The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein.
Fusion of small recombinant antibody fragments to an albumin-binding domain (ABD) from streptococcal protein G strongly extends their plasma half-life. This ABD binds with nanomolar affinity to human (HSA) and mouse serum albumin (MSA). It was speculated that an increase in albumin-binding affinity should lead to a further increase in half-life. In the present study, we analyzed the effects of affinity and valency of the ABD on the pharmacokinetic properties of a bispecific single-chain diabody (scDb), applied previously to investigate various half-life extension strategies. The scDb is directed against carcinoembryonic antigen (CEA) and CD3 capable of mediating T cell retargeting to tumor cells. Two scDb derivatives with increased (scDb-ABD-H) and decreased (scDb-ABD-L) affinity as well as an scDb molecule fused to two ABD (scDb-ABD(2)) were generated and produced in mammalian cells. The altered binding of these constructs to HSA and MSA was confirmed by ELISA and quartz crystal microbalance measurements. All constructs bound efficiently to CEA and CD3-positive cells and were able to activate T cells in a target cell-dependent manner, although T cell activation was reduced in the presence of serum albumin. All three derivatives showed a strongly increased half-life in mice as compared with scDb. Compared with the wild-type scDb-ABD, the half-life of scDb-ABD-H exhibited a prolonged half-life and scDb-ABD-L a reduced half-life, while the half-life scDb-ABD(2) was almost identical to that of scDb-ABD. However, these changes were only moderate, indicating that the half-life-extending property of the ABD in mice is only weakly influenced by affinity for serum albumin or valency of albumin binding.
Hopp
Jonas
J
Institut für Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, Stuttgart, Germany.
Hornig
Nora
N
Zettlitz
Kirstin A
KA
Schwarz
Aline
A
Fuss
Nadine
N
Müller
Dafne
D
Kontermann
Roland E
RE
D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000493:pharmacokinetics*; D017252:CD3 Complex / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000276:immunology; D002460:Cell Line; D006207:Half-Life; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology; D051379:Mice; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000493:pharmacokinetics*; D012709:Serum Albumin / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology
D018033:Antibodies, Bispecific; D017252:CD3 Complex; D002272:Carcinoembryonic Antigen; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin