Camel Nanotechnologies and its Medical Applications

0
0
1486 days ago, 586 views
PowerPoint PPT Presentation
Camel Nanotechnologies and its Restorative Applications. Prof. Dr. Serge Muyldermans. Review of the today's presentation. Auxiliary properties of utilitarian Substantial chain antibodies in Camelidae and Nanobodies Normal era of Overwhelming chain antibodies Choice of antigen-particular Nanobodies

Presentation Transcript

Slide 1

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

Slide 2

Overview of the today's introduction Structural properties of practical Heavy-chain antibodies in Camelidae & Nanobodies Natural era of Heavy-chain antibodies Selection of antigen-particular Nanobodies in restorative applications HCAbs & Nbs in Medicine & Research

Slide 3

Camel Nanotechnology and its therapeutic Applications Structural properties of useful Heavy-chain antibodies in Camelidae & Nanobodies Na tural era of useful Heavy chain antibodies Selection of antigen-particular Nanobodies in restorative applications

Slide 4

Pros & Cons of Antibodies are of restorative intrigue on the grounds that : Limitless assorted qualities High specificity High partiality Yet, a long way from being immaculate on the grounds that : Immunogenicity Unwanted effector work Solubility Stability Yield Size SOLUTION: Look in nature on the best way to make antibodies littler

Slide 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

Slide 6

Serum immunoglobulins 67 43 30 Serum from camelids IgG purging on Protein A/Protein G Protein G: 2.7 - 3.5 Protein A: + + + IgG1 IgG2 IgG3 H 2 L 2 = conv. Stomach muscle H 2 = HCAb H L HCAbs & Nbs in Medicine & Research Hamers et al ., Nature , 1993

Slide 7

Camelid antibodies CH1 VH VL CL scFv CH2 Fc CH3 VHH CH2 Fc CH3 Fv Fab Classical counter acting agent (IgG1) Single space antigen restricting section (15 kDa) NANOBODY Camel Heavy-Chain immune response (IgG2 & IgG3) Monomeric Prolate molecule: Diameter 2.4 nm Height 4 nm HCAbs & Nbs in Medicine & Research Hamers et al ., Nature , 1993

Slide 8

Camel Nanotechnology and its restorative Applications Structural properties of practical Heavy-chain antibodies in Camelidae & Nanobodies Natural era of useful Heavy chain antibodies Selection of antigen-particular Nanobodies in therapeutic applications

Slide 9

VH and VHH contrasts 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

Slide 10

IgH locus 5-6 practical JH qualities J-qualities V-qualities telomer D-qualities ca 50 useful VH qualities more than 1000 kb V-DJ reworking ca 25 useful DH qualities D-J modification centromer Sequential game plan of C-qualities more than 200 kb ATTTGCAT ACAAAAACC L-exon interpretation V-exon 5' 3' CDR2 CDR1 grafting TATA An A CACAGTG 23 bp spacer 3' 5' interpretation switch CH1 pivot CH2 CH3 M1 M2 H-chain HCAbs & Nbs in Medicine & Research

Slide 11

IgH locus of camelids 5-6 useful JH qualities J-qualities V-qualities telomere D-qualities ca 90 useful VH & VHH qualities (more than 1000 kb) ca XX utilitarian DH qualities centromere ATTTGCAT ACAAAAACC L-exon V-exon 5' 3' CDR2 CDR1 TATA CACAGTG 23 bp spacer h C g 3 C g 1 C g 2 C g 1 C m 1 C m 3 C m 4 C m 2 C a 2 C a 1 C a 3 h C g 3 C g 2 Sequential game plan of C-qualities (more than 200 kb) HCAbs & Nbs in Medicine & Research

Slide 12

V-D-J revision produces VHH RSS Ig-promotor Leader flag peptide ~ 50 V H qualities CDR1 CDR2 CDR3 IgG1 D-qualities 6 J H - qualities IgG2 IgG3 ~ 40 V H-qualities 7 subfamilies Position of Cys in CDR1 CDR2 length (VH3) HCAbs & Nbs in Medicine & Research Nguyen et al ., EMBO J ., 2000

Slide 13

HCAbs need CH1 area Dedicated H-chain quality for traditional Ab Poly-A Poly-An AGTGTGG GTAAGT VH CH1 pivot CH2 CH3 M1 M2 Dedicated H-chain quality for Heavy-chain Ab Poly-A Poly-A CGTGTGG A TAAGT VHH CH1 pivot CH2 CH3 M1 M2 HCAbs & Nbs in Medicine & Research Nguyen et al ., Mol. Immunol ., 1999 Zou et al., J.Immunol., 2005

Slide 14

Camel Nanotechnology and its restorative Applications Structural properties of utilitarian Heavy-chain antibodies in Camelidae & Nanobodies Natural era of practical Heavy chain antibodies Selection of antigen-particular Nanobodies in therapeutic applications

Slide 15

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

Slide 16

Dromedary vaccination Serum 1/8000 D2/54 carbonic anhydrase 1.0 OD 405 nm 1.0 0.8 Amylase Lysozyme RNase A Carbon. anhydrase 0.6 IgG1 IgG2a IgG2b IgG3 0.6 0.4 HCAb = 0.4 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

Slide 17

Selection of antigen-particular VHH Isolate lymphocytes Collect blood Extract mRNA RT-PCR Select Ag-particular VHHs by panning Immunize camel Produce dissolvable antigen-particular VHH Make library of ~ 10 7 transformants HCAbs & Nbs in Medicine & Research Ghahroudi et al ., FEBS Letters , 1997 Lauwereys et al ., EMBO J. , 1998

Slide 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 recognizable proof of Ag folios Good expression yields Good soundness Good solvency Antigen particular High partiality for the Ag Easy fitting Nbs target one of a kind epitopes HCAbs & Nbs in Medicine & Research

Slide 19

Tailoring into pluripotent develops 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-protein (ADEPT) : Cortez-Retamozo et al., Can Res 2004 Immuno-poison : Baral et al., Nat Med 2006 Chromobody : Rothbauer et al., Nat Meth 2006 HCAb : Hmila et al., Mol Immunol 2008 Scorpion (bispecific + Fc effector work) VH CH1 CL VL scFv CH2 Fc CH3 VHH Fab CH2 Fc CH3 Single space Antibodies compound VH  poison V NAR C1 NAR VHH or Nanobody V NAR journalist C2 NAR C3 NAR Antibody choice systems C4 NAR C5 NAR Fc

Slide 20

Camel Nanotechnology and its therapeutic Applications Structural properties of useful Heavy-chain antibodies in Camelidae & Nanobodies Natural era of utilitarian Heavy chain antibodies Selection of antigen-particular Nanobodies in restorative applications

Slide 21

Blood maintenance versus Ab measure Residence time in blood a long time Several minutes Fab IgG scFv Size Nb diabody Renal cut off + trimers, tetramers, ... Auxiliary restricting hostile to HSA or Pegylation FcRn Engineering of CHO of IgG or mutagenesis of associating AA (e.g. H310, H435) Pharmacokinetics and immunogenicity

Slide 22

Tumor focusing on OBJECTIVE : Search for maximal tumor stack and quickest blood leeway POSSIBILITIES : scFv have negligible blood maintenance and therefore inadequate tumor stack IgG have any longer blood maintenance, sensible tumor laod however lacking tumor infiltration Minibody, scFv-Fc and HCAb are best entertainers (bivalecy & estimate) Pharmacokinetics and immunogenicity

Slide 23

Fused miniaturized scale PET/CT pictures Mouse with tumor communicating EGFR 2.5 mCi 99m Tc-7C12 1h p.i. CT+SPECT: 30 min Pros and cons of being camelid

Slide 24

Application in treatment (ADEPT rule) Tumor cell  Prodrug CCM CYTOTOXIC AGENT ( PDM ) ENZYME (  - LACTAMASE) Healthy cell Nb-CEA ADEPT= Ab subordinate chemical prodrug treatment  CEA HCAbs & Nbs in Medicine & Research Retamozo et al ., Cancer Res ., 2004

Slide 25

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

Slide 26

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

Slide 27

Antigenic variety Rabbit Poly Ab Nb-An33 AnTat 1.1 MITat 1.4 NbAn33 Hyper variable immunodominant More saved locales Pros and cons of being camelid

Slide 28

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

Slide 29

Tech exchange & turn off Foundation of Ablynx NV (December 2001) 70 M € from investors (3 rounds) Sofinnova, Alto Partners, Abingworth, SR-one GIMV, KBC Private value, (Gilde, VIB, Privak Biotech) November 2007: Introduction at EURONEXT Research coordinated efforts: Proctor & Gamble (July 2004 & April 2006) Genencor (2004), Centocor (2006) Novartis (2006), Kirin (2006) WYETH pharmaceuticals (212 M$, hostile to TNF) BOEHRINGER Ingelheim (265 M$, alzheimer) Merck Serono Boehringer (XXXXXM€) Achievements Phase Ib completed for ALX081 (against thrombotic, December 2008) Phase I started for ALX681 (December 2008) Phase I reported for hostile to 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

Slide 30

VIB-6 joint efforts Loris Remy, Decanniere K. Magez Stefan, Stijlemans B., Toya BN. Revets Hilde (Ablynx), Cortez-Retamozo V., Huang L., De Groeve K., Kindt A. Camel aggregate & accomplices 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 understudies Nguyen Viet Khong (till 2001) Pellis Mireille Vincke Ceçile Deschacht Nick Nguyen Throng Scientists joining Ablynx T. Laeremans, M. Lauwereys, K. Hush H.Revets (2006) Non-VIB coordinated efforts Ablynx D. Altschuh (Strasbourg, QSAR) K. Andersson (

SPONSORS