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Antibodies
Volume 14
Issue 4
10.3390/antib14040084
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

by
Alessia Muzi
1,*,
Roberto Arriga
1,
Giovanni Bulfaro
2,
Francesca Fata
3,
Antonella Costanzo
2,3,
Valerio Chiarini
1,
Manuela Cappelletti
1,
Fabiana Fosca Ferrara
1,
Federica Bucci
1,
Linda Celeste Montemiglio
3,
Carmelinda Savino
3,
Emanuele Marra
1,
Gennaro Ciliberto
1,
Luigi Aurisicchio
1,
Beatrice Vallone
2 and
Giuseppe Roscilli
1,*
1
Takis srl, 00128 Rome, Italy
2
Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy
3
Institute of Molecular Biology and Pathology, located at the Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza University of Rome, National Research Council, P. le Aldo Moro, 5, 00185 Rome, Italy
*
Authors to whom correspondence should be addressed.
Antibodies 2025, 14(4), 84; https://doi.org/10.3390/antib14040084
Submission received: 3 July 2025 / Revised: 19 September 2025 / Accepted: 30 September 2025 / Published: 6 October 2025
(This article belongs to the Section Antibody-Based Therapeutics)
Versions Notes

Abstract

Background/Objectives: The ErbB protein family plays a critical role in the progression of various solid tumors, and HER3 has been implicated in resistance mechanisms to multiple cancer therapies due to its ability to form heterodimers with other ErbB receptors, thereby activating pathways that promote tumor growth and survival. This study aimed to generate and characterize humanized monoclonal antibodies against HER3 to inhibit its function and evaluate their potential as therapeutic agents. Methods: Murine monoclonal antibodies TK-A3 and TK-A4 were humanized and tested for binding to ErbB3 and competition with neuregulin-1β (NRG). Specificity was assessed by ELISA, and epitope identified by X-ray crystallography. Downstream signaling was analyzed by western blot for phosphorylated ErbB3, Akt, and MAPK. Antitumor activity was evaluated in vitro and in a pancreatic cancer xenograft model. A toxicology study was also conducted. Results: TK-hu A3 and TK-hu A4 bound specifically to ErbB3 without cross-reactivity to other ErbB receptors. The ErbB3-TK-hu A3 Fab structure revealed the binding epitope. Both antibodies competed with NRG, inhibiting ErbB3, Akt, and MAPK phosphorylation in a dose-dependent manner. They suppressed cancer cell survival in vitro, and TK-hu A3 significantly delayed tumor growth in vivo. The toxicology study indicated good tolerability. Conclusions: TK-hu A3 emerged as the lead candidate, showing specific HER3 targeting, strong pathway inhibition, and antitumor efficacy in vivo. Beyond standalone use, it could support novel strategies such as T-cell engagers, ADCs, CAR-T, and bispecific antibodies. These findings highlight TK-hu A3 as a promising therapy for HER3-positive, treatment-resistant cancers, meriting further development.
Keywords: humanized antibodies; tumor cell line; anti-tumor activity; HER3 receptor; X-ray crystallography humanized antibodies; tumor cell line; anti-tumor activity; HER3 receptor; X-ray crystallography

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MDPI and ACS Style

Muzi, A.; Arriga, R.; Bulfaro, G.; Fata, F.; Costanzo, A.; Chiarini, V.; Cappelletti, M.; Ferrara, F.F.; Bucci, F.; Montemiglio, L.C.; et al. Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity. Antibodies 2025, 14, 84. https://doi.org/10.3390/antib14040084

AMA Style

Muzi A, Arriga R, Bulfaro G, Fata F, Costanzo A, Chiarini V, Cappelletti M, Ferrara FF, Bucci F, Montemiglio LC, et al. Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity. Antibodies. 2025; 14(4):84. https://doi.org/10.3390/antib14040084

Chicago/Turabian Style

Muzi, Alessia, Roberto Arriga, Giovanni Bulfaro, Francesca Fata, Antonella Costanzo, Valerio Chiarini, Manuela Cappelletti, Fabiana Fosca Ferrara, Federica Bucci, Linda Celeste Montemiglio, and et al. 2025. "Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity" Antibodies 14, no. 4: 84. https://doi.org/10.3390/antib14040084

APA Style

Muzi, A., Arriga, R., Bulfaro, G., Fata, F., Costanzo, A., Chiarini, V., Cappelletti, M., Ferrara, F. F., Bucci, F., Montemiglio, L. C., Savino, C., Marra, E., Ciliberto, G., Aurisicchio, L., Vallone, B., & Roscilli, G. (2025). Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity. Antibodies, 14(4), 84. https://doi.org/10.3390/antib14040084

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