Anti-CD166 single chain antibody-mediated intracellular delivery of liposomal drugs to prostate cancer cells.
ABSTRACT Targeted delivery of small-molecule drugs has the potential to enhance selective killing of tumor cells. We have identified previously an internalizing single chain [single chain variable fragment (scFv)] antibody that targets prostate cancer cells and identified the target antigen as CD166. We report here the development of immunoliposomes using this anti-CD166 scFv (H3). We studied the effects of a panel of intracellularly delivered, anti-CD166 immunoliposomal small-molecule drugs on prostate cancer cells. Immunoliposomal formulations of topotecan, vinorelbine, and doxorubicin each showed efficient and targeted uptake by three prostate cancer cell lines (Du-145, PC3, and LNCaP). H3-immunoliposomal topotecan was the most effective in cytotoxicity assays on all three tumor cell lines, showing improved cytotoxic activity compared with nontargeted liposomal topotecan. Other drugs such as liposomal doxorubicin were highly effective against LNCaP but not PC3 or Du-145 cells, despite efficient intracellular delivery. Post-internalization events thus modulate the overall efficacy of intracellularly delivered liposomal drugs, contributing in some cases to the lower than expected activity in a cell line-dependent manner. Further studies on intracellular tracking of endocytosed liposomal drugs will help identify and overcome the barriers limiting the potency of liposomal drugs.
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ABSTRACT: Since the success of rituximab and trastuzumab for treatment of non-Hodgkin's lymphoma and breast cancer, respectively, a huge therapeutic potential of monoclonal antibodies (mAbs) was realized and development of therapeutic mAbs has been widely tried against various cancers. However, the successful examples are still limited and therapeutic mAbs are not yet available for the majority of human cancers. We established a procedure for comprehensive identification of tumor-associated antigens (TAAs) through the extensive isolation of human mAbs that may become therapeutic. Thirty-twoTAAs have been identified and 555 mAbs that bound to one of the TAAs have been isolated to date. Now we are trying to select TAAs as proper targets for therapy and candidate mAbs as drugs from among them. The immunohistochemical analysis using many fresh lung cancer specimens suggested probabilities of proper targets, and moreover, presence of cancer-specific epitopes that could be distinguished from normal epitopes on the same molecules by mAbs. For Abs to efficiently kill the cancer cells they should have the ability to induce immunological cytotoxicity such as ADCC and/or CDC. They should also be able to inhibit the function mediated by the target Ags. For clinical point of view, the continuous presence of the target molecule on the cell surface until cell death might be essential for successful treatment. Therefore, it will be required for targets TAAs to play essential roles in tumorigenesis. Otherwise the cancer cells that do not express them could selectively survive during treatment and finally become dominant. It was also suggested that even the same molecules could play different roles in tumorigenesis quite often in different patients. Therefore when we develop therapeutic Abs, we should obtain information about the conditions of patients including genetic background to whom the treatment will be effective. I will discuss how we can accomplish this purpose.04/2011; 7(1):14-28.This article is viewable in ResearchGate's enriched formatRG Format enables you to read in context with side-by-side figures, citations, and feedback from experts in your field.
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ABSTRACT: A detailed characterization of the cell surface proteome facilitates the identification of target antigens, which can be used for the development of antibody-based therapeutics for the treatment of hematological malignancies. We have performed cell surface biotinylation of five human myeloid leukemia cell lines and normal human granulocytes, which was used for mass spectrometric analysis and allowed the identification and label-free, relative quantification of 320 membrane proteins. Several proteins exhibited a pronounced difference in expression between leukemia cell lines and granulocytes. We focused our attention on CD166/ALCAM, as this protein was strongly up-regulated on all AML cell lines and AML blasts of some patients. A human monoclonal antibody specific to CD166 (named H8) was generated using phage display technology. H8 specifically recognized AML cells in FACS analysis while demonstrating tumor targeting properties in vivo. After in vitro screening of five potent cytotoxic agents, a duocarmycin derivative was used for the preparation of an antibody-drug conjugate, which was able to kill AML cells in vitro with an IC50 of 8nM. The presented atlas of surface proteins in myeloid leukemia provides an experimental basis for the choice of target antigens, which may be used for the development of anti-AML therapeutic antibodies. The ability to discriminate between malignant and healthy, essential cells represents an important requirement for the development of armed antibodies for the therapy of hematological malignancies. Our proteomic study is, to our knowledge, the first large scale comparison of the accessible cell surface proteome of leukemia cells and normal blood cells, facilitating the choice of a suitable target for the treatment of acute myeloid leukemia (AML). An antibody drug conjugate was generated recognizing the CD166 antigen which was found to be strongly up-regulated in all AML cell lines and AML blasts of some patients. This antibody drug conjugate SIP(H8)-Duo might be further characterized in therapy experiments and might lead to a new targeted treatment option for AML.Journal of proteomics 01/2014; DOI:10.1016/j.jprot.2014.01.022 · 5.07 Impact Factor
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ABSTRACT: We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70) on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding 'healthy' tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG), the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+) tumor cells, in vitro.PLoS ONE 08/2014; 9(8):e105344. DOI:10.1371/journal.pone.0105344 · 3.53 Impact FactorThis article is viewable in ResearchGate's enriched formatRG Format enables you to read in context with side-by-side figures, citations, and feedback from experts in your field.