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A blocking antibody against canine CSF-1R maturated by limited CDR mutagenesis
Breno C B Beirão1, Teresa P Raposo1, Louise M Imamura2
1The Royal (Dick) School of Veterinary Studies and Roslin Institute, The University of Edinburgh-Easter Bush, Midlothian, EH25 9RG, UK.
Abstract:
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.
Insights
Researchers engineered a novel antibody fragment to block Colony-Stimulating Factor 1 Receptor (CSF-1R) in dogs. This enhanced antibody shows improved binding and anti-macrophage activity, offering potential for canine cancer therapies.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Colony-Stimulating Factor 1 Receptor (CSF-1R) is linked to poor prognosis in canine and human cancers.
- Blocking CSF-1R has shown promise in preclinical cancer models.
- Developing targeted therapies for canine cancer requires species-specific reagents.
Purpose of the Study:
- To generate a monoclonal antibody (mAb) capable of blocking CSF-1R in dogs.
- To engineer enhanced antibody variants with improved binding and efficacy for potential therapeutic use.
Main Methods:
- Hybridoma technology was used to raise an initial antibody (mAb3.1) against a CSF-1R fragment.
- Mutagenesis of complementarity-determining regions and phage display screening were employed to engineer improved antibody variants.
- Binding assays and functional assays assessed the activity of engineered single-chain variable fragment (scFv) clones.
Main Results:
- The initial mAb3.1 showed anti-macrophage activity but had limitations in binding native receptor glycoforms and specificity.
- Engineered scFv clones demonstrated enhanced binding to native CSF-1R and increased anti-macrophage potency.
- The developed scFv antibody fragment exhibits potential for treating canine cancer and inflammatory diseases.
Conclusions:
- Engineered antibody fragments targeting CSF-1R can overcome limitations of initial monoclonal antibodies.
- This study presents a promising CSF-1R-blocking antibody fragment for veterinary oncology.
- The findings offer insights into targeted antibody engineering strategies for therapeutic applications.
