Targeting HER2-positive cancer cells with receptor-redirected anthrax protective antigen Andrew J. McCluskey, Andrew J. Olive, Michael N. Starnbach, R. John Collier* Department of Microbiology and Immunobiology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA ARTICLE INFO Article history: Received 12 September 2012 Received in revised form 29 November 2012 Accepted 3 December 2012 Available online 19 December 2012 Keywords: Anthrax toxin HER2/neu Binary toxin Affibody Immunotoxin ABSTRACT Targeted therapeutics have emerged in recent years as an attractive approach to treating various types of cancer. One approach is to modify a cytocidal protein toxin to direct its ac- tion to a specific population of cancer cells. We created a targeted toxin in which the receptor-binding and pore-forming moiety of anthrax toxin, termed Protective Antigen (PA), was modified to redirect its receptor specificity to HER2, a marker expressed at the surface of a significant fraction of breast and ovarian tumors. The resulting fusion protein (mPA-ZHER2) delivered cytocidal effectors specifically into HER2-positive tumor cells, in- cluding a trastuzumab-resistant line, causing death of the cells. No off-target killing of HER2-negative cells was observed, either with homogeneous populations or with mixtures of HER2-positive and HER2-negative cells. A mixture of mPA variants targeting different re- ceptors mediated killing of cells bearing either receptor, without affecting cells devoid of these receptors. Anthrax toxin may serve as an effective platform for developing therapeu- tics to ablate cells bearing HER2 or other tumor-specific cell-surface markers. ª 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction Amplification and/or overexpression of the HER2 gene at the mRNA or protein level occurs in 20e25% of breast, gastric, and ovarian carcinomas (Berchuck et al., 1990; Gravalos and Jimeno, 2008; Arteaga et al., 2012; Slamon et al., 1989). Particu- larly in breast cancer, increased expression of HER2 is associ- ated with an aggressive form of the disease, which shows signs of increased tumor growth, recurrence, and resistance to therapy, all contributing to decreased patient survival (Arteaga et al., 2012). Although the FDA-approved monoclonal antibody, trastuzumab (trade name, Herceptin Ò ), is effective at slowing tumor growth, it remains ineffective at tumor elim- ination. New therapeutics that actively kill tumor cells thus remain a major goal of cancer-related research. A promising example of this strategy is to target the action of cytocidal pro- tein toxins to specific cancer cells (Pastan et al., 2007). Recently, we developed a straightforward way to redirect the receptor specificity of anthrax toxin (Mechaly et al., 2012). First we ablated the native receptor-binding activity of protective an- tigen (PA), the receptor-binding/pore-forming component of an- thrax toxin, and then appended a heterologous, receptor- binding ligand to the C terminus of the mutated protein (mPA). Using this approach we created fusion proteins that direct toxin action specifically to two different receptors: the diphtheria toxin (DT) receptor (HB-EGF) and the epidermal growth factor receptor (EGFR) (Mechaly et al., 2012). In the current study we used this ap- proach to redirect toxin action to cells bearing the HER2 receptor. Anthrax toxin is an ensemble of three nontoxic, monomeric proteins (Young and Collier, 2007). Two of them, the Lethal * Corresponding author. Tel.: þ1 617 432 1930. E-mail addresses: [email protected], [email protected](R.J. Collier). available at www.sciencedirect.com www.elsevier.com/locate/molonc 1574-7891/$ e see front matter ª 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molonc.2012.12.003 MOLECULAR ONCOLOGY 7 (2013) 440 e451
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M O L E C U L A R O N C O L O G Y 7 ( 2 0 1 3 ) 4 4 0e4 5 1
ava i l ab le a t www.sc ienced i rec t . com
www.elsevier .com/locate /molonc
Targeting HER2-positive cancer cells with
receptor-redirected anthrax protective antigen
Andrew J. McCluskey, Andrew J. Olive, Michael N. Starnbach,R. John Collier*
Department of Microbiology and Immunobiology, Harvard Medical School, 77 Avenue Louis Pasteur,
M O L E C U L A R O N C O L O G Y 7 ( 2 0 1 3 ) 4 4 0e4 5 1450
mPA-ZHER2 and mPA-EGF, in combination with LFN-DTA
completely eliminated a panel of tumor cells with different
HER2 and EGF receptor expression levels (Figure 8).
The ability of mPA-ZHER2 to act cooperatively with an
analogous mPA variant targeting a different tumor marker
highlights the adaptability of targeting with mPA. In addition
to combining mPA variants, the ability of the PA pore to trans-
locate any of a variety of intracellular effector enzymes allows
the possibility of using combinations of effectors that kill by
different biochemical mechanisms. The enzymatic destruc-
tion of targeted cells from within by multiple effectors should
minimize the likelihood of resistant escape mutants arising,
a universal problem in chemotherapy.
In summary, our in vitro data indicate that the targeting of
the HER2 receptor by modified, receptor-targeted anthrax
toxin is specific and potent, and displays no off-target toxicity
towards HER2-negative cell lines. The susceptibility of a HER2-
positive trastuzumab-resistant tumor cell line to toxin action
highlights a significant potential advantage of our systemover
current FDA-approved antibody therapies. For these reasons
and the advantages described above, the PA-based targeting
of distinct populations of cancer cells represents a promising
therapeutic strategy for cancer treatment.
Acknowledgements
This research was supported by NIAID grant AI022021 to RJC
and NIAID grant AI062827 to MNS. We thank Dr. Robin Ross,
BenSeiler, and EricaGardner from theNERCEBiomolecule Pro-
duction Core, supported by NIH grant AI057159, for helping
with the production of proteins used in this study. The authors
would also like to thankDrs. TomRoberts and Jean Zhao (Dana
Farber Cancer Institute) for their critical review of the manu-
script. The authorswould also like to thankDr. JeanZhao again
for providing cell lines andDr. GregoryPoon (WashingtonState
Univeristy, Pullman, WA) for the expression plasmids coding
for the ZHER2 proteins and the MDA-MB-231 cell line.
Appendix A.Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.molonc.2012.12.003.
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