Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.
An AB.Fab (albumin-binding Fab) consists of a Fab and a phage-derived albumin-binding peptide. This molecule is capable of binding both antigen and albumin simultaneously. Using a Fab derived from Herceptin we generated a panel of AB.Fab variants with wide-ranging affinities for albumin. An assay that measured AB.Fab binding to albumin in solution was developed to most accurately reflect the binding affinity for albumin in vivo. Affinity varied depending upon the species of albumin tested. For rat and rabbit albumin, affinities ranged from 0.04 to 2.5 microM. Reduced affinity for albumin correlated with a reduced half-life and higher clearance rates in both species; the beta half-life ranged 6-fold while clearance ranged over 50-fold in rats and 20-fold in rabbits. To estimate the pharmacokinetic properties of an AB.Fab in humans, AB.Fab variants with similar affinities for rat and rabbit albumin were selected. Using their pharmacokinetic parameters and the principles of allometric scaling for albumin, we estimate an approximate beta half-life for an AB.Fab with 0.5 microM affinity for albumin of up to 4 days in humans with a clearance of 76 ml/h. These variants demonstrate the ability to modulate the clearance of a Fab fragment in vivo and help to establish guidelines for pharmacokinetic engineering of molecules through albumin binding.
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