Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.     

Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies

Human V_H single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human V_H domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (V_HH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human V_H single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human V_H domains and the selection of binders against a diverse set of antigens. Unlike “camelized” human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the V_H/V_L interface. Structure determination in complex with hen egg white lysozyme revealed an extended V_H binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human V_H domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies.     

Peptide Derived from HIV-1 TAT Protein Destabilizes a Monolayer of Endothelial Cells in an in Vitro Model of the Blood-Brain Barrier and Allows Permeation of High Molecular Weight Proteins

Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3–0.6 μmol/ml of this C-TAT peptide, for a period of 1–2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.