AbCheck Combining affinity, stability, functionality & drugability on customized antibody formats
Company Presentation March - 2011
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Profile AbCheck utilises three synergistic platforms for the discovery and generation of human antibodies: Proprietary Phage Display libraries and Yeast Display of full-length IgG or novel formats combined into the unique AbSieve technology
Highly validated proprietary Phage Display libraries ensure fast and reliable discovery of highly specific and high affinity human antibodies for virtually every possible target protein Track record of more than 25 successful antibody discovery projects Three different proprietary libraries with a combined diversity of 1010 human antibodies
In-licensed Yeast Display technology allows screening of all antibody formats and improves drugability of drug candidates Selects improved drug candidates from a huge number of variants Selection in the most relevant format including full-length IgGs, customer specific and novel antibody formats
Services range from antibody discovery to lead optimization Attractive tailor-made deal structures and with no royalty obligations to its partners 2
Three paths to success Phage Display o o
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Yeast Display
Proven commercially – fast, robust and flexible Proprietary libraries with a combined diversity of 10 10 provide a reliable source of highly specific human antibodies Monovalent display for isolation of high affinity binders Allows cell panning, selection of internalizing antibodies, epitope-focused selections and much more Isolated VH/VL pairs constitute “binding units” and can be used in every antibody format from IgG to customer specific and novel formats
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Displays functional full length IgGs , tandem scFvs , scFvs and in all probability new customer specific and novel antibody formats Eukaryotic ER proof reading mechanism selects-out misfolded antibodies Display efficiency correlates with expression yields and stabilities of soluble antibodies Highest level of control by real-time, quantitative selection of antibody clones by FACS Enables antibody engineering in the relevant antibody format
AbSieve •
Yeast Display of enriched AbCheck phage display libraries in two to three panning rounds • •
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Batch recloning of enriched pool
Yeast Display and FACS-based selection in the most relevant format
Expression of soluble antibodies - including full length IgGs - enables direct screening for functionality •
Apply to existing antibodies for improvement in affinity, expression, stability and functionality 3
AbCheck Workflow - Overview
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AbSieve: Combined strength of Phage and Yeast display
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AbSieve: Increased probability of antibody success AbSieve combines the strength of highly validated phage display libraries with the advanced possibilities of yeast display to address antibody specifications beyond affinity. Ready-to-use and highly validated phage display libraries with 1010 different molecules are optimal tools to discover new antibodies in minimal time. Monovalent display on the phage allows selection of highaffinity antibodies to monomeric, dimeric or oligomeric soluble targets or targets in their native environment on the cell surface.
Significantly increased probability of reaching market by identifying antibodies to exact specifications
Improved drugability through yeast display, selecting for enhanced soluble expression levels and stability of antibodies. The yeast proof reading mechanism guarantees expressability in eukaryotic systems. Yeast Display in combination with FACS allows real-time monitoring and full control of the selection process. Screening in the final drug format avoids any drop outs caused by changes in the format. 6
Proprietary Libraries AbCheck uses three validated distinct human libraries Natural library derived from immune system’s antibody gene repertoire Further info Fully synthetic library Further Info Semi-synthetic library combining aspects of both natural and synthetic antibody libraries Comprising a total of about 1010 sequentially and structurally diverse antibodies.
Further info
Advantages Higher probability of isolating very specific human antibodies against every target High diversity; approximately 1010 not only in sequence, but also in structural diversity Antibodies tuned to best target specific properties
Validation Track record of more than 25 successful antibody discovery projects More than 10 antibodies from AbCheck libraries in pre-clinical programs, one in the clinic Outstanding screening expertise including: Several complex cell surface receptors as well as GPCR Subnanomolar affinities Extreme specificities 7
Library validation + panning procedure Affinities Monovalent binding affinities within the low nanomolar region obtained from primary screens
Low koff rates even for monovalent scFv Transfer into bivalent IgG usually increases binding strength by orders of magnitude Several means of affinity maturation can be applied to enhance the affinity even further
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Library validation + panning procedure Extreme Specificities Efficient counter-selection strategies were successfully applied to isolate antibodies with extreme specificities: Isoform specific antibodies: We have isolated an antibody to the Fcg receptor CD16A (FcgRIIIa) that does not bind the CD16B isoform despite 96% sequence identity between the two isoforms. Specific binding to deletion mutants without binding to the wild type protein: Specific targeting of deletion mutants is challenging, because the only unique epitope is at the fusion site of the fragments upstream and downstream of the deleted part. Conformation-specific antibodies: By depleting the libraries of binders to the non-activated platelet receptor GPIIb/IIIa we were able to isolate antibodies to the activated form of the receptor.
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Library validation + panning procedure Functionality Most of the therapeutic applications require not only binding of the antibody but depend on agonistic or antagonistic activity. From our libraries we have isolated antibodies with different functions beyond binding: Agonistic antibodies: Binding of the anti CD16A antibody to the receptor activates NK cells and is very efficient in inducing ADCC.
Blocking antibodies: The isolated anti GPIIb/IIIa antibody inhibits binding of fibrinogen to activated thrombocytes. Antagonistic antibodies: We have isolated an anti IL13R antibody that interrupts the IL13 signalling cascade.
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Library validation + panning procedure Diversity Our libraries have a combined complexity of 1010 antibodies. In the natural library all VH and VL families are included giving the maximal possible structural diversity. The synthetic library extends the diversity beyond the limits of naturally occurring antibody sequences. A combination of natural and synthetic diversity proved in further increasing the diversity.
In a typical project 5 to 10 different antibodies meet the requested specifications.
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Our Discovery Capabilities De novo isolation of antibodies
Characterisation
Phage Display Ready-to-use highly validated phage display libraries Strong experience in panning strategies including cell panning
Affinity and specificity ELISA & flow cytometry titrations Biacore
AbSieve Change to final drug format during the selection process FACS-based selections
In vitro functionality Target specific cell-based assays
Stability Assessed in thermofluor assays
Lead Optimisation Phage or Yeast Display Sublibraries for lead optimisation can be created for phage or yeast display
Addressing Affinity, expression levels, stability and functionality Possible in different antibody formats Yeast Display platform allows lead optimisation of IgGs or other antibody formats
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Partner Benefits Three discovery platforms ensure three paths to the best antibodies AbSieve is based on highly productive and validated technologies Drugable antibodies delivered in very competitive time frames to virtually any target Three distinct proprietary libraries expressing about 1010 human antibodies Significantly increased probability of identifying antibodies to exact specifications
Proven track record of isolating more than 25 different human antibodies Strong IP position allowing flexible, tailored deal structure No royalty obligations to third parties Senior team with a proven track record in the discovery and research of therapeutic antibodies
Proven, highly enabling technologies combined with attractive cost structures 13
Summary
The Human antibody discovery specialist in commercially validated yeast and phage technologies Antibody discovery for virtually all targets in all formats Higher probability of reaching market by screening in final antibody drug format The unique AbSieve technology delivers drugable antibodies within highly efficient timelines Three commercially validated diversified antibody libraries AbCheck offers tailored deals with no royalty obligations
Thank you
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Senior Management Dr. Volker Lang (Managing Director) 16 years experience in the biotechnology sector, spanning basic research, industry R&D, Business Development and Licensing, commercial operations and management Background as scientist and project manager combined with extensive experience in the commercialization of biotechnology products Considerable CMC/Production experience in his previous positions as Chief Business Officer (Affimed Therapeutics AG) and Vice President Corporate Development (Scil Technology GmbH) Dr. Vera Molkenthin (Chief Scientist and Head of Discovery) Many years experience in the selection of antibodies using phage display PhD from the Faculty of Biology in Mainz with main focus on construction of a phage display library and the selection of stabilizing mutations Head of Screening at Affimed with responsibility for the management of the screening group, with special emphasis on generation of highly diverse antibody libraries, and design and execution of the most appropriate selection procedure for a particular target
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Contact Volker Lang, PhD (Managing Director) Phone: +420 378 05 1500
[email protected] AbCheck s.r.o. Vedeckotechnicky park Plzen Teslova 3 CZ-301 00 Plzen www.AbCheck.eu
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Appendix
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Natural Antibody Library Fully human library In vivo created diversity of antibody sequences comprising sequential as well as structural diversity New combinations of VH and VL chains from multiple healthy donors No bias due to amplification of the VH chains by IgM specific priming Special assembly technology guarantees > 70% functional clones Reliable and rapid selection of highly specific and high affinity antibodies on recombinant proteins or cells
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Fully Synthetic Library Synthetic library Generated by introducing randomized sequences in the antibody binding site of selected frameworks Ensuring reliable folding and high expression yields Diversity generated in vitro thereby Avoiding counter selection against anti-self antibodies
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Semi-Synthetic Library Semi-synthetic library Combining the advantages of the natural and synthetic library Diversity and reliable folding and high expression Increasing the diversity of binding molecules even further
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Yeast Display Explained:
The following four slides define the yeast display library AbCheck utilises for antibody discovery TM SECANT platform is the proprietary technology of Celexion LLC
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SECANT™ Platform Overview • The SECANT™ platform is a next-generation antibody display system that can handle large and complex proteins • First yeast system to display full-length IgG antibodies • Proven to display tandem scFv, bispecific and proprietary scaffolds
• Enables high throughput assay of 108 to 109 variants for • De Novo Antibody Discovery • Complex Scaffold Engineering • Scaffold Reformatting • Affinity Maturation • Stability/Solubility/Folding Control • Humanization
• The SECANT™ platform will accelerate discovery timelines by allowing faster characterization and higher throughput • SECANT™ platform is compatible with many types of proteins and libraries • Full length IgG and scFv, fibronectin, receptor ectodomains, superoxide dismutase, human serum albumin, vitamin D-binding protein Source: Celexion
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SECretion and CApture Tech. (SECANT™) 1. The gene for the variant protein to be engineered (the “gene of interest“) is expressed as a fusion to a biotin acceptor peptide (BAP). A co-expressed biotin ligase attaches biotin to the BAP tag on the expressed protein.
Source: Celexion
2. Avidin is attached to the outer wall of the host cell.
SECANT™ platform (continued) 3. The biotinylated protein of interest is secreted into the extracellular space where it binds to the surface-localized avidin. Approximately 105 proteins can be captured on the yeast surface in this manner.
Source: Celexion
4. The captured proteins can be assayed for binding, expression, or enzymatic activity by labeling with a fluorophoretagged ligand, antibody, or substrate. Protein functionality can then be assayed and the best clones isolated by FACS.
SECANT™ platform can display functional, full-length IgG
Source: Celexion
