Improvement of pharmacokinetics of small recombinant bispecific antibody molecules Von der Fakultät Energie-, Verfahrens- und Biotechnik der Universität Stuttgart zur Erlangung der Würde eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte Abhandlung vorgelegt von Christoph Roland Stork aus Frankfurt am Main Hauptberichter: Prof. Dr. Roland E. Kontermann Mitberichter: Prof. Dr. Peter Scheurich Tag der mündlichen Prüfung: 31.08.2009 Institut für Zellbiologie und Immunologie 2009 Index Index Abbreviations ______________________________________________________________ 4 Summary__________________________________________________________________ 6 Zusammenfassung __________________________________________________________ 8 Introduction ______________________________________________________________ 10 Antibodies in cancer therapy ____________________________________________________ 10 Retargeting of effector cells by bispecific antibodies_________________________________ 12 Improvement of the circulation half-life of small recombinant antibodies _______________ 15 PEGylation ________________________________________________________________________ 17 N-glycosylation _____________________________________________________________________ 18 Exploitation of the Fc-mediated recycling by the neonatal Fc-receptor __________________________ 20 Exploitation of HSA recycling by the neonatal Fc-receptor ___________________________________ 21 Aim of this study ______________________________________________________________ 23 Manuscripts ______________________________________________________________ 27 Short summaries of the manuscripts______________________________________________ 27 Publication I _______________________________________________________________________ 27 Publication II _______________________________________________________________________ 28 Publication III ______________________________________________________________________ 29 A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G_________________________________________________________ 30 Abstract ___________________________________________________________________________ 31 Introduction ________________________________________________________________________ 31 Materials and Methods _______________________________________________________________ 33 Results ____________________________________________________________________________ 36 Discussion _________________________________________________________________________ 42 Acknowledgement___________________________________________________________________ 45 References _________________________________________________________________________ 45 N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single- chain diabodies _______________________________________________________________ 48 Abstract ___________________________________________________________________________ 48 Introduction ________________________________________________________________________ 49 I Index Materials & Methods_________________________________________________________________ 50 Results ____________________________________________________________________________ 55 Discussion _________________________________________________________________________ 62 Footnotes __________________________________________________________________________ 65 References _________________________________________________________________________ 65 Biodistribution of a bispecific single-chain diabody and its half-life extended derivatives __ 69 Abstract ___________________________________________________________________________ 69 Introduction ________________________________________________________________________ 70 Experimental Procedure ______________________________________________________________ 71 Results ____________________________________________________________________________ 74 Discussion _________________________________________________________________________ 80 Footnotes __________________________________________________________________________ 83 References _________________________________________________________________________ 84 Discussion & Perspectives ___________________________________________________ 86 Production of the modified scDb constructs and effects of the modifications on circulation half-life ______________________________________________________________________ 86 Impact of the modifications on the bioactivity of scDbCEACD3 _______________________ 89 Effects of the modifications on tumor accumulation of scDbCEACD3 __________________ 90 Perspectives __________________________________________________________________ 92 Conclusion ___________________________________________________________________ 94 References________________________________________________________________ 95 Acknowledgment _________________________________________________________ 109 Curriculum vitae__________________________________________________________ 110 II Abbreviations Abbreviations aa Amino acid(s) ABD Albumin-binding domain ADCC Antibody dependent cellular cytotoxicity AUC Area under the curve BiTE Bispecific T-cell engager CD Cluster of differentiation CDC Complement dependent cytotoxicity CDR Complementarity determining region CEA Carcinoembryonic antigen CTLA-4 Cytotoxic T-lymphocyte antigen 4 dAbs Human domain antibodies Db Diabody EC 50 Half-maximal effective concentration ECM Extracellular matrix EGF Epidermal growth factor EGFR Epidermal growth factor receptor ELISA Enzyme-linked immunosorbent assay Ep-CAM Epithelial cell adhesion molecule EPO Erythropoietin EPR Enhanced permeability and retention effect Fab Fragment antigen-binding Fc Fragment crystallizable FcRn Neonatal Fc-receptor FcRn hc Heavy chain of the neonatal Fc-receptor FDA US Food and Drug Administration Fv Variable fragment G-CSF Granulocyte colony-stimulating factor GM-CSF Granulocyte macrophage colony-stimulating factor HAMA Human anti-mouse antibody HAP Homo-amino-acid polymer HSA Human serum albumin IC 50 Half-maximal inhibitory concentration Ig Immunoglobulin IL Interleukin KD Dissociation constant KO Knockout mAb Monoclonal antibody MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry MHC Major histocompatibility complex 4 Abbreviations MSA Mouse serum albumin MW Molecular weight NHL Non-Hodgkin Lymphoma PBMC Peripheral blood mononuclear cells PEG Polyethylene glycol PEO Polyethylene oxide PSA Polysialic acid RSA Rat serum albumin scDb Single-chain Diabody scDb-ABD scDb fused to the ABD3 of streptococcal protein G scFv Single-chain variable fragment SEC Size exclusion chromatography TAA Tumor-associated antigen taFv Tandem scFv TNF Tumor necrosis factor TNF-R Tumor necrosis factor receptor VH Heavy chain of the variable domain VHH Single V-like domain of camelid single domain antibodies VL Light chain of the variable domain V-NAR Single V-like domain of shark single domain antibodies 5 Summary Summary The progress in protein engineering of the last two decades led to the development of a variety of small recombinant antibody formats for tumor therapy. These small formats show improved properties compared to the conventional monoclonal antibodies which are commonly IgGs. Small recombinant antibodies penetrate solid tumors more homogenously, cause less side effects for lacking the Fc-region and can be produced more economically in bacteria. However, they exhibit a shorter circulation half-life than IgGs. They are rapidly cleared by renal filtration because of their small size and their lack of the Fc-region, which mediates the recycling after cellular uptake by the neonatal Fc-receptor (FcRn). The short circulation half-life of small antibody formats reduces their anti-tumor efficacy and necessitates frequent infusions, leading to high costs and patient inconvenience. The present study aims at the improvement of pharmacokinetics of a bispecific single-chain diabody (scDb) directed against the tumor-associated antigen CEA and the T-cell receptor complex molecule CD3. Therefore, it is able to retarget cytotoxic T-cells to CEA + tumor cells. Three basically different strategies are compared in this study to improve the pharmacokinetics of scDbCEACD3: N-glycosylation, PEGylation and fusion to a bacterial albumin-binding domain (ABD). N-glycosylation and PEGylation of the scDb aim at an increased hydrodynamic radius and thereby reduced renal clearance. The fusion of the scDb to ABD has an additional effect: As well as IgG, albumin is recycled by the FcRn. Bound to albumin, scDb-ABD can exploit this mechanism and be recycled after cellular uptake. For the N-glycosylation three different derivatives with 3, 6 or 9 N-glycosylation sites (sequons) were engineered, resulting in a moderate enlargement of the scDb, which translated in a moderately prolonged serum half-life. In contrast, PEGylation of the scDb with a large, branched PEG 40 kDa led to a ~ 3-fold enlargement of the hydrodynamic radius, resulting in a drastically increase of the circulation time. The longest circulation time with a ~ 14-fold increased area under the curve (AUC) was observed for scDb-ABD. By comparing its half- life in FcRn heavy chain knockout and in wild-type mice, the contribution of the FcRn- mediated recycling on the prolonged serum half-live of scDb-ABD could be confirmed. Titration of scDb and its derivatives