The product of the HER2 protooncogene, p185HEP2, represents an attractive target for cancer immunotherapies. We The HER2 (c-erbB2, neu) protooncogene and pl85HER2, the growth factor receptor-tyrosine kinase it encodes, appear to play a central role in the pathogenesis of many human cancers.
Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD) HER1 (4,6,20,36). The need to understand the role of the HER2 receptor, and its growth-regulating ligand(s) (15,27,32), is underscored by the prevalence of HER2 DNA amplification in a variety of human epithelial cancers, including those of ovarian, gastrointestinal, and mammary origin, in which the overexpressed receptor contributes to aggressive tumor growth and reduced patient survival (13, 37). Moreover, newly developed anti-HER2 monoclonal antibodies that bind with high affinity to the receptor's extracellular domain (ECD) result in both in vitro and in vivo growth inhibition of HER2-overexpressing tumors (10,14,17,31,38). One of these murine monoclonal antibodies, muMAb4D5, is now involved in clinical testing for the treatment of patients with HER2-overexpressing breast and ovarian cancers (31).Tumors and cell lines overexpressing transmembrane growth factor receptors often release truncated receptor ECD. The truncated forms of these receptors may arise by proteolytic cleavage of the full-length receptor, as for colony-stimulating factor 1 and interleukin-2 receptors (7,26,34), and/or by alternate splicing of receptor transcript into a form that eliminates the transmembrane and cytoplasmic * Corresponding author. domains, as has been shown for interleukin-4, Fc, and EGF receptors (29,30,33). Soluble HER2 ECD is released from HER2-overexpressing tumor cells in vitro and in vivo (1, 24, 41), and the mechanism for this is thought to involve surface proteolysis of the 185-kDa receptor (41). Largely unexplored are the potential physiological effects of truncated growth factor receptors, which might involve extracellular competition for cognate ligand (5) or intracellular dominant-negative suppression of receptor function by heterodimer formation (11,20).We report here the isolation of clones encoding a truncated ECD form of the HER2 receptor from cDNA libraries prepared from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The truncated HER2 ECD transcript appears to be produced by an alternative RNA processing mechanism in which an exon extends through a splice site utilized by the full-length transcript, providing an in-frame stop codon and an alternate poly(A) addition site. The 2.3-kb truncated HER2 transcript is variably expressed in a panel of human epithelial cancer cell lines and produces intracellular HER2 ECD protein of about 100 kDa. Transfection studies suggest that excess production and intracellular retention of 100-kDa HER2 ECD in tumor cells overexpressing 185-kDa HER2 receptor results in resistance to the growth-inhibiting effects of the anti-HER2 monoclonal antibody ...
We describe the assembly of a cationic lipid-nucleic acid nanoparticle from a liquid monophase containing water and a water miscible organic solvent where both lipid and DNA components are separately soluble prior to their combination. Upon removal of the organic solvent, stable and homogenously sized (70-100 nm) lipid-nucleic acid nanoparticles (Genospheres) were formed. The low accessibility (<15%) of the nanoparticle-encapsulated DNA to a DNA intercalating dye indicated well-protected nucleic acids and high DNA incorporation efficiencies. It was demonstrated that Genospheres could be stably stored under a variety of conditions including a lyophilized state where no appreciable increase in particle size or DNA accessibility was observed following reconstitution. Finally, Genospheres were made target-specific by insertion of an antibody-lipopolymer (anti-HER2 scFv (F5)-PEG-DSPE) conjugate into the particle. The target specificity (>100-fold) in HER2 overexpressing SK-BR-3 breast cancer cells was dependent on the degree of PEGylation, where the incorporation of high amounts of PEG-lipid on the particle surface (up to 5 mol%) had only a minor effect on the transfection activity of the targeted Genospheres. In summary, this work describes a novel, readily scalable method for preparing highly stable immunotargeted nucleic acid delivery vehicles capable of achieving a high degree of specific transfection activity.
Background:This phase 1 clinical trial was conducted to determine the safety, maximum-tolerated dose (MTD), and pharmacokinetics of imatinib, bevacizumab, and metronomic cyclophosphamide in patients with advanced colorectal cancer (CRC).Methods:Patients with refractory stage IV CRC were treated with bevacizumab 5 mg kg−1 i.v. every 2 weeks (fixed dose) plus oral cyclophosphamide q.d. and imatinib q.d. or b.i.d. in 28-day cycles with 3+3 dose escalation. Response was assessed every two cycles. Pharmacokinetics of imatinib and cyclophosphamide and circulating tumour, endothelial, and immune cell subsets were measured.Results:Thirty-five patients were enrolled. Maximum-tolerated doses were cyclophosphamide 50 mg q.d., imatinib 400 mg q.d., and bevacizumab 5 mg kg−1 i.v. every 2 weeks. Dose-limiting toxicities (DLTs) included nausea/vomiting, neutropaenia, hyponatraemia, fistula, and haematuria. The DLT window required expansion to 42 days (1.5 cycles) to capture delayed toxicities. Imatinib exposure increased insignificantly after adding cyclophosphamide. Seven patients (20%) experienced stable disease for >6 months. Circulating tumour, endothelial, or immune cells were not associated with progression-free survival.Conclusion:The combination of metronomic cyclophosphamide, imatinib, and bevacizumab is safe and tolerable without significant drug interactions. A subset of patients experienced prolonged stable disease independent of dose level.
Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors.
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