HuCAL Custom Monoclonal Antibodies

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1 HuCAL Custom Monoclonal HuCAL Custom Monoclonal Antibodies Highly Specific, Recombinant Antibodies in 8 Weeks

2 Highly Specific Monoclonal Antibodies in Just 8 Weeks HuCAL PLATINUM (Human Combinatorial Antibody Library) is a synthetic library of 45 billion fully human antibodies in Fab format. HuCAL and CysDisplay (a proprietary, advanced form of phage display) are used to generate high affinity and high specificity fully human antibodies, without immunization of animals. Special features of our monoclonal antibodies include: In vitro technology, no immunization of animals Guided selection strategies to achieve challenging specificities Established technology, over 20,000 unique antibodies generated since 2003 Secure and consistent antibody supply Greater than 90% success rates HuCAL Monoclonal Antibody Generation Process Overview Target antigen supplied by customer, or produced by us, is immobilized on a solid support Guided selection of specific antibodies: 3 rounds of panning using proprietary CysDisplay technology Screening of enriched specific antibodies on the target antigen and control antigens Sequencing of ELISA-positive clones Expression, purification and quality control of unique Fab antibodies (monovalent and bivalent formats) Fab antibodies delivered within 8 weeks Conversion (optional) into full length immunoglobulin; many isotypes available CysDisplay Technology An innovative and efficient phage display method. Fab fragments are linked to phage particles by a disulfide bond allowing elution of phage with reducing agents during antibody selection. Proven, published technology Knappik A et al (2000). J Mol Biol. 296: Prassler J et al (2011). J Mol Biol. 413:

3 HuCAL Technology Leads to Better Antibodies The HuCAL monoclonal antibody technology offers numerous advantages compared to conventional monoclonal antibody generation. Parameter Conventional Monoclonals HuCAL Monoclonals Animals used Yes No Speed of delivery 4-9 months 8 weeks Flexible formats No Fab antibodies Full length Igs, many isotypes Antibody specificity Not predictable Extensive screening required Tailored to customer s needs using Guided Selection (see below) Fully human No Yes, Fc from different species available upon request Technology Immunization, complex cell culture Phage display & bacterial expression Antibody engineering & optimization No Tags for detection Affinity maturation Success rate ~ 75% > 90% Guided Selection Strategies for Superior Specificities Antibody specificity is controlled in vitro by applying guided selection strategies during the selection process to achieve the desired outcome. Guided Selection Strategy Outcome Examples Alternate related antigen(s) during the selection process Selection on immobilized antibody in complex with antigen Pre-incubation of the library with related antigen(s) Antibodies recognizing related antigen(s) Select for antibodies against epitopes different from the immobilized antibody A. Generate antibodies with desired specificities B. Eliminate antibodies with undesired specificities Antibodies recognizing both human & mouse homologs Antibody pair for sandwich ELISA A. Phospho-specific antibodies Anti-idiotypic antibodies B. Antibodies recognizing human but not mouse protein

4 Choose the Best HuCAL Antibody for your Application HuCAL antibodies are produced in just 8 weeks as monovalent or bivalent Fab antibodies with epitope tags. Antibodies can also be converted into full length immunoglobulins with a choice of human, mouse or rat Fc regions. Monovalent Fab and Bivalent Mini-antibodies with Two Epitope Tags Full Length IgA / IgE / IgG / IgM Antibody Format Advantages Mono-, bi - or tetravalent Fab antibodies Full length immunoglobulins Eliminate non-specific binding to cellular Fc receptors & serum factors Avoid cytokine release in cellular assays Monovalency advantageous for competitive assays Co-crystallize with target protein Add various epitope tags for immunodetection or immobilization to a matrix Choice of Fc region: - human IgA, IgE, IgG1, IgG2, IgG3, IgG4, IgM - mouse IgG2a - rat IgG, IgG2a, IgG2b Use Fc region for binding or agglutination reactions Evaluate Fc receptor-mediated effects Use as a fully human standard e.g. as calibrator and/or control Evaluate and Improve Antibody Binding Antibody Selection Based Upon Binding Strength As part of the high throughput process of antibody selection, Fab antibodies can be ranked according to their dissociation rates (k off ). The clones with the preferred k off rates (high, medium, low or mixed) can be selected for further characterization Ylera, F. et al. (2013). Anal Biochem. 441: Affinity Measurement of Purified Antibodies Affinity Maturation of Antibodies - The affinity of selected antibody candidates can be further improved via exchange of selected CDRs against variable CDR cassettes followed by a more stringent selection process Steidl, S. et al. (2008). Mol Immunol. 46: A rapid maturation approach (RapMAT ) of enriched pools of antibodies via diversification of selected CDRs can be included as part of an extended antibody generation Prassler, J. et al. (2009). Immunotherapy. 1:

5 HuCAL - Technology That Works for You Pharmacokinetic and Immunogenicity Studies Shorten Assay Development Time HuCAL technology allows you to develop a single high affinity antibody for use first as a Fab reagent for pharmacokinetic (PK) studies, and later convert into a fully human immunoglobulin positive control for immunogenicity testing. This eliminates the need for patient or animal sera and greatly reduces assay development time and effort (Figure 1). Generate a Standard for ADA Responses Convert Fab antibodies into a range of full length human immunoglobulin isotypes (IgA, IgE, IgG or IgM) to create the ideal standards for the evaluation of anti-drug antibody (ADA) responses in immunogenicity testing. HuCAL Ig antibodies overcome the concerns of limited availability and revalidation of antibodies originating from human or animal sources in clinical studies (Figure 2). Fluorescent Signal ECL signal Adalimumab (ng/ml) in 10% Human Serum Fig. 1: PK Assay using Two HuCAL Anti-Idiotypic Antibodies Anti-ID Antibody (ng/ml) Fig. 2: Anti-Idiotypic HuCAL Antibody as ADA Reference In Vitro Diagnostics Diagnostic tests benefit from the high specificity and sensitivity of HuCAL antibodies and the added reassurance of having a consistent and secure supply. Fully Human Controls and Calibrators HuCAL antibodies can replace patient sera as a source of human control antibodies for immunoassays. HuCAL Igs have been used successfully as control and calibrator materials in quantitative autoimmune and infectious disease assays (Figure 3). Proven Success in Diagnostics Promonitor kits from Proteomika use HuCAL antibodies to measure serum levels of biological drugs or anti-drug antibodies in clinical laboratories. AbD04720-IgA concentration (µg/ml) Fig. 3: Comparison of HuCAL Recombinant IgA in ELISA Assaya

6 Comprehensive Antigen Generation and Assay Services No Antigen? No Problem! We offer high quality custom antigen design geared to generate antibodies to meet your individual needs, including: Gene synthesis of antigen DNA Peptide design, synthesis and conjugation services Bacterial expression of protein fragments using AgX system Human cell line expression of epitope-tagged or Fc-fusion proteins Additional Customization Required? Our antibody specialists will help you customize your antibody project to suit your needs. Additional services available include: Identification of antibody pairs for Sandwich ELISA Antibody production scalable from mg to g amounts Labeling with biotin, FITC, DyLight dyes and more Conversion into an alternate Fab format or to a full length immunoglobulin Ranking of binding strength (k off rate analysis) Affinity determination Affinity maturation Antibody testing in various assays, such as ELISA and Western Blot Terms and Conditions of Use: For More Information abdserotec.com/hucal Americas Europe UK & Worldwide Toll Free: (USA & Canada) Tel: Tel: Tel: Bio-Rad Laboratories, Inc. LIT.HUCALB.V Copyright Bio-Rad Laboratories, Inc. All rights reserved. Published by AbD Serotec, a Bio-Rad Company, Endeavour House, Langford Lane, Langford Business Park, Kidlington, OX5 1GE. AgX, CysDisplay, HuCAL, HuCAL PLATINUM and RapMAT are registered trademarks of MorphoSys AG. DyLight is a registered trademark of ThermoFisher Scientific and its subsidaries. Promonitor is a registered trademark of Proteomika.

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