Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers

Borek, Aleksandra; Sokołowska-Wędzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

doi:10.1371/journal.pone.0192194

Cited by 22 publications

(21 citation statements)

References 36 publications

(39 reference statements)

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“…The high selectivity of scFv-Fc proteins towards FGFR1 can be attributed to the very low homology of FGF receptors within residues 41–76 (13.9% identity, while sequence identity in the full extracellular region of FGF receptor is 36.7%) ( Figure S1 ). To confirm that immobilized fragments of FGF receptors were functional, we employed developed in our group scFvF7 that recognizes FGFR2 [ 19 ] as well as commercial anti-FGFR3 and anti-FGFR4 antibodies as controls ( Figure 2 and Figure S2 ).…”

Section: Resultsmentioning

confidence: 99%

High Affinity Promotes Internalization of Engineered Antibodies Targeting FGFR1

Opaliński

Szymczyk

Szczepara

et al. 2018

IJMS

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Fibroblast growth factor receptor 1 (FGFR1) is a plasma membrane protein that transmits signals from the extracellular environment, regulating cell homeostasis and function. Dysregulation of FGFR1 leads to the development of human cancers and noncancerous diseases. Numerous tumors overproduce FGFR1, making this receptor a perspective target for cancer therapies. Antibody-drug conjugates (ADCs) are highly potent and selective anticancer agents. ADCs are composed of antibodies (targeting factors) fused to highly cytotoxic drugs (warheads). The efficiency of ADC strategy largely depends on the internalization of cytotoxic conjugate into cancer cells. Here, we have studied an interplay between affinity of anti-FGFR1 antibodies and efficiency of their cellular uptake. We have developed a unique set of engineered anti-FGFR1 antibodies that bind the same epitope in the extracellular part of FGFR1, but with different affinities. We have demonstrated that these antibodies are effectively taken up by cancer cells in the FGFR1-dependent manner. Interestingly, we have found that efficiency, defined as rate and level of antibody internalization, largely depends on the affinity of engineered antibodies towards FGFR1, as high affinity antibody displays fastest internalization kinetics. Our data may facilitate design of therapeutically relevant targeting molecules for selective treatment of FGFR1 overproducing cancers.

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Section: Resultsmentioning

confidence: 99%

High Affinity Promotes Internalization of Engineered Antibodies Targeting FGFR1

Opaliński

Szymczyk

Szczepara

et al. 2018

IJMS

Self Cite

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“…Up to date, several ADCs have been approved and are commercially available for treatment of various cancers [18]. Importantly, a number of selective cytotoxic conjugates including ADCs against cancers overproducing FGFRs were generated,however, their in vivo therapeutic potential awaits further evaluation [20–27]. A critical step in the anti‐cancer therapy with ADCs is selective and efficient delivery of the cytotoxic drug to the cell interior [28].…”

Section: Introductionmentioning

confidence: 99%

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

et al. 2020

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Fibroblast growth factor receptor 1 (FGFR1) transmits signals through the plasma membrane regulating essential cellular processes like division, motility, metabolism, and death. Overexpression of FGFR1 is observed in numerous tumors and thus constitutes an attractive molecular target for selective cancer treatment. Targeted anti‐cancer therapies aim for the precise delivery of drugs into cancer cells, sparing the healthy ones and thus limiting unwanted side effects. One of the key steps in targeted drug delivery is receptor‐mediated endocytosis. Here, we show that the efficiency and the mechanism of FGFR1 internalization are governed by the spatial distribution of the receptor in the plasma membrane. Using engineered antibodies of different valency, we demonstrate that dimerization of FGFR1 with bivalent antibody triggers clathrin‐mediated endocytosis (CME) of the receptor. Clustering of FGFR1 into larger oligomers with tetravalent antibody stimulates fast and highly efficient uptake of the receptor that occurs via two distinct mechanisms: CME and dynamin‐dependent clathrin‐independent endocytic routes. Furthermore, we show that all endocytic pathways engaged in FGFR1 internalization do not require receptor activation. Our data provide novel insights into the mechanisms of intracellular trafficking of FGFR1 and constitute guidelines for development of highly internalizing antibody‐based drug carriers for targeted therapy of FGFR1‐overproducing cancers.

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“…primarily relies on the discovery of suitable tumor targets and the development of highly specific agents against these targets [2]. During the last decade the list of tumor antigens available for immunotherapy have gone from dozens to hundreds, allowing us to treat a broader spectrum of human malignancies [3].…”

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confidence: 99%

Novel monoclonal antibodies against thymidine kinase 1 and their potential use for the immunotargeting of lung, breast and colon cancer cells

et al. 2020

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Background: Thymidine kinase 1 (TK1) is a pyrimidine salvage pathway enzyme that is up-regulated in malignant tissues and elevated in the serum of cancer patients. While TK1 has been well established as a tumor biomarker, little has been done to explore its potential as a tumor target. Recently, we reported the membrane expression of TK1 on malignant cells, but not on normal cells. This study explores the possible use of monoclonal antibodies for the targeting of membrane associated TK1 in lung, breast, colon and prostate cancer cells. Methods: We generated and evaluated a panel of monoclonal antibodies against six different epitopes exposed in the tetrameric form of TK1. Antibodies were developed with hybridoma technology and validated with Western blot, siRNA TK1 knockdown, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The therapeutic potential of the antibodies was evaluated in vitro in antibody-dependent cell-mediated-cytotoxicity (ADCC) experiments. Results: Binding of the antibodies to TK1 was confirmed by Western blot in purified recombinant protein, cancer serum, and cell lysate. After a TK1 knockdown was performed, a reduction of TK1 expression was observed with five antibodies. Using indirect ELISA, we identified 3B2E11, 9C10, 7H2, 3B4, 8G2 among the most sensitive antibodies (LOD = 10.73-66.9 pg/ml). Surface expression of TK1 on the membrane of various cancer cell lines was analyzed with flow cytometry. Antibodies 8G2, 3B4, 7HD and 5F7G11 detected TK1 on the membrane of various cancer cell lines, including lung, prostate, colon and breast. No significant binding was detected on normal lymphocytes. Increased cytolysis of lung (~ 70%. p = 0.0001), breast (~ 70%, p = 0.0461) and colon (~ 50% p = 0.0216) cancer cells by effector cells was observed when anti-TK1 antibodies were added during ADCC experiments. Conclusions: The antibodies developed showed potential to be used to detect and target TK1 on the membrane of various tumor cells. The targeting of TK1 in malignant cells using monoclonal antibodies may be a feasible approach for the elimination of high TK1 expressing tumor cells.

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Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers

Cited by 22 publications

References 36 publications

High Affinity Promotes Internalization of Engineered Antibodies Targeting FGFR1

High Affinity Promotes Internalization of Engineered Antibodies Targeting FGFR1

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

Novel monoclonal antibodies against thymidine kinase 1 and their potential use for the immunotargeting of lung, breast and colon cancer cells

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