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3D-Informed Targeting of the Trop-2 Signal-Activation Site Drives Selective Cancer Vulnerability
Emanuela Guerra1,2, Marco Trerotola1,2, Valeria Relli1
1Laboratory of Cancer Pathology, Center for Advanced Studies and Technology (CAST), "G. d'Annunzio" University of Chieti-Pescara, Chieti, Italy.
Abstract:
Next-generation Trop-2-targeted therapy against advanced cancers is hampered by expression of Trop-2 in normal tissues. We discovered that Trop-2 undergoes proteolytic activation by ADAM10 in cancer cells, leading to the exposure of a previously inaccessible protein groove flanked by two N-glycosylation sites. We designed a recognition strategy for this region, to drive selective cancer vulnerability in patients. Most undiscriminating anti-Trop-2 mAbs recognize a single immunodominant epitope. Hence, we removed it by deletion mutagenesis. Cancer-specific, glycosylation-prone mAbs were selected by ELISA, bio-layer interferometry, flow cytometry, confocal microscopy for differential binding to cleaved/activated, wild-type and glycosylation site-mutagenized Trop-2. The resulting 2G10 mAb family binds Trop-2-expressing cancer cells, but not Trop-2 on normal cells. We humanized 2G10 by state-of-the-art complementarity determining region grafting/re-modeling, yielding Hu2G10. This antibody binds cancer-specific, cleaved/activated Trop-2 with Kd < 10-12 mol/L, and uncleaved/wtTrop-2 in normal cells with Kd 3.16×10-8 mol/L, thus promising an unprecedented therapeutic index in patients. In vivo, Hu2G10 ablates growth of Trop-2-expressing breast, colon, prostate cancers, but shows no evidence of systemic toxicity, paving the way for a paradigm shift in Trop-2-targeted therapy.
Insights
Next-generation Trop-2 targeted therapies face challenges due to Trop-2 expression in normal tissues. A novel antibody, Hu2G10, selectively targets activated Trop-2 in cancer cells, offering a promising new therapeutic strategy.
Area of Science:
- Oncology
- Immunology
- Biochemistry
Background:
- Trop-2 targeted therapy for advanced cancers is limited by Trop-2 expression in normal tissues.
- Trop-2 is proteolytically activated by ADAM10 in cancer cells, exposing a unique binding groove.
Purpose of the Study:
- To develop a novel Trop-2-targeted therapy with enhanced cancer selectivity.
- To create antibodies that specifically recognize the activated form of Trop-2 found on cancer cells.
Main Methods:
- Deletion mutagenesis to remove the immunodominant epitope on Trop-2.
- Selection of cancer-specific, glycosylation-prone monoclonal antibodies (mAbs) using various binding assays.
- Humanization of the lead antibody (2G10) to generate Hu2G10.
- In vivo studies in mouse models of breast, colon, and prostate cancer.
Main Results:
- The 2G10 mAb family demonstrated selective binding to activated Trop-2 on cancer cells, not on normal cells.
- Hu2G10 exhibits high affinity for cleaved/activated Trop-2 (Kd < 10-12 mol/L) and low affinity for wild-type Trop-2 (Kd 3.16×10-8 mol/L).
- Hu2G10 effectively inhibited tumor growth in vivo without systemic toxicity.
Conclusions:
- Hu2G10 represents a novel therapeutic antibody with an unprecedented therapeutic index for Trop-2-targeted therapy.
- This approach offers a potential paradigm shift in treating Trop-2-expressing advanced cancers.
- Selective targeting of activated Trop-2 may overcome the limitations of current therapies.
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