1. 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

2. 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

3. 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

4. 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, (1994)
Medesan et al., J. Immunol., 158, (1997)

5. How are IgGs sorted within cells?
Endothelial Cell

6. Endosomal sorting of IgGs
Wild type IgG1
H435A mutant:
does not bind
to FcRn
Transfection Pulse 60’ Wash and
Alexa 546-IgG (37oC) image (37oC)
19-27 hrs
Bars = 1 mm
Ober et al., J. Immunol., 172, (2004)

7. 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, Short in vivo half-life,
good transport poor transport

8. What happens to antigen in complex with antibody?
e.g. antibodies that target cytokines (not immune complexes)

9. Antibody ‘buffering’ of target antigen: the impact of pH dependent binding
engineering pH
pH ~ pH ~7.4
= FcRn
Lysosome
pH independent binding
pH dependent binding

10. Antigen ‘drop-off’ in endosomes by antibodies
with pH dependent binding to IL-6
High affinity at pH
antigen recycling (0222)
Antigen
= IL-6
Histidine
scan of
V regions
pH dependent binding
endosomal ‘drop-off’ (VH4)
Devanaboyina et al., mAbs (2013)

11. Engineering antibodies (‘Abdegs’) to inhibit FcRn
‘Abdeg’ = antibody that
enhances IgG degradation

12. Generation of an inhibitor of FcRn
Thr Glu256
Ser Thr254
Met Tyr252
Asn Phe434
His Lys433
‘MST-HN’
(human
IgG1-
derived)
MST-HN binds to FcRn with:
Increased affinity
Reduced pH dependence
(tight binding at pH 6.0 and 7.4)

13. Abdegs enhance IgG clearance in mice
Wild type (500 mg)
Abdeg (500 mg)
Abdeg (200 mg)
125I-labeled
hIgG1
Unlabeled wild
type hIgG1 or Abdeg
Time (hours)
% Injected dose
Abdeg used:
MST-HN mutant
Vaccaro et al., Nature Biotechnol., 23, (2005)

14. 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

15. Abdeg delivery results in increased
tumor:blood ratios
Swiercz et al., J. Nucl. Med., 55, (2014)

16. Abdeg delivery reduces background during PET
Abdeg WT IgG PBS
124-I
pertuzumab 0h
PET 4h
Abdeg,
WT IgG1
or PBS 8h
PET
24h

17. Abdeg delivery improves contrast during PET

18. Acknowledgements Raimund Ober UTSW (PET) Sripad Ram Ralph Mason
Cruz Martinez Srinivas Chiguru
Prashant Prabhat Xiankai Sun
Jerry Chao Saleh Ramezani
Siva Devanaboyina
Rafal Swiercz MedImmune
Dilip Challa Carl Webster
Amir Tahmasbi Changshou Gao
arGEN-x (Belgium) CIC, UTSW
Hans de Haard Victor Ghetie
Christophe Blanchetot
Funding sources:
NIH, CPRIT, National Multiple Sclerosis Society and
MedImmune