Introduction practical assignment: Identification of VHH/nanobodies against ErbB1/EGFR

Transcription

1 Introduction practical assignment: Identification of VHH/nanobodies against ErbB1/EGFR Sofia Doulkeridou, Rachid El Khoulati, Paul van Bergen en Henegouwen

2 Agenda Introduction to Nanobodies and their properties Phage display Construction of antibody libraries Selection cycle Selection for function Conclusions

3 Agenda Introduction to Nanobodies and their properties Phage display Construction of antibody libraries Selection cycle Selection for function Conclusions

4 Discovery of heavy-chain antibodies Without DTT With DTT kda kda - 43 kda kda kda kda 20 kda Hamers-casterman 1993

5 From heavy chain antibodies to nanobodies The smallest functional fragment of a naturally occurring single-chain antibody Conventional Antibody Heavy and light chains Both chains required for antigen binding and stability Heavy-Chain Antibody Only heavy chains Full antigen binding capacity and highly stable

6 Structure of a nanobody FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 QVQLQESGGGSVQAGGSLKLSCTASGRRFS TYAVG WFRQAPGQDREFVA TISWTNSTDYADSVKG RFTISRDNAKNTGYLQMNGLKPEDTSVYVCAA DKWSSSRRSVDYDY WGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGRTFS GFAMG WFRQAPGKEREFVA AISWSGGSLLYVDSVKG RFTISRDNAKNTVHLQMNSLKPEDTAVYYCAA MVGPPPRSLDYGLGNHYEYDY WGQGTQVTVSS EVQLVESGGGSVQTGGSLRLTCAASGRTSR SYGMG WFRQAPGKEREFVS GISWRGDSTGYADSVKG RFTISRDNAKNTVDLQMNSLKPEDTAIYYCAA AAGSAWYGTLYEYDY WGQGTQVTVSS QVQLQESGGGLVQPGGSLRLSCAASGRTFS SYAMG WFRQAPGKEREFVA AIRWSGGYTYYTDSVKG RFTISRDNAKTTVYLQMNSLKPEDTAVYYCAA TYLSSDYSRYALPQRPLDYDY WGQGTQVTVSS QVQLQESGGGLVQAGGSLLLSCAASGRTFS SYAMG WFRQAPGKEREFVA AINWSGGSTSYADSVKG RFTISRDNTKNTVYLQMNSLKPEDTAAFYCAA TYNPYSRDHYFPRMTTEYDY WGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGRTFT SYAMG WFRQVPGKEREFVA ALSTRSAGNTYYADSVKG RFTISRDNAKNTVYLQMSSLKAEDTAVYYCAA GYMSSDADPSLAASLHPYDY WGQGTQVTVSS

7 Hallmark residues within VHH Hypervariable Regions H1 H2 H3 Hallmark Residues FR2 FR1/3 FR4 37, 44 45, 47 6, ,

8 Properties of nanobodies Small-molecule properties of Nanobodies High Stability Antibody properties of Nanobodies High affinity/potency Alternative routes of administration High target selectivity High tissue penetration Low inherent toxicity Ease of manufacture in bacteria and yeasts Low potential immunogenicity Format flexibility Very low cost of goods Half-life tailoring Unique properties of Nanobodies Wider range of epitopes, including cavities

9 Address targets inaccessible to conventional Ab s: cavity binding and enzyme inhibition 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 Nature Structural Biology 1996, 3 (9)

10 Formatting of VHH To increase half-life from few hrs to 3 weeks To increase potency, gain function due to avid binding/cross-linking To acquire an effector mechanism To expand beyond one specificity

11 Agenda Introduction to Nanobodies and their properties Phage display Construction of VHH libraries Selection cycle Selection for function Conclusions

12 VHH library is made PBL RNA cdna VHH specific amplification PlacZ RBS SS Myc His VHH * giii Library pur8100 E coli Helper phage AMP 12

13 M13 phage 13

14 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)

15 Agenda Introduction to Nanobodies and their properties Phage display Construction of antibody libraries Selection cycle Selection for function Conclusions

16 Phage display Incubation of target with phages 16

17 Phage display Washing away non-specific binders 17

18 Phage display Elution of phages Followed by infection of TG-1 cells TG-1 TG-1 TG-1 18

19 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

20 Agenda Introduction to Nanobodies and their properties Phage display Construction of antibody libraries Selection cycle Selection for function Conclusions

21 Selection for function: isolation of antagonistic antibodies antibody library Analyze phage antibodies Incubate with immobilized cytokine Washing Elution by competition with excess of receptor Removal unbound phage

22 The Her family of RTK as cancer targets Over-expression of Her receptors is often observed in tumors EGFR contributes to different stages of carcinogenesis: Cell proliferation Resistance to apoptosis Cell motility Angiogenesis Several anti-egfr whole antibodies have been successfully introduced into the clinic: e.g. c225 (CetuxiMab) and 425 (MatuzuMab). Resistance to mono-therapy (e.g. EGFR) is associated with increased signalling via other RTK (IGF-1R; Her3)

23 2 different conformations of the EGFR Inactive, auto-inhibited tethered conformation Active, EGF-bound extended dimer Ferguson, KM Bioch. Soc. Trans. 2004

24 Model of activation of the EGFR

25 Structure anti-egfr nanobody and EGFR Schmitz et al 2013

26 Paratope binding to EGFR

27 Footprint binding sites Schmitz et al 2013

28 Mechanisms of EGFR inhibition by nanobodies

29 Phage library EGFR- ECD- Fc An#- Fc Capturing

30 Binding of phages to EGFR-ECD EGFR- ECD- Fc An#- Fc

31 Total elution, e.g. trypsin EGFR- ECD- Fc An#- Fc

32 Specific elution e.g. EGF EGF EGF EGFR- ECD- Fc An#- Fc

33 Agenda Introduction to Nanobodies and their properties Phage display Construction of antibody libraries Selection cycle Selection for function Conclusions

34 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 Phage display permits selection based on function