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Camel Nanotechnologies and its Medical Applications

Camel Nanotechnologies and its Medical Applications. Prof. Dr. Serge Muyldermans. Overview of the today’s presentation. Structural properties of functional Heavy-chain antibodies in Camelidae & Nanobodies Natural generation of Heavy-chain antibodies Selection of antigen-specific Nanobodies

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Camel Nanotechnologies and its Medical Applications

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  1. Camel Nanotechnologies and its Medical Applications Prof. Dr. Serge Muyldermans

  2. Overview of the today’s presentation • Structural properties of functional Heavy-chain antibodies in Camelidae & Nanobodies • Natural generation of Heavy-chain antibodies • Selection of antigen-specific Nanobodies • Nanobodies in medical applications HCAbs & Nbs in Medicine & Research

  3. Camel Nanotechnology and its medical Applications Structural properties of functional Heavy-chain antibodies in Camelidae & Nanobodies Natural generation of functional Heavy chain antibodies Selection of antigen-specific Nanobodies Nanobodies in medical applications

  4. Pros & Cons of antibodies • Antibodies are of medical interest because : • Limitless diversity • High specificity • High affinity • Yet, far from being perfect because : • Immunogenicity • Unwanted effector function • Solubility • Stability • Yield • Size • SOLUTION: Look in nature on how to make antibodies smaller

  5. Artilodactyla Camelus dromedarius Camelus bactrianus Lama glama Lama guanoco Lama alpaca Lama vicugna Tylopoda Camelidae Suidae Hyppopotamidae Tayassuidae Suiformes Tragulidae Cervidae Giraffidae Antilocapridae Bovidae Ruminantia Antilopinae Cephalophinae Hippotraginae Bovinae Caprinae HCAbs & Nbs in Medicine & Research

  6. Serum immunoglobulins 67 43 30 Serum from camelids IgG purification on Protein A /Protein G Protein G: 2.7 - 3.5 Protein A: + + + IgG1IgG2 IgG3 H2L2 = conv. Ab H2 = HCAb H L HCAbs & Nbs in Medicine & Research Hamers et al., Nature, 1993

  7. Camelid antibodies CH1 CH1 VH VH VL CL scFv CH2 Fc CH3 VHH VHH CH2 Fc CH3 Fv Fab Classical antibody (IgG1) Single domain antigen binding fragment (15 kDa) NANOBODY Camel Heavy-Chain antibody (IgG2 & IgG3) • Monomeric • Prolate particle: • Diameter 2.4 nm • Height 4 nm HCAbs & Nbs in Medicine & Research Hamers et al., Nature, 1993

  8. Camel Nanotechnology and its medical Applications Structural properties of functional Heavy-chain antibodies in Camelidae & Nanobodies Natural generation of functional Heavy chain antibodies Selection of antigen-specific Nanobodies Nanobodies in medical applications

  9. VH and VHH differences N CDR1 CDR2 CDR3 VH C N VHH C VH W47 G44 V37 L45 CDR1 CDR2 CDR3 VHH F37 E44 G47 R45 HCAbs & Nbs in Medicine & Research Vu et al., Mol. Immunol., 1997 Desmyter et al., Nat.Struct.Biol., 1996

  10. IgH locus 5-6 functional JH genes J-genes V-genes telomer D-genes ca 50 functional VH genes over 1000 kb V-DJ rearrangement ca 25 functional DH genes D-J rearrangement centromer Sequential arrangement of C-genes over 200 kb ATTTGCAT ACAAAAACC L-exon transcription V-exon 5’ 3’ CDR2 CDR1 splicing TATA A A CACAGTG 23 bp spacer 3’ 5’ translation switch CH1 hinge CH2 CH3 M1 M2 H-chain HCAbs & Nbs in Medicine & Research

  11. IgH locus of camelids 5-6 functional JH genes J-genes V-genes telomere D-genes ca 90 functional VH & VHH genes (over 1000 kb) ca XX functional DH genes centromere ATTTGCAT ACAAAAACC L-exon V-exon 5’ 3’ CDR2 CDR1 TATA CACAGTG 23 bp spacer h Cg3 Cg1 Cg2 Cg1 Cm1 Cm3 Cm4 Cm2 Ca2 Ca1 Ca3 h Cg3 Cg2 Sequential arrangement of C-genes (over 200 kb) HCAbs & Nbs in Medicine & Research

  12. V-D-J rearrangement produces VHH RSS Ig-promotor Leader signal peptide ~ 50 VH genes CDR1 CDR2 CDR3 IgG1 D-genes 6 JH-genes IgG2 IgG3 ~ 40 VHH-genes 7 subfamilies • Position of Cys in CDR1 • CDR2 length (VH3) HCAbs & Nbs in Medicine & Research Nguyen et al., EMBO J., 2000

  13. HCAbs lack CH1 domain Dedicated H-chain gene for conventional Ab Poly-A Poly-A AGTGTGG GTAAGT VH CH1 hinge CH2 CH3 M1 M2 Dedicated H-chain gene for Heavy-chain Ab Poly-A Poly-A CGTGTGGATAAGT VHH CH1 hinge CH2 CH3 M1 M2 HCAbs & Nbs in Medicine & Research Nguyen et al., Mol. Immunol., 1999 Zou et al., J.Immunol., 2005

  14. Camel Nanotechnology and its medical Applications Structural properties of functional Heavy-chain antibodies in Camelidae & Nanobodies Natural generation of functional Heavy chain antibodies Selection of antigen-specific Nanobodies Nanobodies in medical applications

  15. Animalarium : CVRL, Dubaï, UAE HCAbs & Nbs in Medicine & Research

  16. Dromedary immunization Serum 1/8000 D2/54 carbonic anhydrase 1.0 OD 405 nm 1.0 0.8 0.8 Amylase Lysozyme RNase A Carbon. anhydrase 0.6 IgG1 IgG2a IgG2b IgG3 0.6 0.4 HCAb = 0.4 0.2 0.2 1 2 3 4 5 6 7 1 2 3 4 µg/ml WEEKS HCAbs & Nbs in Medicine & Research Lauwereys et al., EMBO J., 1998

  17. Selection of antigen-specific VHH Isolate lymphocytes Collect blood Extract mRNA RT-PCR Select Ag-specific VHHs by panning Immunize camel Produce soluble antigen-specific VHH Make library of ~ 107 transformants HCAbs & Nbs in Medicine & Research Ghahroudi et al., FEBS Letters, 1997 Lauwereys et al., EMBO J., 1998

  18. Nb properties versus scFv and Fab Nb >scFv = Fab Nb >scFv = Fab Nb >Fab > scFv Nb >Fab > scFv Nb = Fab = scFv Nb = Fab = scFv Nb >scFv = Fab Nb≠scFv = Fab • Efficient identification of Ag binders • Good expression yields • Good stability • Good solubility • Antigen specific • High affinity for the Ag • Easy tailoring • Nbs target unique epitopes HCAbs & Nbs in Medicine & Research

  19. Tailoring into pluripotent constructs • Bivalent: • Conrath et al., JBC 2001 • Bispecific: • Conrath et al., JBC 2001 • Pentavalent: • Zhang et al., JMB 2004 • Decavalent/bispecific: • Stone et al., J Imm Meth 2007 • Immuno-enzyme (ADEPT): • Cortez-Retamozo et al., Can Res 2004 • Immuno-toxin: • Baral et al., Nat Med 2006 • Chromobody: • Rothbauer et al., Nat Meth 2006 • HCAb: • Hmila et al., Mol Immunol 2008 • Scorpion (bispecific + Fc effector function) VH CH1 CL VL scFv CH2 Fc CH3 VHH Fab CH2 Fc CH3 Single domain Antibodies enzyme VH  toxin VNAR C1NAR VHH or Nanobody VNAR reporter C2NAR C3NAR Antibody selection strategies C4NAR C5NAR Fc

  20. Camel Nanotechnology and its medical Applications Structural properties of functional Heavy-chain antibodies in Camelidae & Nanobodies Natural generation of functional Heavy chain antibodies Selection of antigen-specific Nanobodies Nanobodies in medical applications

  21. Blood retention vs Ab size Residence time in blood Several weeks Several minutes Fab IgG scFv Size Nb diabody Renal cut off + trimers, tetramers, ... Secondary binding anti-HSA or Pegylation FcRn Engineering of CHO of IgG or mutagenesis of interacting AA (e.g. H310, H435) Pharmacokinetics and immunogenicity

  22. Tumor targeting • OBJECTIVE: • Search for maximal tumor load and fastest blood clearance • POSSIBILITIES: • scFv have minimal blood retention and consequently insufficient tumor load • IgG have much longer blood retention, reasonable tumor laod but insufficient tumor penetration • Minibody, scFv-Fc and HCAb are best performers (bivalecy & size) Pharmacokinetics and immunogenicity

  23. Fused micro PET/CT images • Mouse with tumor expressing EGFR • 2.5 mCi 99mTc-7C12 • 1h p.i. • CT+SPECT: 30 min Pros and cons of being camelid

  24. Application in therapy (ADEPT principle) Tumor cell  Prodrug CCM CYTOTOXICAGENT ( PDM ) ENZYME ( -LACTAMASE) Healthy cell Nb-CEA ADEPT= Ab dependent enzyme prodrug therapy  CEA HCAbs & Nbs in Medicine & Research Retamozo et al., Cancer Res., 2004

  25. Therapeutic effect of ADEPT cAb-Lys3::L + 150 mgCCM/kg cAb-CEA5:: L + 200 mg CCM/kg Control PDM (Toxine) Lys3:bL + 150mg CCM/kg 1800 CEA5:bL + 100mg CCM/kg 1500 CEA5:bL + 150mg CCM/kg CEA5:bL + 200mg CCM/kg 1200 tumor volume (mm³) 900 600 300 0 0 15 30 45 60 75 90 Treatment Days aftertumor implantation HCAbs & Nbs in Medicine & Research Retamozo et al., Cancer Res., 2004

  26. Nbs against African trypanosomes Mammalian host Pros and cons of being camelid

  27. Antigenic variation Rabbit Poly Ab Nb-An33 AnTat 1.1 MITat 1.4 NbAn33 Hypervariable immunodominant More conserved regions Pros and cons of being camelid

  28. Trypanolytic Nbs Targeted Non-targeted Control Pros and cons of being camelid Stijlemans et al., J.Biol.Chem., 2004 Baral et al., Nat Med., 2006

  29. Tech transfer & spin off • Foundation of Ablynx NV (December 2001) • 70 M € from venture capitalists (3 rounds) • Sofinnova, Alto Partners, Abingworth, SR-one • GIMV, KBC Private equity, • (Gilde, VIB, Privak Biotech) • November 2007: Introduction at EURONEXT • Research collaborations: • Proctor & Gamble (July 2004 & April 2006) • Genencor (2004), Centocor (2006) • Novartis (2006), Kirin (2006) • WYETH pharmaceuticals (212 M$, anti-TNF) • BOEHRINGER Ingelheim (265 M$, alzheimer) • Merck Serono Boehringer (XXXXXM€) • Achievements • Phase Ib finished for ALX081 (anti thrombotic, December 2008) • Phase I initiated for ALX681 (December 2008) • Phase I announced for anti-TNF (Wyeth) December2009 • Ablynx • 2002 = 5 man • 2003 = 10 man • 2004 = 20 man • 2005 = 40 man • 2006 = 70 man • 2007 = 90 man • 2008 = 190 man HCAbs & Nbs in Medicine & Research

  30. VIB-6 collaborations Loris Remy, Decanniere K. Magez Stefan, Stijlemans B., Toya BN. Revets Hilde (Ablynx), Cortez-Retamozo V., Huang L., De Groeve K., Kindt A. Camel group & partners Prof. L. Wyns (ULTR) Prof. P. De Baetselier (CIMM) Postdocs • Saerens Dirk (from mid 2005) • Ghloamreza Hassanzadeh (from 2006) • Conrath Katja (till mid 2006) • De Genst Erwin (till 2006) • Pardon Els (till mid 2006) PhD students • Nguyen Viet Khong (till 2001) • Pellis Mireille • Vincke Ceçile • Deschacht Nick • Nguyen Throng Scientists joining Ablynx • T. Laeremans, M. Lauwereys, K. Silence • H.Revets (2006) • Non-VIB collaborations • Ablynx • D. Altschuh (Strasbourg, QSAR) • K. Andersson (Uppsala, QSAR) • M. Brüggemann (Cambridge, camel-mouse) • C. Cambillau (CNRS,Marseilles) • C. Dobson (Oxford) & A. Matagne (Ulg, folding) • F. Frederix, …. (IMEC, biosensors) • R. Harrison (Liverpool, viper toxin) • M. El Ayeb & B. Bouhaouala (Institut Pasteur Tunis) • H. Leonhardt & U. Rothbauer (LMU, München) • M. Przybylski (Constanz, peptibodies) • M. Sara (Vienna, biosensors) Dubaï (Dr Wernery & Kinne) Antibody selection strategies

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