A Human Immunoglobulin G1 Antibody Originating from an in Vitro-Selected Fab Phage Antibody Binds Avidly to Tumor-Associated MUC1 and Is Efficiently Internalized

Henderikx, Paula; Neer, Nicole Coolen-van; Jacobs, Anita; Linden, Edith van der; Arends, Jan–Willem; Mullberg, Jürgen; Hoogenboom, Hennie R.

doi:10.1016/s0002-9440(10)61107-8

Cited by 26 publications

(20 citation statements)

References 31 publications

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“…As shown for bivalent diabodies derived from scFvs with different affinities for binding to ErB2, the increment in binding affinity upon dimerisation was greatest for the lowest affinity scFv (65-fold) and least for the highest affinity scFv (7.7-fold) (Nielsen et al, 2000). In keeping with this observation, a phage display library-selected Fab fragment with low affinity for binding to MUC1 exhibited an even 160-fold improved apparent K d after being re-engineered into a bivalent fully human IgG molecule (Henderikx et al, 2002). Similarly, reformatting the mutant scFv 4.9M characterised in this study into a bivalent diabody resulted in a molecule with tight antigen binding (K d 8 nM; J Krauss, unpublished results).…”

Section: Discussionsupporting

confidence: 68%

Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme

et al. 2004

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Anti-MUC1 single-chain Fv (scFv) fragments generated from the humanised antibody huHMFG1 had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue V H -71, previously described as a key residue for maintaining the CDR-H2 main-chain conformation and thus important for antigen binding, markedly stabilised the scFv while having only a minor effect on the binding affinity of the molecule. Because of its improved stability, the engineered fragment exhibited immunoreactivity with tumour cells even after 7 days of incubation in human serum at 371C. It also showed, in contrast to the wild-type scFv, a concentration-dependent binding to the target antigen when displayed on phage. When fusing the scFv to the recombinant ribonuclease rapLRI, only the fusion protein generated with the stable mutant scFv was able to kill MUC1 þ tumour cells with an IC 50 of 80 nM. We expect this novel immunoenzyme to become a promising tool for the treatment of MUC1 þ malignancies.

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Section: Discussionsupporting

confidence: 68%

Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme

et al. 2004

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“…Numerous antibodies against MUC1, mostly of murine origin, have been developed in the past [10,11,14,16,44] . HMFG-1 and C595 are two antibodies that have been tested in clinical trials unfortunately with limited success.…”

Section: Discussionmentioning

confidence: 99%

“…Two thirds of them were isolated after immunization of mice with protein derived from noncancer-related sources. Attempts have also been made to raise and evolve antibodies of human origin against MUC1 [14][15][16][17][18] as they may be better options at least for the development of therapeutic agents. We have recently described the isolation of an antibody (12ESC-6) by selection on a disulfide-bond-constrained peptide [19] .…”

Section: Introductionmentioning

confidence: 99%

Molecular Evolution of Specific Human Antibody against MUC1 Mucin Results in Improved Recognition of the Antigen on Tumor Cells

Persson

Bäckström²,

Johansson

et al. 2009

Tumor Biol

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The MUC1 mucin is differentially expressed and glycosylated in cancer tissue as opposed to healthy tissue. Due to these differences, MUC1 is considered a potential biomarker suitable for cancer diagnosis and therapy. In a previous study, the human MUC1-specific antibody 12ESC-6 was able to bind a sequence variant of the tandem repeat of MUC1 that is not recognized by many other MUC1-specific antibodies. It was also found to bind efficiently to MUC1-carrying cells. We have now used 12ESC-6 as starting point for random mutagenesis to isolate variants with improved ability to bind MUC1 in human tumor tissue. The resulting 12ESC-6 variants were shown to recognize not only the naked MUC1 tandem repeat but even more so glycosylated variants thereof, in particular those carrying the GalNAc (Tn) glycoform. Selected variants of 12ESC-6 demonstrated improved staining of MUC1 on cell lines using flow cytometry and improved staining of the antigen in breast tumor tissue by immunohistochemistry. Molecular evolution and specific fine-tuning thus have the potential to improve the performance of antibody specificities targeting tumor-associated epitopes on MUC1 mucin.

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“…Biotinylated scFvs were incubated with 10 5 cells for 4 h at 4jC in PBS/0.25% bovine serum albumin. Bound scFvs were detected by streptavidin-phycoerythrin and analyzed by FACS as described previously (30,31). Data was curve fitted and K d values were calculated using GraphPad Prism (GraphPad Software).…”

Section: Methodsmentioning

confidence: 99%

Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies

An¹,

Drummond²,

Wilson³

et al. 2008

Molecular Cancer Therapeutics

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Mesothelioma is a malignancy of the mesothelium and current treatments are generally ineffective. One promising area of anticancer drug development is to explore tumor susceptibility to targeted therapy. To achieve efficient, targeted intracellular delivery of therapeutic agents to mesothelioma cells, we selected a naive human singlechain (scFv) phage antibody display library directly on the surface of live mesothelioma cells to identify internalizing antibodies that target mesothelioma-associated cell surface antigens. We have identified a panel of internalizing scFvs that bind to mesothelioma cell lines derived from both epithelioid (M28) and sarcomatous (VAMT-1) types of this disease. Most importantly, these antibodies stain mesothelioma cells in situ and therefore define a panel of clinically represented tumor antigens. We have further exploited the internalizing function of these scFvs to achieve targeted intracellular drug delivery to mesothelioma cells. We showed that scFv-targeted immunoliposomes were efficiently and specifically taken up by both epithelioid and sarcomatous mesothelioma cells, but not control cells, and immunoliposomes encapsulating the small-molecule drug topotecan caused targeted killing of both types of mesothelioma cells in vitro. [Mol Cancer Ther 2008;7(3):569 -78]

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A Human Immunoglobulin G1 Antibody Originating from an in Vitro-Selected Fab Phage Antibody Binds Avidly to Tumor-Associated MUC1 and Is Efficiently Internalized

Cited by 26 publications

References 31 publications

Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme

Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme

Molecular Evolution of Specific Human Antibody against MUC1 Mucin Results in Improved Recognition of the Antigen on Tumor Cells

Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies

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