Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A

Liu,Hao; Lindbo,Sarah; Ding,Haozhong; Altai,Mohamed; Garousi,Javad; Orlova,Anna; Tolmachev,Vladimir; Hober,Sophia; Gräslund,Torbjörn

doi:10.3892/ijo.2019.4814

Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A

Authors:
- Hao Liu
- Sarah Lindbo
- Haozhong Ding
- Mohamed Altai
- Javad Garousi
- Anna Orlova
- Vladimir Tolmachev
- Sophia Hober
- Torbjörn Gräslund
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Affiliations: Department of Protein Science, KTH Royal Institute of Technology, 114 17 Stockholm, Sweden, Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden, Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden
Published online on: May 28, 2019 https://doi.org/10.3892/ijo.2019.4814
Pages: 309-319

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Abstract

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain‑derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA‑derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half‑life extension, can be used for specific killing of HER2‑expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (KD 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (KD) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50‑values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA‑derivatives are promising agents for targeted cancer therapy.

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1	Alewine C, Hassan R and Pastan I: Advances in anticancer immunotoxin therapy. Oncologist. 20:176–185. 2015. View Article : Google Scholar : PubMed/NCBI
2	Akbari B, Farajnia S, Ahdi Khosroshahi S, Safari F, Yousefi M, Dariushnejad H and Rahbarnia L: Immunotoxins in cancer therapy: Review and update. Int Rev Immunol. 36:207–219. 2017. View Article : Google Scholar : PubMed/NCBI
3	Kreitman RJ, Dearden C, Zinzani PL, Delgado J, Karlin L, Robak T, Gladstone DE, le Coutre P, Dietrich S, Gotic M, et al: Moxetumomab pasudotox in relapsed/refractory hairy cell leukemia. Leukemia. 32:1768–1777. 2018. View Article : Google Scholar : PubMed/NCBI
4	Martin-Killias P, Stefan N, Rothschild S, Plückthun A and Zangemeister-Wittke U: A novel fusion toxin derived from an EpCAM-specific designed ankyrin repeat protein has potent antitumor activity. Clin Cancer Res. 17:100–110. 2011. View Article : Google Scholar
5	Prince HM, Duvic M, Martin A, Sterry W, Assaf C, Sun Y, Straus D, Acosta M and Negro-Vilar A: Phase III placebo-controlled trial of denileukin diftitox for patients with cutaneous T-cell lymphoma. J Clin Oncol. 28:1870–1877. 2010. View Article : Google Scholar : PubMed/NCBI
6	Onda M, Nagata S, FitzGerald DJ, Beers R, Fisher RJ, Vincent JJ, Lee B, Nakamura M, Hwang J, Kreitman RJ, et al: Characterization of the B cell epitopes associated with a truncated form of Pseudomonas exotoxin (PE38) used to make immunotoxins for the treatment of cancer patients. J Immunol. 177:8822–8834. 2006. View Article : Google Scholar : PubMed/NCBI
7	Liu W, Onda M, Lee B, Kreitman RJ, Hassan R, Xiang L and Pastan I: Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes. Proc Natl Acad Sci USA. 109:11782–11787. 2012. View Article : Google Scholar : PubMed/NCBI
8	Onda M, Beers R, Xiang L, Lee B, Weldon JE, Kreitman RJ and Pastan I: Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes. Proc Natl Acad Sci USA. 108:5742–5747. 2011. View Article : Google Scholar : PubMed/NCBI
9	Mazor R, Vassall AN, Eberle JA, Beers R, Weldon JE, Venzon DJ, Tsang KY, Benhar I and Pastan I: Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based on Pseudomonas exotoxin A. Proc Natl Acad Sci USA. 109:E3597–E3603. 2012. View Article : Google Scholar : PubMed/NCBI
10	Onda M, Beers R, Xiang L, Nagata S, Wang Q-C and Pastan I: An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes. Proc Natl Acad Sci USA. 105:11311–11316. 2008. View Article : Google Scholar : PubMed/NCBI
11	Liu H, Seijsing J, Frejd FY, Tolmachev V and Gräslund T: Target-specific cytotoxic effects on HER2-expressing cells by the tripartite fusion toxin Z_HER2:2891-ABD-PE38X8, including a targeting affibody molecule and a half-life extension domain. Int J Oncol. 47:601–609. 2015. View Article : Google Scholar : PubMed/NCBI
12	Altai M, Liu H, Orlova A, Tolmachev V and Gräslund T: Influence of molecular design on biodistribution and targeting properties of an Affibody-fused HER2-recognising anticancer toxin. Int J Oncol. 49:1185–1194. 2016. View Article : Google Scholar : PubMed/NCBI
13	Weldon JE, Skarzynski M, Therres JA, Ostovitz JR, Zhou H, Kreitman RJ and Pastan I: Designing the furin-cleavable linker in recombinant immunotoxins based on Pseudomonas exotoxin A. Bioconjug Chem. 26:1120–1128. 2015. View Article : Google Scholar : PubMed/NCBI
14	Mazor R, Onda M, Park D, Addissie S, Xiang L, Zhang J, Hassan R and Pastan I: Dual B- and T-cell de-immunization of recombinant immunotoxin targeting mesothelin with high cytotoxic activity. Oncotarget. 7:29916–29926. 2016. View Article : Google Scholar : PubMed/NCBI
15	Wurch T, Pierré A and Depil S: Novel protein scaffolds as emerging therapeutic proteins: From discovery to clinical proof-of-concept. Trends Biotechnol. 30:575–582. 2012. View Article : Google Scholar : PubMed/NCBI
16	Zielinski R, Lyakhov I, Jacobs A, Chertov O, Kramer-Marek G, Francella N, Stephen A, Fisher R, Blumenthal R and Capala J: Affitoxin - a novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors. J Immunother. 32:817–825. 2009. View Article : Google Scholar : PubMed/NCBI
17	Zielinski R, Lyakhov I, Hassan M, Kuban M, Shafer-Weaver K, Gandjbakhche A and Capala J: HER2-affitoxin: A potent therapeutic agent for the treatment of HER2-overexpressing tumors. Clin Cancer Res. 17:5071–5081. 2011. View Article : Google Scholar : PubMed/NCBI
18	Simon M, Stefan N, Borsig L, Plückthun A and Zangemeister-Wittke U: Increasing the antitumor effect of an EpCAM-targeting fusion toxin by facile click PEGylation. Mol Cancer Ther. 13:375–385. 2014. View Article : Google Scholar
19	Ham S, Min KA, Yang JW and Shin MC: Fusion of gelonin and anti-insulin-like growth factor-1 receptor (IGF-1R) affibody for enhanced brain cancer therapy. Arch Pharm Res. 40:1094–1104. 2017. View Article : Google Scholar : PubMed/NCBI
20	Nilvebrant J and Hober S: The albumin-binding domain as a scaffold for protein engineering. Comput Struct Biotechnol J. 6:e2013030092013. View Article : Google Scholar : PubMed/NCBI
21	Alm T, Yderland L, Nilvebrant J, Halldin A and Hober S: A small bispecific protein selected for orthogonal affinity purification. Biotechnol J. 5:605–617. 2010. View Article : Google Scholar : PubMed/NCBI
22	Nilvebrant J, Åstrand M, Löfblom J and Hober S: Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3. Cell Mol Life Sci. 70:3973–3985. 2013. View Article : Google Scholar : PubMed/NCBI
23	Nilvebrant J, Åstrand M, Georgieva-Kotseva M, Björnmalm M, Löfblom J and Hober S: Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin. PLoS One. 9:e1030942014. View Article : Google Scholar : PubMed/NCBI
24	Hynes NE and MacDonald G: ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol. 21:177–184. 2009. View Article : Google Scholar : PubMed/NCBI
25	Tai W, Mahato R and Cheng K: The role of HER2 in cancer therapy and targeted drug delivery. J Control Release. 146:264–275. 2010. View Article : Google Scholar : PubMed/NCBI
26	Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, et al: Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 344:783–792. 2001. View Article : Google Scholar : PubMed/NCBI
27	von Minckwitz G, Procter M, de Azambuja E, Zardavas D, Benyunes M, Viale G, Suter T, Arahmani A, Rouchet N, Clark E, et al: APHINITY Steering Committee and Investigators: Adjuvant pertuzumab and trastuzumab in early HER2-positive breast cancer. N Engl J Med. 377:122–131. 2017. View Article : Google Scholar : PubMed/NCBI
28	Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J, Pegram M, Oh DY, Diéras V, Guardino E, et al EMILIA Study Group: Trastuzumab emtansine for HER2-positive advanced breast cancer. N Engl J Med. 367:1783–1791. 2012. View Article : Google Scholar : PubMed/NCBI
29	Lindbo S, Garousi J, Åstrand M, Honarvar H, Orlova A, Hober S and Tolmachev V: Influence of histidine-containing tags on the biodistribution of ADAPT scaffold proteins. Bioconjug Chem. 27:716–726. 2016. View Article : Google Scholar : PubMed/NCBI
30	Garousi J, Lindbo S, Nilvebrant J, Åstrand M, Buijs J, Sandström M, Honarvar H, Orlova A, Tolmachev V and Hober S: ADAPT, a novel scaffold protein-based probe for radionuclide imaging of molecular targets that are expressed in disseminated cancers. Cancer Res. 75:4364–4371. 2015. View Article : Google Scholar : PubMed/NCBI
31	Hopp J, Hornig N, Zettlitz KA, Schwarz A, Fuss N, Müller D and Kontermann RE: The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein. Protein Eng Des Sel. 23:827–834. 2010. View Article : Google Scholar : PubMed/NCBI
32	Makrides SC, Nygren PÅ, Andrews B, Ford PJ, Evans KS, Hayman EG, Adari H, Uhlén M and Toth CA: Extended in vivo half-life of human soluble complement receptor type 1 fused to a serum albumin-binding receptor. J Pharmacol Exp Ther. 277:534–542. 1996.PubMed/NCBI
33	Orlova A, Jonsson A, Rosik D, Lundqvist H, Lindborg M, Abrahmsen L, Ekblad C, Frejd FY and Tolmachev V: Site-specific radiometal labeling and improved biodistribution using ABY-027, a novel HER2-targeting affibody molecule-albumin-binding domain fusion protein. J Nucl Med. 54:961–968. 2013. View Article : Google Scholar : PubMed/NCBI
34	Jonsson A, Dogan J, Herne N, Abrahmsén L and Nygren PÅ: Engineering of a femtomolar affinity binding protein to human serum albumin. Protein Eng Des Sel. 21:515–527. 2008. View Article : Google Scholar : PubMed/NCBI
35	Gunneriusson E, Nord K, Uhlén M and Nygren P: Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling. Protein Eng. 12:873–878. 1999. View Article : Google Scholar : PubMed/NCBI
36	Garousi J, Lindbo S, Honarvar H, Velletta J, Mitran B, Altai M, Orlova A, Tolmachev V and Hober S: Influence of the N-terminal composition on targeting properties of radiometal-labeled anti-HER2 scaffold protein ADAPT₆. Bioconjug Chem. 27:2678–2688. 2016. View Article : Google Scholar : PubMed/NCBI
37	Pastan I, Beers R and Bera TK: Recombinant immunotoxins in the treatment of cancer. Methods Mol Biol. 248:503–518. 2004.PubMed/NCBI
38	Zahnd C, Kawe M, Stumpp MT, de Pasquale C, Tamaskovic R, Nagy-Davidescu G, Dreier B, Schibli R, Binz HK, Waibel R, et al: Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: Effects of affinity and molecular size. Cancer Res. 70:1595–1605. 2010. View Article : Google Scholar : PubMed/NCBI
39	Thurber GM, Schmidt MM and Wittrup KD: Antibody tumor penetration: Transport opposed by systemic and antigen-mediated clearance. Adv Drug Deliv Rev. 60:1421–1434. 2008. View Article : Google Scholar : PubMed/NCBI
40	Heldin CH, Rubin K, Pietras K and Östman A: High interstitial fluid pressure - an obstacle in cancer therapy. Nat Rev Cancer. 4:806–813. 2004. View Article : Google Scholar : PubMed/NCBI
41	Roopenian DC and Akilesh S: FcRn: The neonatal Fc receptor comes of age. Nat Rev Immunol. 7:715–725. 2007. View Article : Google Scholar : PubMed/NCBI
42	Schmidt MM and Wittrup KD: A modeling analysis of the effects of molecular size and binding affinity on tumor targeting. Mol Cancer Ther. 8:2861–2871. 2009. View Article : Google Scholar : PubMed/NCBI
43	Nielsen R, Christensen EI and Birn H: Megalin and cubilin in proximal tubule protein reabsorption: From experimental models to human disease. Kidney Int. 89:58–67. 2016. View Article : Google Scholar : PubMed/NCBI