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A critical comparison of three internalization assays applied to the evaluation of a given mAb as a toxin-carrier
P Casalini1, M Caldera, S Canevari
1Department of Experimental Oncology E, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Abstract:
In the attempt to define a strategy for screening new monoclonal antibodies (mAb) that could be appropriate for clinical application in oncology, we evaluated the suitability of three methods: a direct internalization assay (DIA), an indirect internalization assay (IIA) and an indirect cytotoxicity assay (ICA), by applying them to already selected mAb. The latter were directed against three antigenic systems [38-kDa glycoprotein (gp38), epidermal growth factor receptor, and the neu oncogene product], which, according to their tumor selectivity, could be considered suitable for mAb-guided therapy. The dose-dependent and time-dependent binding, as well as the low intra-assay variability, demonstrated the reliability of the three tests. However, a certain degree of inter-assay variability was observed in each one, the highest value being that found when IIA was applied. Furthermore, the degree of variability, as well as the predictability, seemed to be more related to the mAb/antigen (Ag) combination used rather than to the test applied. From the overall data we suggest a procedure to be applied for screening purposes. As a first approach applied to the raw material, ICA is only suitable for screening in the case of an already selected toxin whereas IIA may be helpful to eliminate the true negative mAb. After purification of the relevant mAb a repeated analysis using DIA could allow the selection of true internalizing mAb. However, this second screening should be followed by a further analysis of the fate of the Ag-Ab complex after internalization.
Insights
This study evaluates three assays for screening monoclonal antibodies (mAbs) for oncology. A sequential approach using indirect cytotoxicity assay, indirect internalization assay, and direct internalization assay is proposed for selecting effective cancer therapies.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Developing monoclonal antibodies (mAbs) for clinical oncology requires robust screening methods.
- Targeting tumor-specific antigens is crucial for effective mAb-guided therapy.
- Existing screening assays may have limitations in predicting clinical efficacy.
Purpose of the Study:
- To evaluate the suitability of three assays for screening clinical-grade monoclonal antibodies (mAbs) in oncology.
- To define an optimized screening strategy for identifying internalizing mAbs against tumor-associated antigens.
Main Methods:
- Evaluation of three assays: Direct Internalization Assay (DIA), Indirect Internalization Assay (IIA), and Indirect Cytotoxicity Assay (ICA).
- Application of assays to selected mAbs targeting gp38, epidermal growth factor receptor, and neu oncogene product.
- Assessment of dose-dependent and time-dependent binding, intra-assay, and inter-assay variability.
Main Results:
- All three assays demonstrated reliable performance with low intra-assay variability.
- Inter-assay variability was observed, with IIA showing the highest degree.
- Variability and predictability were influenced more by the mAb/antigen combination than the assay itself.
Conclusions:
- A sequential screening strategy is proposed: ICA for initial toxin screening, IIA for eliminating non-internalizing mAbs, and DIA for selecting internalizing mAbs.
- Further analysis of the fate of the antigen-antibody complex post-internalization is recommended after DIA.
- The proposed strategy aims to improve the selection of clinically relevant monoclonal antibodies for cancer therapy.