IgG-Engineered Protective Antigen for Cytosolic Delivery of Proteins into Cancer Cells

Zeyu Lu; Nicholas L Truex; Mariane B Melo; Yiran Cheng; Na Li; Darrell J Irvine; Bradley L Pentelute

doi:10.1021/acscentsci.0c01670

IgG-Engineered Protective Antigen for Cytosolic Delivery of Proteins into Cancer Cells

ACS Cent Sci. 2021 Feb 24;7(2):365-378. doi: 10.1021/acscentsci.0c01670. Epub 2021 Feb 4.

Authors

Zeyu Lu¹, Nicholas L Truex¹, Mariane B Melo^{2

3}, Yiran Cheng¹, Na Li², Darrell J Irvine^{2

3

4

5

6}, Bradley L Pentelute^{1

2

7

8}

Affiliations

¹ Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
² The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, Massachusetts 02142, United States.
³ Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
⁴ Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
⁵ Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
⁶ Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20815, United States.
⁷ Center for Environmental Health Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
⁸ Broad Institute of MIT and Harvard, 415 Main Street, Cambridge, Massachusetts 02142, United States.

Abstract

Therapeutic immunotoxins composed of antibodies and bacterial toxins provide potent activity against malignant cells, but joining them with a defined covalent bond while maintaining the desired function is challenging. Here, we develop novel immunotoxins by dovetailing full-length immunoglobulin G (IgG) antibodies and nontoxic anthrax proteins, in which the C terminus of the IgG heavy chain is connected to the side chain of anthrax toxin protective antigen. This strategy enabled efficient conjugation of protective antigen variants to trastuzumab (Tmab) and cetuximab (Cmab) antibodies. The conjugates effectively perform intracellular delivery of edema factor and N terminus of lethal factor (LF_N) fused with diphtheria toxin and Ras/Rap1-specific endopeptidase. Each conjugate shows high specificity for cells expressing human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), respectively, and potent activity across six Tmab- and Cmab-resistant cell lines. The conjugates also exhibit increased pharmacokinetics and pronounced in vivo safety, which shows promise for further therapeutic development.