Anti-Metastatic and Anti-Invasion Effects of a Specific Anti-MUC18 scFv Antibody on Breast Cancer Cells (original) (raw)
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Selection and characterization of anti-MUC-1 scFvs intended for targeted therapy
Clinical cancer research : an official journal of the American Association for Cancer Research, 2003
The selection and characterization of anti-MUC-1 single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer molecules designed for optimal blood clearance and tumor penetration. The mucin MUC-1 was chosen as an antigen because it is abundantly expressed on epithelial cancers in an aberrantly glycosylated form, making it structurally and antigenically distinct from MUC-1 expressed on normal cells. A previously constructed anti-MUC-1 phage display library from hyperimmunized mice, with 5 x 10(5) calculated variants, was screened for the selection of anti-MUC-1 scFvs. Selection criteria were high binding to a MUC-1 peptide containing 4 tandem repeats of 20 amino acids and to MUC-1-positive MCF-7 (human breast cancer) cell lysates in ELISA. Six anti-MUC-1 scFv clones were selected and characterized. Nucleotide sequencing showed that four of them were full length scFv genes (variable heavy chain + variable light chain), whereas the remaining two cont...
International Journal of Cancer, 1999
The products of the MUC1 gene are known to be highly expressed in human breast cancer cells. The best characterized MUC1 protein is a polymorphic, type 1 transmembrane molecule containing a large extracellular domain composed primarily of a variable number of 20 amino acid tandem repeats. We have recently identified a novel protein product of the MUC1 gene, the MUC1/Y protein, that is also a transmembrane protein but is devoid of the tandem repeat array and its immediate flanking sequences. To analyze its expression in tumor cells we generated monoclonal antibodies directed against the MUC1/Y extracellular domain (anti-MUC1/Yex MAbs). Epitope mapping identified the MAb, 6E6, which recognized the MUC1/Y isoform with exquisite specificity-the repeat-array-containing MUC1 isoform could not compete out this immunoreactivity. A 30mer peptide which is unique for MUC1/Y and corresponds to the ''join'' region generated by the MUC1/Y specific splice, abrogated all 6E6
International Journal of Molecular Sciences
Mucin1 (MUC1) is aberrantly glycosylated and overexpressed in various cancers, and it plays a crucial role in cancerogenesis. MUC1 is a type I membranous protein composed of α and β subunits. MUC1-α can be cleaved in cancers, exposing MUC1-β (MUC1-C). MUC1-C is involved with multiple cancer cellular functions, which makes it an attractive target for cancer treatment. However, its multifunctional mechanisms have not been fully elucidated and there has not been a successful therapeutic development against MUC1-C. Through a phage display process, we isolated the specific antibodies for the extracellular domain of MUC1-C. The relevant full IgG antibodies were produced successfully from mammalian cells and validated for their MUC1-C specificities through ELISA, dual FACS analysis, BLI assay, and confocal image analysis. In the comparison with reference antibody, elected antibodies showed characteristic bindings on target antigens. In the functionality assessment of high-ranking antibodie...
Development of a novel bi-specific monoclonal antibody approach for tumour targeting
1999
To overcome the disadvantages of bi-specific antibody methodologies in vivo, a novel antibody approach has been designed to improve tumour targeting and effector to target ratio. The technique involves biotinylated anti-CD3 Fab fragments and streptavidinylated anti-tumour monoclonal antibodies (mAbs) that can spontaneously form cross-links. We describe here a method for the direct cross-linking of sulphydryl-conjugated HMFG1 (anti-MUC1 mucin mAb) to streptavidin by sulphosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate. Fab fragments generated by papain digestion of the 1452C11 antibody (anti-CD3 mAb without Fc to avoid peripheral activation of T-cells) were biotinylated with NHS-Iminobiotin. MUC1-transfected BALB/c breast cancer cell lines 413BCR and 425CCR and the parental cell line (410.4) were labelled with streptavidinylated mouse anti-MUC1 mucin mAb. BALB/c effector T-cells were separately labelled with biotinylated anti-CD3 Fab fragments (1452C11) and mixed with tumour cells in different effector to target ratios. Percentage of killing was assessed using the 51 Cr cytotoxicity assay. Seventy per cent lysis was measured in the case of 413BCR (high MUC1 mucin expressor) and 40% in the case of 425CCR (low expressor) cell line. No lysis was apparent in the MUC1 negative cell line. These results demonstrate that the novel T-cell redirecting approach we have developed can produce effective immune lysis of target cells in vitro. T Cell CD3 MUC1 Tumour cell Key : Biotinylated anti-CD3 Fab fragment Streptavidinylated HMFG1 Figure 1 The strategy of the novel antibody approach for the redirection of T-cells against tumour cells. MUC1 mucin-expressing tumour cells labelled with streptavidinylated anti-MUC1 MAb (HMFG1 SA) cross-linking biotinylated anti-CD3 Fab fragments (Fab Bio) bound on ConA/IL-2 preactivated T-cells by biotin-streptavidin bond 436 AA Koumarianou et al
The usage of monoclonal antibodies (mAbs), as a matter associated with the biopharmaceutical industry, is increasingly growing. Harmonious with this concept, we designed the exquisitely modeled anti-MET scFv against breast cancer by gene cloning, and expression using a bacterial host. Herein, we developed a recombinant scFv against MET and examined its preclinical efficacy for the reduction of tumor growth, invasiveness and angiogenesis in vitro and in vivo. Expressed anti-MET scFv demonstrated high binding capacity (48.8%) toward MET-overexpressing cancer cells. The IC50 value of anti-MET scFv against MET-positive human breast cancer cell line (MDA-MB-435) was 11.4 nM whereas this value was measured as 47.01 nM in MET-negative cell line BT-483. Similar concentrations could also effectively induce apoptosis in MDA-MB-435 cancer cells. Moreover, this antibody fragment could reduce migration and invasion in MDA-MB-435 cells. Grafted breast tumors in Balb/c mice showed significant tumo...
Cancer Immunology, Immunotherapy, 2010
Screening a phage-display single-chain antibody library for binding to the breast cancer cell line PM-1 an antibody, scFv173, recognising activated leukocyte cell adhesion molecule (ALCAM, CD166) was isolated and its binding profile was characterized. Positive ALCAM immunohistochemical staining of frozen human tumour sections was observed. No ALCAM staining was observed in the majority of tested normal human tissues (nine of ten). Flow cytometry analyses revealed binding to 22 of 26 cancer cell lines of various origins and no binding to normal blood and bone marrow cells. Antibody binding inhibited invasion of the breast cancer cell line MDA-MB-231 by 50% in an in vitro Matrigel-coated membrane invasion assay. Reduced growth of tumours in nude mice was observed in an in vivo model in which the mice were injected subcutaneously with colorectal carcinoma HCT 116 cells and treated with scFv173 when compared to control. In summary, we have characterized a novel fully human scFv antibody recognising ALCAM on cancer cells and in tumour tissues that reduces cancer cell invasion and tumour growth in accordance with the hypothesised role for ALCAM in cell growth and migration control. Keywords Single-chain antibody Á CD166 Á ALCAM Á Matrigel invasion Á In vivo tumour growth Electronic supplementary material The online version of this article (
In vivo anti-MUC1+ tumor activity and sequences of high-affinity anti-MUC1-SEA antibodies
Cancer Immunology, Immunotherapy, 2020
Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha-beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1 + malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1 + Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.
International Journal of Cancer, 1999
Multivalency is a recognized means of increasing the functional affinity of single-chain Fvs (scFvs) for optimizing tumor uptake. A unique divalent single-chain Fv protein [sc(Fv) 2 ], based on the variable regions of the monoclonal antibody (MAb) CC49, has been generated that differs from other dimeric single-chain constructs in that a linker sequence (L) is encoded between the repeated V L and V H domains (V L -L-V H -L-V L -L-V H ). This construct was expressed in soluble form in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Purity and immunoreactivity were determined by SDS-PAGE, HPLC and competitive RIA. sc(Fv) 2 exhibited a relative K A (3.34 ؋ 10 7 M ؊1 ) similar to that of the native IgG (1.14 ؋ 10 8 M ؊1 ) as determined by BIAcore analysis. Pharmacokinetic studies showed rapid blood clearance for sc(Fv) 2 , with a T 1/2 less than 40 min. Whole-body clearance analysis also revealed rapid clearance, suggesting no significant retention in the extravascular space or normal tissues. Biodistribution studies of radiolabeled sc(Fv) 2 showed tumor uptake greater than 6% ID/g after 30 min, which remained at this level for 6 hr. High tumor uptake and retention of sc(Fv) 2 coupled with rapid blood and whole-body clearance makes this dimeric scFv of MAb CC49 a strong candidate for imaging and therapeutic applications. Int. J. Cancer 81:911-917, 1999.
Human Antibodies, 2012
With PR81 as a murine monoclonal antibody (mAb) that was prepared against the human breast cancer, the MUC1 receptor specific targeting is possible. In this study, PR81-conjugated bovine serum albumin (BSA) nanoparticles loaded with anticancer drug 5-fluorouracil (5-FU) were developed. Enzyme linked immunosorbant assay (ELISA) results showed high immunoreactivity of PR81 mAb conjugated to nanoparticles towards MUC1 related peptide or native cancerous MUC1 and almost no cross-reaction to non-specific proteins. In vitro experiments were performed to determine the ability of this new drug delivery system on overcoming MCF-7 breast cancer cells in comparison with four other systems. The results revealed that these cell-type specific drug loaded nanoparticles could achieve more cell death as compared to when the 5-FU was used with no carriers. Stability studies of produced drug delivery system proved high immunoreactivity of conjugated PR81 even after 11 days of storage in room temperature.