Improved Pharmacokinetics of Recombinant Bispecific Antibody Molecules by Fusion to Human Serum Albumin

Abstract

Recombinant bispecific antibodies such as tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb) have shown to be able to retarget T lymphocytes to tumor cells, leading to their destruction. However, therapeutic efficacy is hampered by a short serum half-life of these small molecules having molecule masses of 50 -60 kDa. Thus, improvement of the pharmacokinetic properties of small bispecific antibody formats is required to enhance efficacy in vivo. In this study, we generated several recombinant bispecific antibody-albumin fusion proteins and analyzed these molecules for biological activity and pharmacokinetic properties. Three recombinant antibody formats were produced by fusing two different scFv molecules, bispecific scDb or taFv molecules, respectively, to human serum albumin (HSA). These constructs (scFv 2 -HSA, scDb-HSA, taFv-HSA), directed against the tumor antigen carcinoembryonic antigen (CEA) and the T cell receptor complex molecule CD3, retained full binding capacity to both antigens compared with unfused scFv, scDb, and taFv molecules. Tumor antigen-specific retargeting and activation of T cells as monitored by interleukin-2 release was observed for scDb, scDb-HSA, taFv-HSA, and to a lesser extent for scFv 2 -HSA. T cell activation could be further enhanced by a target cell-specific costimulatory signal provided by a B7-DbCEA fusion protein.Furthermore, we could demonstrate that fusion to serum albumin strongly increases circulation time of recombinant bispecific antibodies. In addition, our comparative study indicates that single chain diabody-albumin fusion proteins seem to be the most promising format for further studying cytotoxic activities in vitro and in vivo.Bispecific antibodies are designed to target two different antigens simultaneously (1). In the context of a tumor therapy they can be applied to selectively recruit potent effector cells of the immune system such as cytotoxic T lymphocytes to tumor cells (2). This is achieved by binding on the one side to a tumorassociated antigen and on the other side to a trigger molecule on the effector cell, leading to the activation of the effector cell and tumor cell destruction. To reduce potential side effects elicited by the Fc part of antibodies (3) small recombinant bispecific antibody formats composed only of the variable regions, which define the binding unit of an antibody, have been developed (1, 4). These formats include bispecific diabodies (Db), 4 single chain diabodies (scDb), and tandem scFv (taFv) molecules, which have been applied successfully in vitro and also in vivo for the retargeting of cytotoxic T lymphocytes (via T-cell receptor molecule CD3) to tumor cells (e.g. recognizing CEA, EpCAM, or CD19) (5-8).However, these small bispecific antibody molecules with molecular masses between 50 and 60 kDa are rapidly cleared from circulation with an initial half-life of less than 30 min (9, 10). This puts some obstacles on therapeutic applications, e.g. requirement of high doses and repeated injections or infusion...

Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding

“…Recent reports have addressed the in vivo half-life of HSA fused or targeted molecules in mice (25,28) and argued that the increase in half-life observed is a consequence of FcRn-mediated rescue. Improved tumor imaging in rodents has been obtained using antitumor antigen antibody fragments genetically fused to HSA-or HSA-binding proteins (26,29,55 binds MSA with a K D of ϳ10 M. The affinity for the endogenous ligand is 10-fold higher than that for HSA, a fact that nicely correlates with the inhibition data where smFcRn WT was shown to prefer MSA over HSA.…”

An Antibody Fusion Protein for Cancer Immunotherapy Mimicking IL-15 trans-Presentation at the Tumor Site

“…Then the fusion proteins were added to the cells. After 3 (CTLL-2) or 4 (Mo7e) days, proliferation of the cells was measured by MTT assay (28). The cytokine activity of the fusion proteins presented in an antibody-mediated membrane-bound form was analyzed in the following setting: 10,000 cells per well (B16-FAP/B16) were seeded in a 96-well tissue culture plate.…”

“…Then previously starved CTLL-2 cells (20,000 cells per well) were added and incubated for 3 days. Finally, the cells in suspension (CTLL-2) were carefully transferred to a new 96-well plate, followed by the MTT assay (28).…”

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

“…Molecular weights range from »55 kDa to over 300 kDa, their valences for antigen binding are from two to six, and serum half-lives are reported to be between »0.5 hours and »2 weeks. 1,2,[5][6][7] Each format presents a particular strength in physicochemical properties, manufacturing, formulation, pharmacokinetics, or pharmacodynamics while being limited in the other evaluation criteria. 1 Sustained structural and functional homogeneity throughout manufacture, storage, and in vivo exposure is critical to their successful therapeutic application, but recognized in many bispecific formats as limiting factor.…”