Transcript Engineering Antibodies for Diagnostics and Therapy

Antibody engineering for diagnosis and
therapy
E. Sally Ward
Texas A&M Health Science Center
Disclosures:
E.S.W. is a (co-)inventor on UT Southwestern-owned patents describing engineered
antibodies that are licensed to MedImmune and arGEN-x
Regulation of antibody (IgG) levels and distribution
 Fundamental aspect of humoral immunity
Regulation of antibody concentrations in the body
 Antibodies as therapeutic agents
Optimized delivery of antibodies; antigen clearance strategies
 Modulation of endogenous antibody levels
Treatment of antibody-mediated disease
Clearing background during diagnostic imaging
FcRn: a global regulator of antibody levels
and transport
Endothelial Cell
 FcRn expression is ubiquitous
e.g. endothelial, epithelial and APCs such
as dendritic cells, B cells and monocytes/
macrophages
Region of IgG that interacts with FcRn
His310
Ile253
His435, His436
(Tyr436 in
humans)
 Histidines mediate pH dependence of the interaction
 The site is distinct from the ‘classical’ FcgR and complement binding sites
Kim et al., Eur. J. Immunol., 24, 542-548 (1994)
Medesan et al., J. Immunol., 158, 2211-2217 (1997)
How are IgGs sorted within cells?
Endothelial Cell
Endosomal sorting of IgGs
Wild type IgG1
H435A mutant:
does not bind
to FcRn
Transfection 19-27 hrs Pulse 60’
Alexa 546-IgG (37oC)
Wash and
image (37oC)
Bars = 1 mm
Ober et al., J. Immunol., 172, 2021-2029 (2004)
Endosomal sorting correlates with
whole body behavior
IgG
FcRn-GFP
Played at
acquisition
speed
Human IgG1
H435A mutant
(does not bind to FcRn)
Long in vivo half-life,
good transport
Short in vivo half-life,
poor transport
What happens to antigen in complex with
antibody?
e.g. antibodies that target cytokines (not immune complexes)
Antibody ‘buffering’ of target antigen:
the impact of pH dependent binding
Antibody
engineering
pH 6.0-7.4
pH ~6.0
pH ~7.4
= FcRn
Lysosome
pH independent binding
pH dependent binding
Antigen ‘drop-off’ in endosomes by antibodies
with pH dependent binding to IL-6
High affinity at pH 6.0-7.4
antigen recycling (0222)
Antigen
= IL-6
Histidine
scan of
V regions
pH dependent binding
endosomal ‘drop-off’ (VH4)
Devanaboyina et al., mAbs (2013)
Engineering antibodies (‘Abdegs’) to inhibit FcRn
 ‘Abdeg’ = antibody that
enhances IgG degradation
Generation of an inhibitor of FcRn
Thr256
Ser254
Met252
Glu256
Thr254
Tyr252
Asn434
Phe434
His433
Lys433
‘MST-HN’
(human
IgG1derived)
MST-HN binds to FcRn with:
 Increased affinity
 Reduced pH dependence
(tight binding at pH 6.0 and 7.4)
Abdegs enhance IgG clearance in mice
125I-labeled
hIgG1
% Injected dose
Unlabeled wild
type hIgG1 or Abdeg
 Abdeg used:
MST-HN mutant
Wild type (500 mg)
Abdeg (200 mg)
Abdeg (500 mg)
Time (hours)
Vaccaro et al., Nature Biotechnol., 23, 1283-1288 (2005)
Can Abdegs be used to improve contrast
during PET?
Inject 124-I or 125-I labeled pertuzumab
into tumor bearing mice
4 hours
PET
4 hours
Inject Abdeg (MST-HN), wild type IgG1 or PBS
16 or 40 hours
PET or biodistribution
Abdeg delivery results in increased
tumor:blood ratios
Swiercz et al., J. Nucl. Med., 55, 1204-1207 (2014)
Abdeg delivery reduces background
during PET
Abdeg
124-I
pertuzumab
0h
PET
4h
Abdeg,
WT IgG1
or PBS
8h
PET
24h
WT IgG1
PBS
Abdeg delivery improves contrast during PET
Acknowledgements
Raimund Ober
Sripad Ram
Cruz Martinez
Prashant Prabhat
Jerry Chao
Siva Devanaboyina
Rafal Swiercz
Dilip Challa
Amir Tahmasbi
arGEN-x (Belgium)
Hans de Haard
Christophe Blanchetot
UTSW (PET)
Ralph Mason
Srinivas Chiguru
Xiankai Sun
Saleh Ramezani
MedImmune
Carl Webster
Changshou Gao
CIC, UTSW
Victor Ghetie
Funding sources:
NIH, CPRIT, National Multiple Sclerosis Society and
MedImmune