HPMA copolymer–anticancer drug–OV-TL16 antibody conjugates. II. Processing in epithelial ovarian carcinoma cells in vitro

Omelyanenko, V.; Gentry, Christine; Kopečková, Pavla; Kopeček, Jindřich

doi:10.1002/(sici)1097-0215(19980209)75:4<600::aid-ijc18>3.0.co;2-c

Cited by 86 publications

(72 citation statements)

References 13 publications

(25 reference statements)

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“…In vitro, free DOX accumulates in the nucleus (39,44) as does DOX bound to HPMA copolymers via lysosomally degradable spacers (45). DOX bound to PEG via an acid-sensitive linker was found in the cytoplasm (44).…”

Section: Discussionmentioning

confidence: 99%

The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in hep G2 cells

et al. 2001

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The development of macromolecules as drugs and drug carriers requires knowledge of their fate in cells. To this end, we studied the internalization and subcellular fate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers in Hep G2 (human hepatocellular carcinoma) cells. Semiquantitative fluorometry confirmed that galactose was an effective ligand for receptormediated endocytosis for Hep G2 cells. The rate of internalization of a galactose-targeted copolymer was almost 2 orders of magnitude larger than that of the nontargeted copolymer. Confocal fluorescent microscopy of both fixed and live cells revealed that the polymer entered the cells by endocytosis. After longer incubation times (typically >8 hours), polymer escaped from small vesicles and distributed throughout the cytoplasm and nuclei of the cells. Polymer that entered the cytoplasm tended to accumulate in the nucleus. Microinjection of the HPMA copolymers into cells' cytoplasm and nuclei indicated that the polymers partitioned to the nucleus. The data from fixed cells was confirmed by microscopy of live, viable cells. To examine the effect of the fluorescent dye on the intracellular fate, polymers with fluorescein, Oregon Green 488, Lissamine rhodamine B, and doxorubicin were tested; no significant differences were observed.

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Section: Discussionmentioning

confidence: 99%

The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in hep G2 cells

et al. 2001

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“…The attachment of polylysine to a conjugate containing mAb OC125 directed against ovarian cancer cells enhances PS uptake up to 17-fold, 70 and similarly, internalization of meso-chlorin e6 mono(N-2-aminoethylamide) by OVCAR-3 ovarian carcinoma cells could be achieved by coupling it to MAb OV-TL16 using N-(2-hydroxypropyl)methacrylamide. 71 Furthermore, it has recently been shown that m-tetrahydroxyphenylchlorin attached to internalizable mAb 425 specific for head and neck squamous cell carcinoma is more effective than a conjugate with a non-internalizable antibody (U36). 72 There certainly appears to be much promise for enhancing PDT in using tumour cell-specific internalizable antibodies.…”

Section: Systems For Photosensitizer Delivery Into Cellsmentioning

confidence: 99%

Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency

Jans²,

2000

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IntroductionPhotodynamic therapy (PDT) is a relatively new cytotoxic treatment, predominantly used in anticancer approaches, that depends on the retention of photosensitizers (PS) in tumour cells and their activation within the tumour through irradiation with light of the appropriate wavelength. Photoactivated PS generate reactive oxygen species (singlet oxygen, 1 O 2 , and free radicals, such as ·OH, HO· 2 and ·O 2 -) which are able to damage cellular structures, meaning that PDT is particularly attractive as an alternative means to kill drug-and radioresistant tumour cells.1,2 Normal cells, however, are also able to accumulate PS and be damaged by them, so that prolonged skin photosensitization, light-sensitivity of the eye and other side-effects have proved to be severe limitations of PDT. When photoactivated, PS inflict damage on many types of biomolecules without any specificity, their action being mediated largely via the reactive oxygen species mentioned, none of which travel more than several tens of nanometers before reacting with a biomolecule. Keeping in mind that cell dimensions are of the order of micrometres or tens of micrometres, it is clear that the intracellular action of PS is restricted to the site of their subcellular location and the surrounding radius of not more than 40 nm.3-5 That uneven intracellular distribution of PS can lead to differences in toxicity has been shown using laser microbeam irradiation. 6In contrast to cell membranes and other cytoplasmic organelles, the cell nucleus 7-9 is known to be a very sensitive target for reactive oxygen species. In order to reduce the dose of PS administered to patients and hence minimize the harmful side-effects of PDT, new approaches have been devised to increase the effectiveness of tumour-cell killing through targeted delivery of PS to hypersensitive subcellular sites. These approaches are the focus of the present review. Subcellular distribution of photosensitizersInsomuch as most mammalian cells span tens of micrometres, it is clear that PS efficiency will depend not only on the relative distribution of PS between the tumour and surrounding tissues and between malignant and normal cells, but also on the intracellular distribution of the PS. Furthermore, membranes divide the interior of the eucaryotic cell into compartments that differ markedly in their sensitivity to reactions induced by PS-generated reactive oxygen species, in the ability of damaged molecules to be replaced/recycled and in the extent to which such damage affects cell viability and/or the capability of the cell to divide. It is noteworthy that preferential PS accumulation in tumours is itself not a guarantee of selective photoinduced tumour damage and successful PDT. In experiments on rats with gliosarcoma 9L, 10 for example, it has been found that despite a 13-fold higher accumulation of the PS Photofrin in the tumour compared with Summary Photodynamic therapy (PDT) is a novel treatment, used mainly for anticancer therapy, that depends on the retention of photosensitizer...

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“…2 The unmet medical need for more effective anticancer therapeutics, especially for strategies that can focus toxicity to tumor cells and away from normal tissues, has lead to the development of monoclonal antibodies (mAbs) 3 linked to immunotoxins, radionuclides, or cytotoxic drugs, to provide selective elimination of antigen-positive target cells. The first such clinically approved agent, Gemtuzumab ozogamicin (Mylotarg), an anti-CD33 mAb linked to the potent DNA damaging agent calicheamicin, is used for the treatment of patients with relapsed acute myeloid leukemia (1).…”

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Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-Auristatin Conjugates

Sutherland

Sanderson

Gordon

et al. 2006

Journal of Biological Chemistry

267

180

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The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valinecitrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibodydrug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrinmediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.

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HPMA copolymer–anticancer drug–OV-TL16 antibody conjugates. II. Processing in epithelial ovarian carcinoma cells in vitro

Cited by 86 publications

References 13 publications

The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in hep G2 cells

The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in hep G2 cells

Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency

Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-Auristatin Conjugates

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