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 Pulse 60’ Wash and Alexa 546-IgG (37oC) image (37oC) 19-27 hrs 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, Short in vivo half-life, good transport 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 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 Glu256 Ser254 Thr254 Met252 Tyr252 Asn434 Phe434 His433 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)

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, 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 WT IgG1 PBS 124-I pertuzumab 0h PET 4h Abdeg, WT IgG1 or PBS 8h PET 24h

Abdeg delivery improves contrast during PET

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