Introducing antigen-binding sites in structural loops of immunoglobulin constant domains: Fc fragments with engineered HER2/neu-binding sites and antibody properties.

Yeast surface display libraries of human IgG1 Fc regions were prepared in which loop sequences at the C-terminal tip of the CH3 domain were randomized. A high percentage of these library members bound to soluble CD64 and Protein A indicating that the randomization step did not grossly interfere with the overall structure of the displayed Fc. Sorting these libraries by FACS for binders against HER2/neu yielded antigen-specific Fc binders (Fcab; Fc antigen binding) of which one was affinity matured, resulting in Fcab clone H10-03-6 which showed >10-fold improvement in antigen-binding activity versus the parental clone. Pre-equilibrium surface plasmon resonance experiments revealed a K(D) value of 69 nM. H10-03-6 did not react with other members of the HER family and specifically interacted with HER2-positive but not with HER2-negative cells. Importantly, Fcab H10-03-6 elicited potent antibody-dependent cellular cytotoxicity in vitro. Finally, the in vivo half-life in mice was similar to wild-type Fc indicating that the amino acid changes in the CH3 domain did not affect the pharmacokinetic behavior of the recombinant Fc. Our data demonstrate that the Fcab scaffold combines all features of normal antibodies in a small 50 kD homodimeric protein: antigen binding, effector functions and long half-life in vivo.

Effects of Hydergine on platelet deposition on "active" human carotid artery lesions and platelet function.

Effects of co-dergocrine mesylate (Hydergine), a drug widely used for the therapy of cerebral vascular disease on local platelet accumulation in the carotid artery region was studied by means of the platelet uptake ratio (PUR) and on the systemic platelet-vascular wall interaction as calculated from platelet half-life were investigated. A placebo controlled, double blind, randomised protocol was used, 18 patients were treated with co-dergocrine and compared to placebo (n = 18). Co-dergocrine treatment resulted in a significant decrease in platelet deposition, PUR decreased from 1.28 +/- 0.05 before treatment to 1.25 +/- 0.06 on day 5 of therapy with a statistically significant (p less than 0.001) in the paired comparison. In the control group the corresponding changes from 1.29 +/- 0.04 before to 1.28 +/- 0.04 did not show a p-value of less than 0.05 in paired comparison. Platelet half-life (72 +/- 11 before vs. 76 +/- 11 hours after 5 days of co-dergocrine treatment) showed a statistically significant (p less than 0.001) prolongation, whereas in the placebo group no relevant change of T/2 was observed (71 +/- 10 before vs. 72 +/- 10 hours on day 5, p greater than 0.10). No relevant effects on ADP-induced platelet aggregation, platelet-release reaction, platelet aggregate ratio, TXB2 plasma levels and thrombin-induced MDA-formation could be detected. These results indicate that co-dergocrine decreased in-vivo platelet residence time to atherosclerotic lesions of the carotid artery. Co-dergocrine may thereby be of benefit in prevention of mural thrombus formation and prevention of transient ischemic attacks, but also of atherosclerosis in man.