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