A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.

• MeSH
• antigeny CD20 imunologie   MeSH
• chromatografie kapalinová MeSH
• imunoglobulin G chemie   MeSH
• kinetika MeSH
• lehké řetězce imunoglobulinů genetika   metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky chemie   genetika   MeSH
• nádorové buněčné linie MeSH
• peptidová knihovna MeSH
• psi MeSH
• rekombinantní fúzní proteiny MeSH
• sekvence aminokyselin MeSH
• tandemová hmotnostní spektrometrie MeSH
• těžké řetězce imunoglobulinů genetika   metabolismus   MeSH
• vazebná místa protilátek MeSH
• vodík/deuteriová výměna a hmotnostní spektrometrie * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

CSF-1R is a receptor mostly associated with the mononuclear phagocytic system. However, its expression within tumors has been linked with poor prognosis in both humans and dogs. Accordingly, several reports have demonstrated the beneficial effects of blocking CSF-1R in model systems of cancer. In this study, we generated a monoclonal antibody that could block CSF-1R in dogs as the first step to develop an anticancer drug for this species. Initially, an antibody was raised by the hybridoma methodology against the fragment responsible for receptor dimerization. mAb3.1, one of the resulting hybridoma clones, was able to bind macrophages in fixed tissues and was shown to inhibit cells of the mononuclear phagocytic line. Nevertheless, mAb 3.1 could not bind to some glycoforms of the receptor in its native form, while also demonstrating cross-reactivity with other proteins. To enhance binding properties of the mAb, five amino acids of the complementarity-determining region 2 of the variable heavy chain of mAb3.1 were mutated by PCR, and the variant scFv clones were screened by phage display. The selected scFv clones demonstrated improved binding to the native receptor as well as increased anti-macrophage activity. The resulting scFv antibody fragment presented here has the potential for use in cancer patients and in inflammatory diseases. Furthermore, this work provides insights into the use of such restricted mutations in antibody engineering.

• Publikační typ
• časopisecké články MeSH