HER4 Expression Correlates with Cytotoxicity Directed by a Heregulin-Toxin Fusion Protein

doi:10.1074/jbc.270.13.7625

HER4 Expression Correlates with Cytotoxicity Directed by a Heregulin-Toxin Fusion Protein (*)

From the (1)Molecular Immunology Department and the BioProcess Research Department, Bristol-Myers Squibb, Pharmaceutical Research Institute, Seattle, Washington 98121 and
(2)Advanced Cellular Diagnostics, Inc., Elmhurst, Illinois 60126

^§ To whom correspondence should be addressed.
Molecular Immunology Dept., Bristol-Myers Squibb, Pharmaceutical Inst., 3005 First Ave., Seattle, WA 98121
. Tel.: 206-727-3542; Fax: 206-727-3603.

↵^¶ Present address: SUGEN Corp., 515 Galveston Dr., Redwood City, CA 94063.

Abstract

We have constructed, expressed, and purified a fusion protein, HAR-TX β2, consisting of heregulin-β2 fused to a binding-defective form of Pseudomonas exotoxin A, PE40. The fusion protein was found to induce receptor tyrosine phosphorylation in CEM cells transfected with HER4 alone or in combination with HER2 but not in cells transfected with HER2 or HER1 alone. The phosphorylation of receptor tyrosines was both dose-dependent and saturable in amounts similar to those shown to be active for native heregulin. HAR-TX β2 was specifically cytotoxic toward a variety of carcinoma cell lines in the ng/ml range. However, some tumor cell lines were found to be insensitive to the cytotoxic action of the fusion protein even at >2 μg/ml. Relative amounts of HER4, HER3, and HER2 were determined on seven cell lines sensitive and four cell lines insensitive to HAR-TX β2. All lines that express HER4 were killed by HAR-TX β2, while none lacking HER4 were affected. HAR-TX β2 was able to bind to and signal via tyrosine phosphorylation in cell lines that co-express HER2 and HER3 in the absence of HER4 without inducing cytotoxicity. Thus HAR-TX β2 may prove to be a useful reagent for the targeting and elimination of HER4-positive tumor cells.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EGF

epidermal growth factor

HER

human EGF receptor

PE

Pseudomonas exotoxin

HAR-TX β2

heregulin/amphiregulin leader sequence-PE40 fusion protein

NDF

neu differentiation factor

FBS

fetal bovine serum

CHO

Chinese hamster ovary

AR

amphiregulin

IPTG

isopropyl-1-thio-β-D-galactopyranoside

mAb

monoclonal antibody

CAS

cellular analysis system.
- Received December 2, 1994.