Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells

Epidermal growth factor receptor-2 (EGFR-2) has been implicated in the pathogenesis of breast and other carcinomas. In this report, we tested the ability of a bacteriophage selected peptide KCCYSL, radiolabeled with 64 Cu, to image EGFR-2 expressing breast tumors in vivo by positron emission tomography (PET). We evaluated and compared the in vivo tissue distribution and imaging properties of 64 Cu-X-(Gly-Ser-Gly)-KCCYSL peptide (X ¼ 1,4,7,10, tetraazacyclododecane-N,N',N'',N'''-tetracetic acid, [DOTA] 1,4,8, hexadecane-4,11-diacetic acid [CB-TE2A], and 1,4,7-triazacyclononane-1,4,7-triacetic acid [NOTA] chelators) in a human breast cancer xenograft mouse model using dual modality PET and computed tomography (CT). The synthesized peptides DO3A-GSG-KCCYSL, CB-TE2A-GSG-KCCYSL, and NO2A-GSG-KCCYSL were purified, radiolabeled with 64 Cu, and evaluated for human breast cancer cell (MDA-MB-435) binding, 50% inhibitory concentration, and serum stability. In vivo pharmacokinetic and small animal PET/CT imaging studies were performed using SCID mice bearing MDA-MB-435 xenografts. The radiolabeled peptides bound with an 50% inhibitory concentration of 42.0 AE 10.2 nM (DO3A), 31 AE 7.9 nM (CB-TE2A), and 44.2 AE 6.6 nM (NO2A) to cultured MDA-MB-435 cells. All of the radiolabeled peptides were stable in vitro. The tumor uptake of DO3A, CB-TE2A, and NO2A peptides were 0.73 AE 0.15 percent injected dose per gram (%ID/g), 0.64 AE 0.08%ID/g, and 0.52 AE 0.04%ID/g, respectively at 1 hour. Liver uptake for the 64 Cu-DO3A-peptide (1.68 AE 0.42%ID/g) was more than that of the 64 Cu-CB-TE2A-peptide (0.52 AE 0.02% ID/g) and 64 Cu-NO2A-peptide (0.48 AE 0.05%ID/g) at 2 hours. PET/CT studies revealed successful tumor uptake of 64 Cu-peptides at 2 hours postinjection. In vivo kidney retention was observed with all of the radiolabeled peptides. The optimization of bifunctional chelators improves the pharmacokinetic properties of the 64 Cu-labeled GSG-KCCYSL peptide, which enables the selection of a suitable peptide homolog as a PET imaging agent for EGFR-2 expressing breast carcinomas.

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“…In the past few years, several groups have developed antibodies, scFV fragments, and peptides [7][8][9][10] that bind to EGFR-2.…”

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confidence: 99%

In VitroandIn VivoEvaluation of⁶⁴Cu-Radiolabeled KCCYSL Peptides for Targeting Epidermal Growth Factor Receptor-2 in Breast Carcinomas

Kumar

Gallazzi

Ferdani

et al. 2010

Cancer Biotherapy and Radiopharmaceuticals

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“…This antibody-mediated uptake has been successfully used to deliver plasmid DNA and antisense oligonucleotides. [13][14][15][16][17] Recently, Song et al 18 demonstrated that siRNA targeted delivered to human immunodeficiency virus-infected cells and HER2 positive cells by this strategy executed gene silencing. However, whether this promising strategy for siRNA delivery is applicable to other diseases remains unknown.…”

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confidence: 99%

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

et al. 2007

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RNA interference is highly effective at inhibiting HBV gene expression and replication. However, before small interfering RNA (siRNA) can be used in the clinic, it is essential to develop a system to target their delivery. Antibody-mediated delivery is a novel approach for targeting siRNA to appropriate cells. In this report, we asked whether this siRNA delivery strategy would be effective against HBV. Of 5 candidates, a specific siRNA that effectively inhibited HBV gene expression and replication was determined. Two fusion proteins, s-tP and sC-tP, were constructed to contain a single chain of the human variable fragment, scFv, against hepatitis B surface antigen (HBsAg), a truncated protamine (tP), and in the case of sC-tP, a constant region of the chain (C). S-tP and sC-tP were developed to provide targeted delivery of the siRNA, siRNA expressing cassettes (SEC), and siRNA-producing plasmids. Fluorescein isothiocyanate-siRNA, fluorescein isothiocyanate-SEC, and plasmid DNA were specifically delivered into HBsAg-positive cells using the sC-tP fusion protein, and effectively inhibited HBV gene expression and replication. HBV gene expression was also inhibited by siRNA or siRNA-producing plasmids in HBV transgenic mice. Conclusion: Our results describe a potential method for the targeted delivery of siRNA or siRNAproducing plasmids against HBV, using anti-HBsAg fusion proteins. (HEPATOLOGY 2007;46: 84-94.) R ibonucleic acid interference is a naturally occurring mechanism for silencing homologous genes. 1,2 Synthetic and plasmid-based siRNA expression systems are both effective at suppressing HBV infection, in vitro and in vivo, [3][4][5][6][7][8][9] providing the basis for a new therapeutic strategy against HBV. However, the absence of a suitable delivery system presents a major obstacle against their clinical use. [10][11][12] Targeted delivery of small interfering RNA (siRNA) to relevant cell populations should increase the therapeutic index and minimize potential side effects and cellular toxicity.Antibody-mediated delivery is an effective method of targeting siRNA to particular cells. This involves fusing the nucleic acid-binding domain to a Fab fragment or scFv that recognizes a membrane receptor, resulting in a fusion protein that possesses cell recognition and nucleic acid-binding abilities. The fusion protein can then bind nucleic acids and deliver them into target cells through receptor-mediated endocytosis. This antibody-mediated uptake has been successfully used to deliver plasmid DNA and antisense oligonucleotides. [13][14][15][16][17] Song et al. 18 demonstrated that siRNA targeted delivered to human immunodeficiency virus-infected cells and HER2 positive cells by this strategy executed gene silencing. However, whether this promising strategy for siRNA delivery is applicable to other diseases remains unknown. 19 In this report, we provide evidence that antibody-mediated siRNA delivery is effectively targeted to HBV-infected cells.In a previous study, we have obtained 5 human antihepatitis B sur...

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“…1). In contrast, the previous study reported the selective delivery of ML39 ScFv complexed with DNAs into HER2/ neu-positive cells (Li et al, 2001). It seems likely that the association of cells with ML39 ScFv-presenting bacteria is not sufficient to trigger the receptor-mediated entry mechanism.…”

Section: Discussionmentioning

confidence: 76%

Engineering of Escherichia coli for targeted delivery of transgenes to HER2/neu‐positive tumor cells

Chang

Cheng

Chen

et al. 2011

Biotech & Bioengineering

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Targeting of non-phagocytic tumor cells and prompt release of gene cargos upon entry into tumors are two limiting steps in the bacterial gene delivery path. To tackle these problems, the non-pathogenic Escherichia coli strain BL21(DE3) was engineered to display the anti-HER2/neu affibody on the surface. After co-incubation with tumor cells for 3 h, the anti-HER2/neu affibody-presenting E. coli strain was selectively internalized into HER2/neu-positive SKBR-3 cells. The invasion efficiency reached as high as 30%. Furthermore, the bacteria were equipped with the phage ϕX174 lysin gene E-mediated autolysis system. Carrying the transgene (e.g., eukaryotic green fluorescent protein, GFP), the tumor-targeting bacteria were subjected to the thermal shock to trigger the autolysis system upon entry into HER2/neu-positive cells. Flow cytometric analysis revealed that 3% of infected cells expressed GFP 24 h post thermal induction. Overall, the results show a promise of the proposed approach for developing bacteria as a delivery carrier.

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Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells

Cited by 68 publications

References 38 publications

In VitroandIn VivoEvaluation of⁶⁴Cu-Radiolabeled KCCYSL Peptides for Targeting Epidermal Growth Factor Receptor-2 in Breast Carcinomas

In VitroandIn VivoEvaluation of⁶⁴Cu-Radiolabeled KCCYSL Peptides for Targeting Epidermal Growth Factor Receptor-2 in Breast Carcinomas

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

Engineering of Escherichia coli for targeted delivery of transgenes to HER2/neu‐positive tumor cells

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Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells

Cited by 68 publications

References 38 publications

In VitroandIn VivoEvaluation of64Cu-Radiolabeled KCCYSL Peptides for Targeting Epidermal Growth Factor Receptor-2 in Breast Carcinomas

In VitroandIn VivoEvaluation of64Cu-Radiolabeled KCCYSL Peptides for Targeting Epidermal Growth Factor Receptor-2 in Breast Carcinomas

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

Engineering of Escherichia coli for targeted delivery of transgenes to HER2/neu‐positive tumor cells

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Product

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In VitroandIn VivoEvaluation of⁶⁴Cu-Radiolabeled KCCYSL Peptides for Targeting Epidermal Growth Factor Receptor-2 in Breast Carcinomas

In VitroandIn VivoEvaluation of⁶⁴Cu-Radiolabeled KCCYSL Peptides for Targeting Epidermal Growth Factor Receptor-2 in Breast Carcinomas