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Neuregulin Receptor-Mediated Gene Transfer by Human Epidermal Growth Factor Receptor 2-Targeted Antibodies and Neuregulin-1

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Neuregulin Receptor-Mediated Gene Transfer by Human Epidermal Growth Factor Receptor 2-Targeted Antibodies and Neuregulin-1 Neuregulin receptor-mediated gene transfer by human epidermal growth factor receptor 2-targeted antibodies and neuregulin-1 Jeffrey A. Kern,1 Robert Wakita,2 and Mark X. Sliwkowski2 1Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242; and 2Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080. The human epidermal growth factor receptors 2, 3, and 4 (HER2, HER3, and HER4, respectively) are frequently overexpressed in many human cancers, and therefore may be potential targets for receptor-mediated gene transfer. To evaluate this possibility, we constructed a series of HER-targeted gene transfer vehicles by covalently linking poly-L-lysine polymers (pLYS) to the epidermal growth factor-like domain of the HER ligand neuregulin-1 (NRG1177–244), a HER2 antibody (Ab), and the Fab fragment of the HER2 Ab. In vitro, pLYS modification of NRG1177–244 decreased the affinity of the ligand for HER3 or HER4 homodimer receptors by 6- to 7-fold. DNA loading of the pLYS-modified NRG1177–244 had a minimal additional affect on the affinity of the complex for its receptor. In cell lines engineered to solely express HER2, HER3, or HER4, each vehicle correctly targeted the receptors; the NRG1177–244 construct transferred a luciferase gene only into cells expressing HER4, whereas the HER2 Ab and Fab constructs transferred the reporter gene only into cells expressing HER2. The most efficient gene transfer occurred using the intact HER2 Ab as a gene transfer vehicle, whereas the Fab fragment of the HER2 Ab was the least efficient, and NRG1177–244 was intermediate. These studies suggest that the NRG receptor or HER2, a component of the receptor, can be pursued as targets for gene transfer. Key words: Gene transfer; neuregulin-1; human epidermal growth factor receptor 2; human epidermal growth factor receptor 3; human epidermal growth factor receptor 4. elective gene transfer to cancer cells is a barrier to tion of breast,8–10 ovary,9 lung,11 and gastric epithelial Sthe implementation of gene therapy as a cancer cells. In addition, patients whose tumors overexpress treatment strategy. Receptor-mediated gene transfer is HER2 have a poor prognosis,9,12 with shorter overall one method that may have the potential to achieve the survival compared with patients whose tumors do not necessary selectivity. Reports of targeting the transferrin express HER2. Less is known about the function and receptor,1 asialooromucoid,2 folate,3 the polymeric im- clinical importance of HER3 and HER4. Increased 4 munoglobulin (Ig) receptor, and the epidermal growth HER3 expression has been reported in a subset of 5 factor receptor (EGFR) have shown the feasibility of human mammary, pancreatic, prostate, and oral can- such an approach. Recently, we demonstrated that a cers.13–15 HER3 also enhances the transforming activity member of the human epidermal growth factor receptor of HER2 when coexpressed in NIH3T3 cells.16,17 HER4 (HER) family, HER2, could also be used as a target for is expressed in several established tumor cell lines,18 but receptor-mediated gene transfer using an antibody (Ab) 6 little is known about its expression in primary tumor directed against HER2 as a gene transfer agent. The cells. HER proteins are growth factor receptors with intrinsic There are a number of different peptides that bind tyrosine kinase activity, termed subclass 1 growth factor and activate these receptors, as the receptor proteins are receptors. Members of the family include EGFR, HER2 7 modular, with homo- or heterodimerization possible (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). This within the family. Each combination, however, alters family of receptors has been implicated in the pathogen- ligand binding specificity. EGF, transforming growth esis of many human tumors. Overexpression of HER2 factor-a, amphiregulin, betacellulin, and epiregulin bind due to gene amplification is involved in the transforma- EGFR homodimers.19 HER2/HER3 heterodimers bind neuregulin 1 (NRG1) (also called heregulin-b1). HER3 homodimers bind NRG1 and also the recently described 20,21 Received July 8, 1998; accepted November 1, 1998. NRG2. HER4 homodimers can bind NRG1, 14,15 Address correspondence and reprint requests to Dr. Jeffrey A. Kern, NRG2, betacellulin, epiregulin, HB-EGF, and a Department of Internal Medicine, C-33, GH, University of Iowa, Iowa third member of the NRG family, NRG3. This report City, IA 52242. focuses on NRG1 and HER2. The functional effect of © 1999 Stockton Press 0929-1903/99/$12.00/10 the interaction of NRG1 with its receptor(s) is not Cancer Gene Therapy, Vol 6, No 6, 1999: pp 537–545 537 538 KERN AND SLIWKOWSKI: NRG RECEPTOR-MEDIATED GENE TRANSFER completely understood, but appears to be tissue-specific Preparation of poly-L-lysine DNA complexes and can be classified into three broad categories. Recep- The humanized HER2 Ab (rhuMAbHER2),36 the Fab of this tor activation regulates neuron-Schwann cell interac- Ab (rhuMAbHER2.Fab),37 and the EGF-like domain of tions, notably Schwann cell differentiation and prolifer- NRG1 (NRG1 )38 have been described previously. Poly- 22,23 177–244 ation. It also regulates neuron-muscle cell L-lysine polymers (pLYS) of different chain lengths (158 and interactions in the differentiation of neuromuscular syn- 251 lysines) were obtained from Sigma (St. Louis, Mo). apses,24 activation of synapse-specific gene expression,25 Chemical conjugation was performed by resuspending the and expression of ion channels in skeletal muscle.26 targeting protein (rhuMAbHER2, rhuMAbHER2.Fab, Finally, receptor activation also causes epithelial cell NRG1177–244)in1mLof0.1M2-(N-morpholino)ethanesul- differentiation and proliferation.27–32 fonic acid (pH 5.0), a 10-fold excess of the desired pLYS Because of the preferential expression of these recep- polymer, and a 500-fold excess of 1-ethyl-3-(3-dimethylami- nopropyl)carbodiimide hydrochloride (Pierce, Rockford, Ill). tor proteins in certain tumors, therapy directed to The reaction mix was incubated at room temperature over- HER2-, HER3-, or HER4-expressing cancers may be night. Underivatized protein was separated from the reaction possible using NRGs or receptor-specific Abs. NRG1, mixture by cation exchange chromatography. The reaction the first member of the NRG family, exists in its mature mixture was adjusted to a pH of 8.5 using 20 mM of sodium form as a 45-kDa protein derived by the proteolytic borate and loaded onto an S Sepharose Fast Flow column; cleavage of a 645-amino acid (aa) precursor in the next, 1-mL fractions were collected over a 30-minute gradient endoplasmic reticulum or at the cell surface.33 Structur- (buffer A 5 20 mM of sodium borate (pH 8.5); buffer B 5 ally it is a multimotif protein containing an IgG domain, buffer A plus 4 M NaCl). Further purification of derivatized a carbohydrate spacer, an EGF-like domain, and a rhuMAbHER2 included removing unbound pLYS by centrif- juxtamembrane region.34 Receptor binding is mediated ugation through a Centriprep-100 filter/concentrator (Amicon, Beverly, Mass) to yield pLYS-rhuMAbHER2. Purification of through a specific 67-aa motif in the protein, called the derivatized rhuMAbHER2.Fab and NRG1177–244 required re- EGF-like domain. This domain interacts with low-affin- versed-phase high performance liquid chromatography ity binding sites formed by HER3 or HER4 homodimers (HPLC) to remove unbound pLYS. Fractions with 280 nm and high-affinity sites formed by HER2/HER3 and absorbance obtained by cation exchange chromatography were HER2/HER4 heterodimers. We hypothesized that a combined, adjusted to 0.1% trifluoroacetic acid, and loaded minimal NRG1 ligand consisting of the 67-aa EGF-like onto a C-18 reversed phase column (SynChropak RP-P, Syn- domain (NRG1177–244) could be derivatized to form an chrom, Lafeyette, Ind). Fractions (1 mL) were collected over a 30-minute gradient (buffer A 5 0.1% trifluoroacetic acid; effective gene transfer complex, thereby targeting tumors 5 expressing HER3/HER3 or HER4/HER4 homodimers buffer B buffer A plus 100% acetonitrile). The eluant was and HER2/HER3 or HER2/HER4 heterodimers. In addi- monitored at 280 nm and peaks were pooled, lyophilized, and resuspended in water. Protein concentration was determined tion, because stimulation and internalization of the recep- by bicinchoninic acid assay (Pierce). Amino acid analysis was tor/NRG1177–244 gene transfer complex would occur performed to determine the molar ratio of carrier to pLYS. through a normal physiologic process, NRG1177–244 would Samples were hydrolyzed in 5.7 N HCl for 24 hours at 110°C be a more efficient gene transfer agent than Abs targeting and analyzed using a Beckman 6300 Amino Acid Analyzer the receptor. Here we describe the development of the (Beckman Instruments, Fullerton, Calif). minimal NRG1 ligand as a gene transfer vehicle, and directly compare Ab-mediated gene transfer directed DNA binding of poly-L-lysine protein conjugates against HER2 with ligand-mediated gene transfer using To create the DNA complex, pLYS-conjugated proteins were NRG1 binding sites. mixed with DNA at room temperature in N-2-hydroxyeth- ylpiperazine-N9-2-ethanesulfonic acid (HEPES)-buffered sa- MATERIALS AND METHODS line (HBS) (15 mM HEPES, 150 mM NaCl) and allowed to incubate for 15 minutes. The ability of the poly-L-lysine Cell culture conjugate to bind DNA was tested by electrophoretic mobility through a 1% agarose gel containing ethidium bromide (EB). The human breast adenocarcinoma cell lines BT474, SKBR-3, Plasmid DNA (1 mg) was incubated for 15 minutes in HBS at MCF-7, and MDA-MB-468; the ovarian carcinoma cell line room temperature with varying amounts of pLYS-carrier CaOV3; and the human erythroleukemia cell line K562 were conjugate. Samples were loaded onto the gel, and electro- obtained from the American Type Culture Collection (Man- phoresis was performed at 50 V for 2 hours.
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