Abstract P5-05-08: Comparative evaluation of a trastuzumab biosimilar or originator trastuzumab in association with pertuzumab: Binding and biological activities in cell culture-based assays

Abstract

Background: trastuzumab (TZB) is approved for the treatment of HER2-positive (HER2+) breast cancer in different indications and schemes. The association of pertuzumab (PTZ) in some regimens has shown to improve the clinical benefit. Recently, biosimilars (BS) of trastuzumab were approved after extensive exercises of comparison with the originator, proving that these new biologicals are highly similar in structure and biological activity, and inherent microheterogeneity is not clinically relevant, since efficacy, safety and immunogenicity were found to be equivalent in pivotal phase 3 trials. In this study, we aimed to compare binding and biological activities of biosimilar trastuzumab (BS-TZB) or trastuzumab originator in association with PTZ in cell culture-based assays. Methods: two cancer cell lines were used to perform all the tests: BT474 (HER2+/ progesterone (PR) and estrogen receptor (ER)- positive) and HCC1954 (HER2+/PR and ER-negative); fluorescent labeling of trastuzumab originator (Herceptin®- Produtos Roche Químicos e Farmacêuticos S.A.) with Alexa 488, biosimilar of trastuzumab (trastuzumab-dkst, Biocon Limited/ Zedora- Libbs Farmacêutica Ltda.) with Alexa 488 and pertuzumab (Perjeta®- Produtos Roche Químicos e Farmacêuticos S.A.) with Alexa 647; FACS-based antigen binding evaluations and live cell imaging were performed (TZB or BS-TZB, and PTZ at concentrations of: 0; 10; 100; 1,000 ng/mL in every combinations. Predefined acceptance criteria for ligand-binding assay (LBA) was ±20% (25% at lower limit of quantification - LLOQ). Proliferation and CDC (complement-dependent cytotoxicity) assays: TZB or BS-TZB were tested at concentrations of: 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 100 μg/mL combined with PTZ at concentrations 1; 10; 100 μg/mL; the number of viable cells were estimated by quantitation of the ATP present using the CellTiter-Glo® kit. Equivalence was stablished with 95% confidence if 90% confidence interval (CI) of difference between means (DBM) lies within the zone of indifference (±20%). Results: tests were done using different concentrations and combinations of antibodies. For LBA, results are shown with concentrations in which the effect was neither minimal nor maximal (for both cell lines: 100 ng/mL for PTZ; 1,000 ng/mL for TZB or BS-TZB) (Table 1). For proliferation and CDC analysis, results at concentrations 100 μg/mL for PTZ, and TZB or BS-TZB (for both cell lines) are shown as a ratio to control. Table 1: Ligand-binding, proliferation and CDC assays. SD: standard deviation.Cell lineAnti-bodiesLBA (% cells) mean ± SDDBM 90% CIProliferation (ratio) mean ±SDDBM 90% CICDC (% cells) mean ± SDDBM 90% CIBT474BS-TZB + PTZ69.6 ±0.3-4.5 to -3.465.3 ±2.5-7.2 to -0.358.2 ±0.1-5.8 to -3.2TZB + PTZ73.5 ±0.369.6 ±1.262.8 ±1.1HCC1954BS-TZB + PTZ94.2 ±0.35.8 to 6.480.5 ±1.9-14.3 to -0.349.8 ±0.1-4.3 to -1.9TZB + PTZ88.1 ±0.187.8 ±5.452.9 ±1.0 Conclusions: trastuzumab and pertuzumab bind to HER2 receptor at different subdomains acting synergistically and, ultimately, improving the clinical benefit. In this study we comparatively evaluated the ligand and biological activities of the biosimilar BS-TZB and the TZB originator in the presence of PTZ. In addition to the previous findings of highly similar structure and biological activities of biosimilar to its originator, this study showed that not only PTZ binding to HER2 domain II but also biological activity showed the same pattern in the presence of BS-TZB as well as in the presence of TZB. Herein, in evaluated mechanisms, we have showed that the combination of BS-TZB and PTZ are highly similar to the combination of TZB and PTZ. Citation Format: Franklin F Pimentel, Juliano S Toledo, João Gonçalves, Leandro L Alves, Mayara I de Paula, Jurandyr M de Andrade, Daniel G Tiezzi, Roger Chammas. Comparative evaluation of a trastuzumab biosimilar or originator trastuzumab in association with pertuzumab: Binding and biological activities in cell culture-based assays [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-08.