5/15/13. Agenda. Agenda. Introduction practical assignment: Identification of VHH against ErbB1/ EGFR. Structure of heavy chain antibodies

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1 Introduction practical assignment: Identification of VHH against ErbB1/ EGFR Erik Hofman, Alex Klarenbeek, Rachid El Khoulathi Structure of heavy chain antibodies C H 1 C L V H V L V HH C H 2 C H 3 Conventional Antibody Heavy and light chains Both chains required for antigen binding and stability C H 2 C H 3 Heavy-Chain Antibody Only heavy chains Full antigen binding capacity and highly stable V HH Ablynx s Nanobody The smallest functional fragment of a naturally occurring single-chain antibody 1

2 Properties of VHH Hallmark residues within VHH Small-molecule properties of Nanobodies High Stability Alternative routes of administration High tissue penetration Antibody properties of Nanobodies High affinity/potency High target selectivity Low inherent toxicity Hypervariable Regions H1 H2 H3 Ease of manufacture in Low potential bacteria and yeasts immunogenicity Format flexibility Very low cost of goods Half-life tailoring Unique properties Wider range of epitopes, of Nanobodies including cavities Hallmark Residues FR2 FR1/3 FR4 37, 44 45, 47 6, , Address targets inaccessible to conventional Ab s: cavity binding and enzyme inhibition Formatting of VHH Extended CDR3 loop inhibits enzymes and binds in receptor clefts and canyon sites of viral envelopes Access to small-molecule targets but with antibody specificity and affinity Most known therapeutic targets are either enzymes (28%) or receptors (45%) Lysozyme Active site CDR3 loop Nanobody To increase half-life from few hrs to 3 weeks To acquire an effector mechanism To increase potency, gain function due to avid binding/cross-linking To expand beyond one specificity Nature Structural Biology 1996, 3 (9) 2

3 VHH library is made Construction of VHH libraries PlacZ RBS SS Myc His PBL RNA cdna VHH specific amplification VHH pur8100 AMP * giii Library E coli Helper phage 10 M13 phage Phage biology: replication cycle Bacteriophage F1 (M13) infects E coli by binding to sexpilus (encoded by F episome) via gene 3 Double stranded (RF) DNA genome replicated within cell Single strand DNA packaged into phage particle and released from cells No lytic event, E coli cells survive, but grow slower (plaques) Phenotype-Genotype linkage. Rigid and infective after selection 11 3

4 Phage display Incubation of target with phages 14 Phage display Phage display Washing away non-specific binders Elution of phages Followed by infection of TG-1 cells TG-1 TG-1 TG

5 Eluted phages are rescued and multiplied by infection of E.coli. Each phage results in one E.coli colony on a selective plate through the ampicillin resistance encoded in the phagemid DNA. Titration of the phages gives an estimate of the number of phages in the elution Selection for function: isolation of antagonistic antibodies The Her family of RTK as cancer targets Over-expression of these growth factor receptors is often observed in epithelial tumours antibody library Analyze phage antibodies EGFR is a well studied RTK that contributes to different stages of carcinogenesis: Cell proliferation Resistance to apoptosis Cell motility Angiogenesis Incubate with immobilized cytokine Washing Removal unbound phage Elution by competition with excess of receptor Several anti-egfr whole antibodies have been successfully introduced into the clinic: e.g. c225 (CetuxiMab) and 425 (MatuzuMab). Their mode of action is being increasingly well understood. Resistance to mono-therapy (e.g. EGFR) is associated with increased signalling via other RTK (IGF-1R; Her3) 5

6 5/15/13 2 different conformations of the EGFR Inactive, auto-inhibited tethered conformation Model of activation of the EGFR Active, EGF-bound extended dimer Ferguson, KM Bioch. Soc. Trans Structure Fab Erbitux-sEGFR Ferguson et al., 2005 Mechanism of action Erbitux 1. Blocking EGF binding sites on domain III 2. Steric hindrance of conformational change 6

7 Binding of phages to EGFR-ECD Phage library EGFR- ECD- Fc An#- Fc Capturing EGFR- ECD- Fc An#- Fc Total elution, e.g. trypsin Specific elution e.g. EGF EGF EGF EGFR- ECD- Fc EGFR- ECD- Fc An#- Fc An#- Fc 7

8 Conclusions Nanobodies (VHH) originate from Camelid heavy chain only antibodies Nanobodies combine advantages of antibodies and small molecules Nanobodies can be easily formatted into multivalent construct Nanobodies can be identified from immune libraries via phage display using PBLs from immunized animals permits selection based on function 8