E. Sally WardTexas 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

Antibody engineering for diagnosis and therapy

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

Endothelial Cell

FcRn expression is ubiquitous e.g. endothelial, epithelial and APCs such as dendritic cells, B cells and monocytes/macrophages

FcRn: a global regulator of antibody levels and transport

His310

Ile253

His435, His436 (Tyr436 in humans)

Region of IgG that interacts with FcRn

Histidines mediate pH dependence of the interaction

The site is distinct from the ‘classical’ FcR 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

Wild type IgG1

H435A mutant:does not bind to FcRn

Endosomal sorting of IgGs

Bars = 1 mTransfection Pulse 60’ Wash and Alexa 546-IgG (37oC) image (37oC)

19-27 hrs

Ober et al., J. Immunol., 172, 2021-2029 (2004)

Endosomal sorting correlates with whole body behavior

Human IgG1 H435A mutant(does not bind to FcRn)

IgGFcRn-GFP

Long in vivo half-life, Short in vivo half-life, good transport poor transport

Played atacquisitionspeed

What happens to antigen in complex with antibody?

e.g. antibodies that target cytokines (not immune complexes)

pH independent binding pH dependent binding

Antibody ‘buffering’ of target antigen:

the impact of pH dependent binding

pH 6.0-7.4 pH ~6.0 pH ~7.4

Antibodyengineering

= FcRn

Lysosome

Antigen ‘drop-off’ in endosomes by antibodieswith pH dependent binding to IL-6

High affinity at pH 6.0-7.4 antigen recycling (0222)

pH dependent binding endosomal ‘drop-off’ (VH4)

Histidine scan ofV regions

Devanaboyina et al., mAbs (2013)

Antigen= IL-6

Engineering antibodies (‘Abdegs’) to inhibit FcRn

‘Abdeg’ = antibody that enhances IgG degradation

Generation of an inhibitor of FcRn

Thr256 Glu256

Ser254 Thr254Met252 Tyr252

Asn434 Phe434His433 Lys433

‘MST-HN’(humanIgG1-derived)

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

Wild type (500 g)

Abdeg (500 g)Abdeg (200 g)

125I-labeled hIgG1

Unlabeled wildtype hIgG1 or Abdeg

Time (hours)

% I

nje

cte

d d

ose

Vaccaro et al., Nature Biotechnol., 23, 1283-1288 (2005)

Abdeg used: MST-HN mutant

Inject 124-I or 125-I labeled pertuzumabinto tumor bearing mice

PET

Inject Abdeg (MST-HN), wild type IgG1 or PBS

PET or biodistribution

4 hours

4 hours

16 or 40 hours

Can Abdegs be used to improve contrast during PET?

Abdeg delivery results in increased tumor:blood ratios

Swiercz et al., J. Nucl. Med., 55, 1204-1207 (2014)

Abdeg delivery reduces backgroundduring PET

124-I pertuzumab 0h

4h

8h

24h

PET

PET

Abdeg,WT IgG1or PBS

Abdeg WT IgG1 PBS

Abdeg delivery improves contrast during PET

Acknowledgements

Raimund OberUTSW (PET)

Sripad Ram Ralph MasonCruz Martinez Srinivas ChiguruPrashant Prabhat Xiankai SunJerry Chao Saleh RamezaniSiva DevanaboyinaRafal Swiercz MedImmuneDilip Challa Carl WebsterAmir Tahmasbi Changshou Gao

arGEN-x (Belgium) CIC, UTSWHans de Haard Victor GhetieChristophe Blanchetot

Funding sources: NIH, CPRIT, National Multiple Sclerosis Society and MedImmune