 40% 60% 80% 100% ǁ A1 ǁ B1 ǁ C1 ǁ D1 ǁ E1 # F1 ǁ G1 ǁ H1 ǁ A2 ǁ B2 ǁ C2 ǁ D2 ǁ E2 ǁ F2 ǁ G2 # H2 A3 ǁ B3 C3 ǁ D3 ǁ E3 ǁ F3 ǁ G3 # H3 ǁ A4 ǁ B4 ǁ C4 ǁ D4 ǁ E4 ǁ F4 ǁ G4 H4 A5 ǁ B5 ǁ C5 ǁ D5 ǁ E5 F5 ǁ G5 ǁ H5 ǁ A6 B6 ǁ C6 ǁ D6 3-20/3-23 3-20/3-23 TBS TBS = In-frame 0% 20% 40% 60% 80% 100% ǁ A1 ǁ B1 ǁ C1 ǁ D1 E1 ǁ F1 ǁ G1 H1 A2 ǁ B2 ǁ C2 ǁ D2 ǁ E2 ǁ F2 ǁ G2 # H2 ǁ A3 ǁ B3 ǁ C3 ǁ D3 # E3 ǁ F3 ǁ G3 ǁ H3 ǁ A4 ǁ B4 ǁ C4 ǁ D4 ǁ E4 ǁ F4 G4 # H4 ǁ A5 ǁ B5 ǁ C5 ǁ D5 ǁ E5 # F5 ǁ G5 ǁ H5 ǁ A6 ǁ B6 ǁ C6 D6 4-01/3-… 4-01/3-… TBS TBS Biopanning and Screening Selection Specific Clones Amplification Further Characterization RAM kit™ if needed Positive Clones Screening ALTHEA Gold Libraries™ L1 L2 L3 L1 L2 L3 Diversified Synthetic Scaffolds Sequence Diversity H1 H2 H1 H2 In - frame indels 0 0.2 0.4 0.6 0.8 1 1.2 4.00E+08 4.00E+09 4.00E+10 4.00E+11 R1 R2 R3 R4 3-20/3-23 (-) 0 0.2 0.4 0.6 0.8 1 1.2 4.00E+08 4.00E+09 4.00E+10 4.00E+11 R1 R2 R3 R4 3-20/3-23 (-) cfu x 10 5 cfu x 10 5 OD Φ/mL OD Anti-protein Anti-peptide Anti-peptide Anti-protein Summary of the Biopanning Results: Hit rate 80% after 3 rounds BSA HEL Titre Polyclonal ELISA Amplicon 2 SL1 % SL2 % Total sequences 1,077,746 100.00 1,227,176 100.00 Accepted sequences 853,327 79.0 990,864 80.7 Unique sequences 851,681 99.8 989,005 99.8 Unique CDR-H3 sequences 522,939 61.3 592,317 59.8 0.048 0.599 0.045 0.045 0.045 0.045 0.81 0.873 0.868 0.85 0.742 0.68 0.041 0.041 0.041 0.041 0.546 0.042 0.783 0.87 0.882 0.851 0.583 0.69 0.05 0.059 0.046 0.047 0.055 0.047 0.826 0.902 1.03 0.865 0.753 0.723 0.047 0.045 0.045 0.045 0.561 0.743 0.819 0.92 0.919 0.841 0.571 0.726 0.048 0.046 0.045 0.045 0.044 0.717 0.601 0.933 0.894 0.851 0.795 0.727 0.042 0.04 0.691 0.039 0.041 0.04 0.838 0.919 0.751 0.851 0.806 0.782 0.17 0.044 0.044 0.044 0.044 0.045 0.239 0.917 0.892 0.826 0.818 0.801 0.044 0.042 0.043 0.043 0.048 0.295 0.982 0.951 0.914 0.907 0.853 0.275 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 1 2 3 4 5 6 7 8 9 10 11 12 Clone 11 Clone 5 Clone 3 (-) Dilutions 3 clones EC50 40-100 nM by ELISA Best clones HSA-B3 Kd 3.3 nM as Fab