The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells

Abstract

Background Antibody drug conjugates (ADCs) represent one of the most promising approaches in the current immuno-oncology research. The precise delivery of cytotoxic drugs to the cancer cells using ADCs specific for tumor-associated antigens enables sparing the healthy cells and thereby reduces unwanted side effects. Overexpression of fibroblast growth factor receptor 1 (FGFR1) has been demonstrated in numerous tumors and thereby constitutes a convenient molecular target for selective cancer treatment. We have recently engineered tetravalent anti-FGFR1 antibody, T-Fc, and have demonstrated that it displays extremely efficient internalization into FGFR1 producing cells, a feature highly desirable in the ADC approach. We have revealed that T-Fc mediates clustering of FGFR1, largely enhancing the uptake of FGFR1-T-Fc complexes by induction of clathrin-independent endocytic routes. The aim of this study was to obtain highly internalizing cytotoxic conjugate of the T-Fc for specific delivery of drugs into FGFR1-positive cancer cells. Methods Conjugation of the T-Fc to a cytotoxic payload, vcMMAE, was carried out via maleimide chemistry, yielding the T-Fc-vcMMAE. The specific binding of the T-Fc-vcMMAE conjugate to FGFR1 was confirmed in vitro with BLI technique. Confocal microscopy and flow cytometry were applied to determine FGFR1-dependence of the T-Fc-vcMMAE internalization. Western blot analyses of FGFR1-dependent signaling were conducted to assess the impact of the T-Fc-vcMMAE on FGFR1 activation and initiation of downstream signaling cascades. Finally, using FGFR1-negative and FGFR1-possitive cell lines, the cytotoxic potential of the T-Fc-vcMMAE was evaluated. Results We have performed the efficient conjugation of the tetravalent engineered antibody with a cytotoxic drug and generated FGFR1-specific ADC molecule, T-Fc-vcMMAE. We have demonstrated that T-Fc-vcMMAE conjugate exhibits high selectivity and affinity for FGFR1, similarly to T-Fc. Furthermore, we have shown that T-Fc constitutes an effective drug delivery vehicle as T-Fc-vcMMAE was efficiently and selectively internalized by FGFR1-producing cells leading to their death. Interestingly, we show that the efficiency of the uptake of T-Fc-vcMMAE corresponds well with the cytotoxicity of the conjugate, but doesn’t correlate with the FGFR1expression level. Conclusion Our results show that T-Fc-vcMMAE fulfills the key criteria for the successful cytotoxic drug carrier in a targeted approach against FGFR1-positive cancer cells. Furthermore, our data implicate that not solely expression level of the receptor, but rather its cellular trafficking should be taken into account for selection of suitable molecular targets and cancer models for successful ADC approach.

Modular self-assembly system for development of oligomeric, highly internalizing and potent cytotoxic conjugates targeting fibroblast growth factor receptors

“…Human osteosarcoma cell line (U2OS) and human breast adenocarcinoma cell line (SKBR3) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). U2OS cells stably expressing FGFR1 (U2OS-R1) were obtained by transfection of U2OS cells with expression plasmid encoding FGFR1 [ 27 ]. Cells were cultured in 5% CO 2 atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (Biowest, Nuaille, France) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), antibiotics mix (100 U/mL penicillin and 100 μg/mL streptomycin) (Thermo Fisher Scientific), for U2OS-R1 additionally supplemented with 0.5 mg/mL geneticin (Thermo Fisher Scientific).…”

Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers

“…The human osteosarcoma cell line (U2OS) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and U2OS cells stably expressing FGFR1 (U2OS-R1) were obtained by transfection of U2OS cells with expression plasmids encoding FGFR1. 23 U2OS cells were cultivated in DMEM (Biowest, Nuaille, France) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). For U2OSR1 cells, growth media were additionally supplemented with geneticin (0.5 mg/mL) (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…NIH3T3 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). The human osteosarcoma cell line (U2OS) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and U2OS cells stably expressing FGFR1 (U2OS-R1) were obtained by transfection of U2OS cells with expression plasmids encoding FGFR1 . U2OS cells were cultivated in DMEM (Biowest, Nuaille, France) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin).…”

“…All these features make FGFR1 an attractive molecular target for selective anticancer therapies. To date, a few cytotoxic conjugates with antibody fragments or natural ligands as targeting molecules have been developed for the selective treatment of FGFR1-overproducing tumors. − However, novel FGFR1-targeting molecules are still urgently needed to improve the efficiency and selectivity of drug delivery and to enable simultaneous visualization of the conjugates during their action.…”

FGF1 Fusions with the Fc Fragment of IgG1 for the Assembly of GFPpolygons-Mediated Multivalent Complexes Recognizing FGFRs

“…The human osteosarcoma cell line (U2OS) was obtained from American Type Culture Collection (ATCC), and U2OS, stably expressing FGFR1 (U2OS-R1), were obtained as described previously [23]. Cells were cultured in 5% CO 2 atmosphere at 37 • C in Dulbecco's Modified Eagle's Medium (Biowest, Nuaille, France) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), antibiotics mix (100 U/mL penicillin and 100 µg/mL streptomycin) (Thermo Fisher Scientific), for U2OS-R1, additionally supplemented with 1 mg/mL geneticin (Thermo Fisher Scientific).…”