Target cell-restricted apoptosis induction by 528scFv-TRAIL fusion protein specific for human EGFR and expressed in Escherichia coli.

We report the preparation and functional characterization of an Escherichia coli-expressed recombinant fusion protein, 528scFv-TRAIL, specific for the human epidermal growth factor receptor (EGFR) and empowered by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 528scFv-TRAIL, expressed as insoluble inclusion bodies in E. coli, was solubilized and then refolded by using a modified stepwise dialysis method. Treatment with 528scFv-TRAIL resulted in the specific binding to the cell surface of EGFR-positive cells with concomitant deployment of the TRAIL moiety to DR-5 receptor in a manner comparable to a commercially available form of recombinant TRAIL (cTRAIL). 528scFv-TRAIL, prepared by either of three refolding processes described herein, showed potent cytotoxic activity against EGFR-positive TFK-1 cell line and was superior to its parental 528scFv; a recombinant variable fragment with single specificity against human EGFR. Narrow variations in the cytotoxic potential of 528scFv-TRAIL were ascribed to manipulation of redox conditions during the refolding process. Together, our findings point to the potential value of 528scFv-TRAIL for treatment of EGFR-expressing cancers. Furthermore, preparation of 528scFv-TRAIL from insoluble aggregates in a prokaryotic cell based expression system by means of in vitro refolding introduces a feasible cost-benefit, time-efficient approach for industrial-scale production.

Integration of PEGylation and refolding for renaturation of recombinant proteins from insoluble aggregates produced in bacteria--application to a single-chain Fv fragment.

The conjugation of polyethylene glycol (PEGylation) with downsized compact antibodies is an effective method for overcoming the problem of rapid elimination of the compact antibodies from the body. We integrated site-specific PEGylation with the refolding of a single-chain Fv (scFv) of humanized monoclonal antibody 528 with affinity for the epidermal growth factor receptor, to prepare active PEGylated scFv from insoluble aggregates produced in an Escherichia coli expression system. The insertion of a cysteine residue at the C-terminus of scFv to serve as the conjugation site for PEG led to the formation of highly multimeric scFv during the refolding process; however, PEGylation after refolding drastically dispersed the multimer into monomeric active scFv fragments. Further, the PEGylation of partially refolded scFv during the refolding process improved the PEGylation efficiency and suppressed the formation of highly multimeric scFv; consequently, monomeric active scFv fragments were obtained directly from the insoluble aggregates in E. coli. We show that in vitro refolding of PEGylated scFv should be useful for improving downstream processing performance in the production of clinically useful small antibodies from insoluble fractions.

Strategies to endow cytotoxic T lymphocytes or natural killer cells with antibody activity against carcinoembryonic antigen.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are main effecter cells in cellular immunity against tumor cells. T-cell immunotherapy is based on the assumption that tumor(-associated) antigen (TA) peptides are correctly presented by HLA class I molecules on target tumor cells, and NK cell immunotherapy is based on the hypothesis that cell surface TAs or ligands for NK receptors are widely expressed in tumor cells. However, human tumor cells often lose HLA class I molecules, and target cell ligands for NK receptors are not always expressed in human tumor cells. These altered HLA class I phenotypes and non-ubiquitous expression of NK receptor ligands constitute the major tumor escape mechanism facing tumor-specific CTL and/or NK cell mediated responses. These facts also indicate that it is not easy to eliminate the target tumors only by activating tumor-specific CTLs or NK cells with cancer vaccine treatments. On the other hand, it is easily confirmed by immunohistochemistry whether or not antibody-recognized TAs exist on the cell surface of target tumor cells. Therefore, endowing CTLs or NK cells with antigen-binding specificity of anti-TA antibody is a promising approach for re-targeting the activities of these effector cells to tumor cells in an HLA-independent manner. This review summarizes the following four new strategies for re-targeting CTLs or NK cells to carcinoembryonic-antigen-expressing tumor cells: (1) bispecific antibody technology; (2) antibody-cytokine fusion protein technology; (3) chimeric immune receptor technology, and (4) antibody-HLA/peptide complex technology.

Cloning and sequencing of variable region cDNAs of a novel human monoclonal antibody to carcinoembryonic antigen, and generation of a single chain variable fragmented antibody.

BACKGROUND: Taking into consideration immunogenicity in humans, human antibodies and their derivatives have potential advantages for cancer immunotherapy and/or gene therapy over those from different species such as mouse. Recently, we generated 22 human monoclonal antibodies (HmAbs) specific for human carcinoembryonic antigen (CEA) using KM mouse, which carries human antibody genes. In the present study, we tried to clone the variable (V) region genes of the C2-45 HmAb, with the highest affinity for CEA and to generate a human single-chain variable fragmented (scFv) antibody. MATERIALS AND METHODS: Using RT-PCR methods, we cloned and sequenced cDNAs encoding V regions of C2-45, and constructed an scFv gene including a signal sequence for secreting into the periplasmic space of E. coli. RESULTS: Soluble scFv antibody, designated 45KHscFv, was secreted in the periplasmic space and purified by Ni2+ affinity column from the supernatant of E. coli after osmotic shock. When analyzed by SDS-PAGE, the 45KHscFv antibody showed a band corresponding to a calculated molecular weight of 30 kDa. The 45KHscFv antibody also retained a high reactivity with CEA in ELISA using immobilized CEA. CONCLUSION: These results indicate that the nonimmunogenic 45KHscFv antibody could be advantageously used for fusing with other human functional proteins such as cytokines.

Tumor-targeting of viral vectors for cancer gene therapy by using antibodies or their genes against tumor-associated antigens.

Gene therapy is a promising modality for the treatment of malignant tumors for which conventional therapies are often inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. The aim of this review is to summarize the tumor-targeting viral vectors for cancer gene therapy by utilizing antibodies or genes against tumor-associated antigens.

Identification of a novel splice variant of the human anti-apoptopsis gene survivin.

Survivin, a new member of the inhibitor of apoptosis protein (IAP) family, has been reported to be expressed in many cancers but not in differentiated normal tissues. In the present study, we describe the identification of a novel alternatively spliced survivin transcript, designated as survivin-3B. It comprises 5 exons including novel exon 3B derived from a 165-bp long portion of intron 3. Acquisition of a new in-frame TGA stop codon within the novel exon 3B results in an open reading frame (ORF) of 363 nucleotides, predicting a truncated 120 amino acid protein. Expression of survivin-3B was detected in human colon and gastric adenocarcinoma cell lines as well as mononuclear cells prepared from patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Survivin-3B contains a single baculovirus IAP repeat (BIR), which is critical for apoptosis inhibition. However, it lacks a carboxyl-terminal coiled-coil domain, suggesting that survivin-3B may not be associated with G2/M phase. These data indicate that the function of survivin-3B may be different from that of regular survivin.

Expression of the antiapoptotic gene survivin in chronic myeloid leukemia.

Survivin, a unique member of the inhibitor of the apoptosis protein (IAPs) family, is over-expressed in many cancers but not in normal differentiated adult tissues. Using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated patterns of survivin gene expression in a group of 12 patients with chronic myeloid leukemia (CML) representing both chronic and blastic phases of the disease. All 6 patients in chronic phase CML were uniformly negative for the survivin transcript, in contrast to 4 Philadelphia chromosome-positive (Ph+) CML patients in blastic crisis, all of whom (100%) were positive for survivin with tangible levels of expression. However, survivin expression was markedly down-regulated in 2 atypical CML patients with Philadelphia chromosome-negative (Ph-) blastic crisis. Our data indicates that up-regulation of survivin expression may be involved in typical CML evolution from the chronic into the blastic phase of the disease.

Atypical nuclear apoptosis downstream to caspase-3 activation in ara-C treated CCRF-CEM cells.

The necessity of internucleosomal DNA fragmentation for the complete execution of apoptosis is still controversial. While investigating the apoptotic pathway induced by 1-beta-D-arabinofuranosylcytosine (ara-C) in the human T-lymphoblastic leukemia CCRF-CEM (CEM) cells, we could easily retrieve high molecular weight (HMW) DNA fragments with a predominant size of 50 kb. However, under the same circumstances, internucleosomal DNA fragmentation characteristic of apoptosis was undetectable despite estimated heightened caspase-3 activity. Paradoxically, generation of low molecular weight DNA fragments was readily demonstrable by flow cytometric and immunohistochemical evidence in the absence of any detectable DNA ladder formation. These findings present a proof that, within certain contexts, small-sized DNA fragmentation occurring in apoptosis may not necessarily be of the ladder yielding internucleosomal integer multiples' pattern. Our data also add to the evidence that the machinery underlying HMW DNA fragmentation is distinct from that responsible for the internucleosomal one.

Expression of the anti-apoptotic gene survivin in myelodysplastic syndrome.

Survivin is a member of the inhibitor of apoptosis protein (IAPs) family and considered to play a pivotal role in oncogenesis. We present the first report of survivin expression profile in myelodysplastic syndrome (MDS). Expression of survivin messenger RNA was evaluated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in patients with MDS and acute myeloid leukemia (AML). Eleven out of 12 patients with refractory anemia (RA) (91.6%), and all 3 patients with refractory anemia with excess blasts in transformation (RAEBt) (100%), were positive for survivin expression with the majority of cases showing abundant levels of the survivin transcript. On the other hand, expression of survivin was undetectable in the 4 patients with chronic myelomonocytic leukemia (CMMoL). The level and frequency of survivin expression in patients with refractory anemia were compared to those in patients with AML. Out of 12 patients with de novo AML, 5 patients (41.7%) showed detectable levels of survivin expression. Abundant survivin expression in RA was also confirmed by immunohistochemistry. In contrast, survivin was almost absent in two cases with aplastic anemia. We propose that high levels of survivin expression can serve as a reliable diagnostic marker of RA in MDS.

Doxycycline induces apoptosis by way of caspase-3 activation with inhibition of matrix metalloproteinase in human T-lymphoblastic leukemia CCRF-CEM cells.

Evidence for nonantibiotic activity displayed by tetracycline has been extensively reported in the field of antiinflammation. Here, we report a growth-inhibitory effect of doxycycline on CCRF-CEM, a T-lymphoblastic human leukemic cell line. Cells were incubated with doxycycline at concentrations ranging from zero to 50 micromol/L. We examined the hypothesis that induction of apoptosis is one of the mechanisms by which doxycycline inhibits CCRF-CEM proliferation. Caspase-3 activity of cells grown in the presence of 10 micromol/L and 50 micromol/L doxycycline increased dose-dependently after 24 hours in culture. The demonstration that doxycycline induces APO 2.7 expression in CCRF-CEM cells in vitro also supports its capacity for induction of apoptosis. The level of matrix metalloproteinase-2 was significantly lower in the medium cultured with 50 micromol/L doxycycline than the control. These phenomena suggest that this well-tolerated oral agent has the potential to be of value in antileukemic therapy.