Recombinant humanized Fab fragments targeting the CFC domain of human Cripto-1
Annamaria Sandomenico1, Fabio Selis2, Jwala P Sivaccumar1
1Institute of Biostructures and Bioimaging, CNR, Via P. Castellino, 111, 80131, Napoli, Italy.
Abstract:
In the era of immunotherapy, the targeting of disease-specific biomarkers goes hand in hand with the development of highly selective antibody-based reagents having optimal pharmacological/toxicological profiles. One interesting and debated biomaker for several types of cancers is the onco-fetal protein Cripto-1 that is selectively expressed in many solid tumours and has been actively investigated as potential theranostic target. Starting from previously described anti-CFC/Cripto-1 murine monoclonal antibodies, we have moved forward to prepare the humanized recombinant Fabs which have been engineered so as to bear an MTGase site useful for a one-step site-specific labelling. The purified and bioconjugated molecules have been extensively characterized and tested on Cripto-1-positive cancer cells through in vitro binding assays. These recombinant Fab fragments recognize the target antigen in its native form on intact cells suggesting that they can be further developed as reagents for detecting Cripto-1 in theranostic settings.
Insights
Researchers developed humanized antibody fragments targeting the Cripto-1 onco-fetal protein. These engineered Fabs can be site-specifically labeled for potential use in cancer theranostics.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Cripto-1 is an onco-fetal protein and a debated biomarker for various cancers.
- Targeting cancer-specific biomarkers with selective reagents is crucial for immunotherapy.
- Cripto-1 is actively investigated as a potential theranostic target due to its selective expression in solid tumors.
Purpose of the Study:
- To engineer humanized recombinant antibody fragments (Fabs) targeting Cripto-1.
- To create Fabs with a site-specific labeling capability for theranostic applications.
- To characterize and validate the binding affinity of these Fabs to Cripto-1 on cancer cells.
Main Methods:
- Humanization of anti-Cripto-1 murine monoclonal antibodies to create recombinant Fabs.
- Engineering Fabs with an MTGase site for one-step, site-specific labeling.
- Purification, bioconjugation, and characterization of the engineered Fabs.
- In vitro binding assays using Cripto-1-positive cancer cells.
Main Results:
- Successfully prepared humanized recombinant Fabs targeting Cripto-1.
- Engineered Fabs possess a site for one-step, site-specific labeling.
- The Fabs demonstrated binding to the native Cripto-1 antigen on intact cancer cells.
- Extensive characterization confirmed the properties of the purified and bioconjugated molecules.
Conclusions:
- The developed recombinant Fab fragments recognize Cripto-1 in its native form on cancer cells.
- These engineered Fabs show promise as reagents for detecting Cripto-1 in theranostic settings.
- Further development of these Cripto-1-targeting Fabs could advance cancer theranostics.
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