Control of nanopore formation using external triggers
Mutter, Natalie
DOI:
10.33612/diss.131163011
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2020
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Mutter, N. (2020). Control of nanopore formation using external triggers. University of Groningen.
https://doi.org/10.33612/diss.131163011
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5
SUMMARY
Cancer is one of the leading causes of death worldwide.1 Currently, common cancer therapies
include surgery, radiation therapy and chemotherapy. These common cancer treatments suffer from serious disadvantages such as a lack of effectiveness and severe side effects due to off-target toxicity.2,3
Chapter 1 provides a general introduction of current approaches to overcome the limitations of
common cancer treatments. Latterly, researchers focused on developing targeted cancer therapies to surpass the efficacy of treatments currently used in the clinics and to avoid damage of healthy cells.4,5 Targeted cancer therapeutics are drugs developed to target cancer specific molecules
that are involved in carcinogenesis, tumor growth, or metastasis.6 In particular, protein-based
drugs are under investigation as new promising group of potential anticancer pharmaceuticals. Advances in genetic as well as protein engineering provide possibilities to optimize protein properties to facilitate their therapeutic potential.7,8 A main focus in cancer drug research is
the development of monoclonal antibodies (mAbs) which bind with high selectivity to tumor antigens, thus blocking their biological function and marking the cells for the body’s immune system.9,10 However, their antitumor activity is limited, therefore they were utilized as delivery
vehicles and conjugated to drugs11, toxins12, cytokines13 or radioactive particles14. Taking all
these aspects into consideration, the selectivity of cell-killing drugs toward target cells could be increased.6 One example to improve selectivity are immunotoxins consisting of a cell-targeting
element fused to a potent toxin.15
In Chapter 2 we describe the preparation of immunotoxins comprising of pore-forming toxins (PFTs). Originally, immunotoxins were based on plant toxins like ricin16 or bacterial toxins
like Pseudomonas exotoxin A17 and diphtheria toxin18, which lyse cells by inhibiting protein
synthesis. Efficiency of these potent toxins depends on their capability to penetrate tumor tissue and adequate internalization rates of the target receptor to allow sufficient accumulation of toxin causing cell death.19,20 Alternatively, immunotoxins can be built from PFTs, which assemble into
oligomeric pores in membranes of target cells leading to cell death.21 We chemically conjugated
cytolysin A (ClyA) from Salmonella typhi with the small molecule folate to target cancer cells overexpressing folate receptors. Bioorthogonal chemical linkages can be instable that might result in heterogeneous samples. Therefore, we also genetically fused ClyA to a nanobody, the variable domain of a single-domain camelid antibody, against the epidermal growth factor
receptor (EGFR). However, the cytotoxicity of PFTs depends on the membrane composition,22
from Actina fragacea. Conjugation to cell-targeting elements resulted in two-fold increased cytotoxicity toward cell lines overexpressing the cognate receptors. The main limitation of PFTs for pharmacological applications is their basal toxicity toward all cells. We looked into nature and adopted the principle of synthesizing toxins as protoxin. The activity of FraC could be completely abolished by introducing a polypeptide segment at the transmembrane N-terminus via a linker containing a furin cleavage site. Cancer cells often overexpress proteases, such as furin or cathepsin B, which promote invasion and metastasis by digesting the extracellular matrix and penetrating the basal lamina.23,24 This allowed in situ proteolysis by the cancer-associated
protease furin and resulted in activation of FraC. We combined the two approaches, attachment of a nanobody and addition of a polypeptide segment to the N-terminus, to prepare a PFT with a two-degree control of toxin activity. Additionally, we could use directed evolution to fine-tune the properties of our protein-based therapeutic agents. Directed evolution mimics the process of natural selection and optimizes protein properties in iterative cycles. The gene encoding the protein of interest is diversified by mutagenesis and expressed in a suitable host. Then proteins with desired functions are identified by appropriate screening or selection methods. Finally, selected variants are amplified and serve as template for subsequent rounds of directed evolution.25
We used directed evolution to improve the soluble expression, to increase the target affinity and to reduce off-target cytotoxicity of our immunotoxin. Finally, our designed targeted cancer therapeutic consists of three elements that can be easily exchanged: a targeting element, a pore-forming toxin and a protease trigger.
Addition of an external trigger could achieve enhanced control over PFT activity. An particular attractive external trigger is light, because it is noninvasive, biorthogonal and allows spatiotemporal control.26
In Chapter 3 we describe the preparation of a photocontrolled nanopore. FraC was equipped with photoswitchable azobenzene molecules to remotely control nanopore assembly by light. Azobenzenes have two isomers, the thermally stable trans state, and upon irradiation with light it interconverts into the metastable cis state. We chose azobenzenes due to their favorable photophysical properties, such as high photostationary states and quantum yields as well as fast
photoisomerization and low rates of photobleaching.27 Azobenzene pendants were covalently
attached to cysteine-modified FraC. Pore-formation of FraC depends on sphingomyelin,28
therefore we introduced cysteines at various locations near the sphingomyelin binding pocket, hoping to substitute the role of sphingomyelin as membrane anchor. We expected that switching between the two isoforms modulates the affinity of FraC monomers for the lipid membrane.
hemolytic activity assays. Rewardingly, we found several constructs that were inactive in the thermal resting state (trans isomer), but active in the light excited state (cis isomer). Notably, FraC modified at position Y138 and attached to a para-sulfonated azobenzene (FraC-Y138C-C) showed five-fold higher hemolytic activity in the cis state compared to the trans state. Importantly, at selected concentrations FraC-Y138C-C was completely inactive in the trans isoform while inducing complete red blood cell lysis in the cis isoform. Finally, the cytotoxicity of FraC-Y138C-C could be completely stopped by irradiation with white light. The reversible control of nanopore formation provides potential for the use in clinical applications. The inactive toxin can get locally activated at the tumor site by irradiation, and the damage of healthy neighboring tissue can be prevented by inactivating diffusing active monomers with white light. Except for their potential use as therapeutics, light-responsive FraC might also be advantageous in biosensing applications. FraC nanopores can be used to sense and detect single molecules, such as peptides, and proteins.29
In particular, thousands of individual nanopores are now integrated in portable and low-cost devices to sequence DNA.30 The ability to control nanopore assembly with light would facilitate
precise preparation of arrays of single nanopores, thus reducing the cost of multiplexing. A lingering challenge in cancer drug design is the lack of selectivity leading to severe side-effects. In Chapter 2 and 3 we showed two different approaches to reduce off-target toxicity of PFTs. The next step is to combine the concepts, implementing a dual activation mechanism to further improve the protein therapeutic. Hence, in Chapter 4 we describe the preparation of a genetically encoded PFT activated by both proteases and light of a variety of wavelengths. The photoresponsive light-oxygen-voltage domain of Avena sativa (AsLOV2) was used to regulate the function of FraC by light. In the dark the AsLOV2 domain binds noncovalently its flavin mononucleotide (FMN) cofactor. Upon blue light irradiation a covalent bond is formed between
the FMN and a conserved cysteine of the AsLOV2 domain.31 Adduct formation promotes
unfolding and detachment of the C-terminal α-helix from the β-sheet core of the AsLOV2 domain.32 Therefore, we fused the C-terminus of the AsLOV2 domain to the N-terminus of FraC
via a 10 amino acid long linker including a protease cleavage site. In the dark the AsLOV2 domain buries the protease cleavage site, and the fusion protein was inactive. Irradiation with blue light results in structural rearrangement of the AsLOV2 domain and exposure of the protease cleavage site. Therefore, simultaneous irradiation with light and in situ proteolytic cleavage resulted in activation of FraC and cytotoxicity. However, blue light suffers from cytotoxicity and limited tissue penetration abilities.33,34 Activation of AsLOV2 domains with higher wavelength light could
enable for further applications in optogenetics such as multiplexing. Notably, our fusion protein could be used as screening platform to evolve AsLOV2 domains with enhanced photoactivating properties. A few rounds of directed evolution could reduce the dark state activity of
AsLOV2-FraC and additional random mutagenesis and DNA shuffling with subsequent screening on red blood cells could identify variants activated by green or even red light. Interestingly, our results indicate that irradiation with higher wavelength light induce conformation changes by a parallel mechanism independent of the FMN-cysteine adduct formation. Blue and green responding AsLOV2-FraC variants were successfully employed to target specifically cancer cells expressing the corresponding protease and could be possible candidates for targeted cancer therapy. In summary, this thesis describes a method to design targeted cancer drugs on the basis of pore-forming toxins. We presented a modular approach to equip pore-forming toxins with different targeting and trigger mechanisms. Additionally, we used directed evolution to fine--tune the properties of our protein-based therapeutics.
OUTLOOK
The applications of pore-forming toxins (PFTs) as drugs are in an early stage and a variety of challenges remain to be addressed. The development of targeted cancer therapeutics, such as immunotoxins, face three major challenges, their specificity toward cancer cells, their ability to penetrate solid tumors, and their immunogenicity.35
In this thesis the focused was to increase the selectivity and controlling the activity of pore-forming toxin based targeted cancer therapeutics. In Chapter 2 the toxicity toward target cells could be increased up to 4-fold by conjugation with a cell-targeting element. A possibility to further increase and fine-tune cytotoxicity toward target cells is to utilize the oligomeric nature of the pores. It allows individual modification of the protomeric subunits with multiple different targeting elements, such as nanobodies, aptamers or small molecules. Additionally, the pores could be investigated as drug delivery systems by increasing the permeability of target cells for suicidal cancer gene therapy36 or to import impermeable drugs37.
A main limitation of PFTs in the development of drugs is there basal toxicity towards all cells due to targeting of specific widespread lipids, such as sphingomyelin or cholesterol. However, it also can be a chance, because some cancers have altered lipid compositions. For example, phosphatidylserine is located in the inner leaflet of the membrane bilayer of healthy cells, in contrast in cancer cells it is exposed in the outer leaflet.38 Therefore, changing the lipid specificity
of PFTs to phosphatidylserine, could be a possibility to increase selectivity.
The recently emerging field of photopharmacology offers fascinating possibilities in chemical biology, pharmaceutical research and medicine to control biological functions. In Chapter 3 the PFT FraC was equipped with photoswitchable azobenzene molecules to reversibly control toxicity by UV light. The photochromic compounds need to fulfill a set of requirements, such as high photostationary states and quantum yields, high water solubility, low toxicity, and sufficient metabolic stability.39 The main limitation of our system is the use of UV light for activation of the
toxin, because irradiation with UV light is not ideal mainly because of the limited penetration depth, potential damage to living tissue and interference with switching in biological samples.40,41
Therefore, a remaining challenge is to develop new photoswitches, which can be operated with visible light. Furthermore, the light-activated FraC could be combined with the triggered immunotoxin prepared in Chapter 2, to further target cytotoxicity of FraC.
In Chapter 4 a light- and protease-activated PFT was prepared to reduce off-target toxicity. However, to control activity of the PFT with light a photosensory domain was used in place of a chemical photoswitch. Thus, the protein-based drug could be completely genetically encoded and chemical attachment and sophisticated purification was avoided. Although the structural difference between light and dark state is distinctive, activated AsLOV-FraC cannot be inactivated again. Advantagous is that multiple photosensory domains are available responding to visible light, but the majority of photoreceptors absorb blue light.42 One example is the heavily studied
AsLOV2 domain. Directed evolution was used to prepare AsLOV2 domains selectively activated by green or red light. The next step is to test these evolved AsLOV2 domains as optogenetic
and imaging tool, in particular in experiments where deep tissue penetration is required.34
Additionally, it would be interesting to test their suitability in optogenetic applications were multiple and orthogonal excitation is required. Furthermore, the new variants could give new insights into the photocycle of LOV domains. Apart from that our PFT-based screening platform for directed evolution could be used to evolve other proteins, for example proteases with tailored cleavage sites.
In this thesis several methods to increase specificity of PFTs are explored, but additional questions need to be addressed to develop effective targeted cancer therapies. On the one hand, it is important to determine the pharmacokinetic properties, such as the stability, absorption and distribution of the proteins.43 A known limitation of immunotoxins is their molecular size
resulting in poor tumor penetration.44 Therefore, it could be necessary to reduce the size of our
immunotoxin by using different cell-targeting elements and smaller proteins for the protease trigger.
On the other hand, the immunogenicity needs to be determined, a major limitation for common protein therapeutics. Previous research discovered that PFTs can cause immune reactions.45,46
Therefore, it will be most properly necessary to investigate appropriate methods to prevent immunogenicity. Various strategies were developed to reduce immunogenicity of classical immunotoxins such as combination therapy with immunosuppressive molecules47 or polyethylene
glycol modifications48. More promising seems to be humanization49,50, or the mapping and removal
of B-and T-cell epitopes51–53. In particular, immunotoxins showed lower immunogenicity after
mutating of B- and T-cell epitopes and remained high cytotoxicity toward target cells.53
This thesis presents first steps towards the development of pore-forming toxins as targeted cancer therapies, however for successful implementation is further research required.
REFERENCES
(1) Dagenais, G. R.; Leong, D. P.; Rangarajan, S.; Lanas, F.; Lopez-Jaramillo, P.; Gupta, R.; Diaz, R.; Avezum, A.; Oliveira, G. B. F.; Wielgosz, A.; Parambath, S. R.; Mony, P.; Alhabib, K. F.; Temizhan, A.; Ismail, N.; Chifamba, J.; Yeates, K.; Khatib, R.; Rahman, O.; Zatonska, K.; Kazmi, K.; Wei, L.; Zhu, J.; Rosengren, A.; Vijayakumar, K.; Kaur, M.; Mohan, V.; Yusufali, A.; Kelishadi, R.; Teo, K. K.; Joseph, P.; Yusuf, S. Variations in Common Diseases, Hospital Admissions, and Deaths in Middle-Aged Adults in 21 Countries from Five Continents (PURE): A Prospective Cohort Study. Lancet
2019. https://doi.org/10.1016/s0140-6736(19)32007-0.
(2) Sereno, M.; Brunello, A.; Chiappori, A.; Barriuso, J.; Casado, E.; Belda, C.; de Castro, J.; Feliu, J.; González-Barón, M. Cardiac Toxicity: Old and New Issues in Anti-Cancer Drugs. Clin. Transl. Oncol.
2008, 10 (1), 35–46.
(3) Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P. G. Cancer Drug Resistance: An Evolving Paradigm. Nature Reviews Cancer. October 2013, pp 714–726. https://doi.org/10.1038/ nrc3599.
(4) Aggarwal, S. Targeted Cancer Therapies. Nature Reviews Drug Discovery. June 2010, pp 427–428. https://doi.org/10.1038/nrd3186.
(5) Chari, R. V. J. Targeted Cancer Therapy: Conferring Specificity to Cytotoxic Drugs. Acc. Chem. Res.
2008, 41 (1), 98–107. https://doi.org/10.1021/ar700108g.
(6) Baudino, T. Targeted Cancer Therapy: The Next Generation of Cancer Treatment. Curr. Drug Discov.
Technol. 2015, 12 (1), 3–20. https://doi.org/10.2174/1570163812666150602144310.
(7) Vasserot, A. P.; Dickinson, C. D.; Tang, Y.; Huse, W. D.; Manchester, K. S.; Watkins, J. D. Optimization of Protein Therapeutics by Directed Evolution. Drug Discovery Today. January 21, 2003, pp 118–126. https://doi.org/10.1016/S1359-6446(02)02590-4.
(8) Stryjewska, A.; Kiepura, K.; Librowski, T.; Lochyñski, S. Biotechnology and Genetic Engineering in the New Drug Development. Part I. DNA Technology and Recombinant Proteins. Pharmacol. Reports
2013, 65 (5), 1075–1085. https://doi.org/10.1016/S1734-1140(13)71466-X.
(9) Sela-Culang, I.; Kunik, V.; Ofran, Y. The Structural Basis of Antibody-Antigen Recognition. Front.
Immunol. 2013, 4 (OCT). https://doi.org/10.3389/fimmu.2013.00302.
(10) Scott, A. M.; Wolchok, J. D.; Old, L. J. Antibody Therapy of Cancer. Nature Reviews Cancer. March 22, 2012, pp 278–287. https://doi.org/10.1038/nrc3236.
(11) Lambert, J. M.; Berkenblit, A. Antibody-Drug Conjugates for Cancer Treatment. Annu. Rev. Med.
2018, 69 (1), 191–207. https://doi.org/10.1146/annurev-med-061516-121357.
(12) Alewine, C.; Hassan, R.; Pastan, I. Advances in Anticancer Immunotoxin Therapy. Oncologist 2015,
20 (2), 176–185. https://doi.org/10.1634/theoncologist.2014-0358.
(13) Pasche, N.; Neri, D. Immunocytokines: A Novel Class of Potent Armed Antibodies. Drug Discovery
Today. June 2012, pp 583–590. https://doi.org/10.1016/j.drudis.2012.01.007.
(14) Kitson, S. L.; Cuccurullo, V.; Moody, T. S.; Mansi, L. Radionuclide Antibody-Conjugates, a Targeted Therapy towards Cancer. Curr. Radiopharm. 2013, 6 (2), 57–71.
(15) Pastan, I.; Hassan, R.; Fitzgerald, D. J.; Kreitman, R. J. Immunotoxin Therapy of Cancer. Nat. Rev.
Cancer 2006, 6 (7), 559–565. https://doi.org/10.1038/nrc1891.
(16) Krolick, K. A.; Uhr, J. W.; Slavin, S.; Vitetta, E. S. In Vivo Therapy of a Murine B Cell Tumor (BCL1) Using Antibody-Ricin A Chain Immunotoxins. J. Exp. Med. 1982, 155 (6), 1797–1809. https://doi. org/10.1084/jem.155.6.1797.
(17) Mansfield, E.; Amlot, P.; Pastan, I.; FitzGerald, D. J. Recombinant RFB4 Immunotoxins Exhibit Potent Cytotoxic Activity for CD22-Bearing Cells and Tumors. Blood 1997, 90 (5), 2020–2026. https://doi. org/10.1182/blood.v90.5.2020.
(18) Williams, D. P.; Parker, K.; Bacha, P.; Bishai, W.; Borowski, M.; Genbauffe, F.; Strom, T. B.; Murphy, J. R. Diphtheria Toxin Receptor Binding Domain Substitution with Interleukin-2: Genetic Construction and Properties of a Diphtheria Toxin-Related Interleukin-2 Fusion Protein. Protein Eng. 1987, 1 (6), 493–498. https://doi.org/10.1093/protein/1.6.493.
(19) Polito, L.; Mercatelli, D.; Bortolotti, M.; Maiello, S.; Djemil, A.; Battelli, M.; Bolognesi, A. Two Saporin-Containing Immunotoxins Specific for CD20 and CD22 Show Different Behavior in Killing Lymphoma Cells. Toxins (Basel). 2017, 9 (6), 182. https://doi.org/10.3390/toxins9060182.
(20) Shan, L.; Liu, Y.; Wang, P. Recombinant Immunotoxin Therapy of Solid Tumors: Challenges and Strategies. J. Basic Clin. Med. 2013, 2 (2), 1–6.
(21) Cosentino, K.; Ros, U.; García-Sáez, A. J. Assembling the Puzzle: Oligomerization of α-Pore Forming Proteins in Membranes. Biochim. Biophys. Acta - Biomembr. 2016, 1858 (3), 457–466. https://doi. org/10.1016/j.bbamem.2015.09.013.
(22) Rojko, N.; Anderluh, G. How Lipid Membranes Affect Pore Forming Toxin Activity. Acc. Chem. Res.
2015, 48 (12), 3073–3079. https://doi.org/10.1021/acs.accounts.5b00403.
(23) Mohamed, M. M.; Sloane, B. F. Cysteine Cathepsins: Multifunctional Enzymes in Cancer. In Nature
Reviews Cancer; 2006; Vol. 6, pp 764–775. https://doi.org/10.1038/nrc1949.
(24) Jaaks, P.; Bernasconi, M. The Proprotein Convertase Furin in Tumour Progression. Int. J. Cancer
2017, 141 (4), 654–663. https://doi.org/10.1002/ijc.30714.
(25) Packer, M. S.; Liu, D. R. Methods for the Directed Evolution of Proteins. Nat. Rev. Genet. 2015, 16 (7), 379–394. https://doi.org/10.1038/nrg3927.
(26) Hüll, K.; Morstein, J.; Trauner, D. In Vivo Photopharmacology. Chemical Reviews. November 2018, pp 10710–10747. https://doi.org/10.1021/acs.chemrev.8b00037.
(27) Beharry, A. A.; Woolley, G. A. Azobenzene Photoswitches for Biomolecules. Chem. Soc. Rev. 2011,
40 (8), 4422–4437. https://doi.org/10.1039/c1cs15023e.
(28) Tanaka, K.; Caaveiro, J. M. M.; Morante, K.; González-Manãs, J. M.; Tsumoto, K. Structural Basis for Self-Assembly of a Cytolytic Pore Lined by Protein and Lipid. Nat. Commun. 2015, 6, 6337. https:// doi.org/10.1038/ncomms7337.
(29) Huang, G.; Willems, K.; Soskine, M.; Wloka, C.; Maglia, G. Electro-Osmotic Capture and Ionic Discrimination of Peptide and Protein Biomarkers with FraC Nanopores. Nat. Commun. 2017, 8 (1), 935. https://doi.org/10.1038/s41467-017-01006-4.
(30) Bayley, H. Nanopore Sequencing: From Imagination to Reality. Clin. Chem. 2015, 61 (1), 25–31. https://doi.org/10.1373/clinchem.2014.223016.
(31) Salomon, M.; Eisenreich, W.; Dürr, H.; Schleicher, E.; Knieb, E.; Massey, V.; Rüdiger, W.; Müller, F.; Bacher, A.; Richter, G. An Optomechanical Transducer in the Blue Light Receptor Phototropin from Avena Sativa. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (22), 12357–12361. https://doi.org/10.1073/ pnas.221455298.
(32) Harper, S. M.; Neil, L. C.; Gardner, K. H. Structural Basis of a Phototropin Light Switch. Science (80-.
). 2003, 301 (5639), 1541–1544. https://doi.org/10.1126/science.1086810.
(33) Yoshida, A.; Shiotsu-Ogura, Y.; Wada-Takahashi, S.; Takahashi, S.; Toyama, T.; Yoshino, F. Blue Light Irradiation-Induced Oxidative Stress in Vivo via ROS Generation in Rat Gingival Tissue. J.
Photochem. Photobiol. B. 2015, 151, 48–53. https://doi.org/10.1016/j.jphotobiol.2015.07.001.
(34) Ash, C.; Dubec, M.; Donne, K.; Bashford, T. Effect of Wavelength and Beam Width on Penetration in Light-Tissue Interaction Using Computational Methods. Lasers Med. Sci. 2017, 32 (8), 1909–1918. https://doi.org/10.1007/s10103-017-2317-4.
(35) Pastan, I.; Hassan, R.; FitzGerald, D. J.; Kreitman, R. J. Immunotoxin Treatment of Cancer *. Annu.
Rev. Med. 2007, 58 (1), 221–237. https://doi.org/10.1146/annurev.med.58.070605.115320.
(36) Yang, W. S.; Park, S. O.; Yoon, A. R.; Yoo, J. Y.; Kim, M. K.; Yun, C. O.; Kim, C. W. Suicide Cancer Gene Therapy Using Pore-Forming Toxin, Streptolysin O. Mol. Cancer Ther. 2006, 5 (6), 1610–1619. https://doi.org/10.1158/1535-7163.MCT-05-0515.
(37) Soletti, R. C.; de Faria, G. P.; Vernal, J.; Terenzi, H.; Anderluh, G.; Borges, H. L.; Moura-Neto, V.; Gabilan, N. H. Potentiation of Anticancer-Drug Cytotoxicity by Sea Anemone Pore-Forming Proteins in Human Glioblastoma Cells. Anticancer. Drugs 2008, 19 (5), 517–525. https://doi.org/10.1097/ CAD.0b013e3282faa704.
(38) Sharma, B.; Kanwar, S. S. Phosphatidylserine: A Cancer Cell Targeting Biomarker. Seminars in Cancer
Biology. Academic Press October 1, 2018, pp 17–25. https://doi.org/10.1016/j.semcancer.2017.08.012.
(39) Velema, W. A.; Szymanski, W.; Feringa, B. L. Photopharmacology: Beyond Proof of Principle. J. Am.
Chem. Soc. 2014, 136 (6), 2178–2191. https://doi.org/10.1021/ja413063e.
(40) Lerch, M. M.; Hansen, M. J.; van Dam, G. M.; Szymanski, W.; Feringa, B. L. Emerging Targets in Photopharmacology. Angew. Chemie Int. Ed. 2016, 55 (37), 10978–10999. https://doi.org/10.1002/ anie.201601931.
(41) Kalka, K.; Merk, H.; Mukhtar, H. Photodynamic Therapy in Dermatology. J. Am. Acad. Dermatol.
2000, 42 (3), 389–413; quiz 414–416.
(42) Losi, A.; Gardner, K. H.; Möglich, A. Blue-Light Receptors for Optogenetics. Chemical Reviews. American Chemical Society November 14, 2018, pp 10659–10709. https://doi.org/10.1021/acs. chemrev.8b00163.
(43) Protein Pharmacokinetics and Metabolism; Ferraiolo, B. L., Mohler, M. A., Gloff, C. A., Eds.; Pharmaceutical Biotechnology; Springer US: Boston, MA, 1992; Vol. 1992. https://doi.org/10.1007/978-1-4899-2329-5.
(44) Sung, C.; Dedrick, R. L.; Hall, W. A.; Johnson, P. A.; Youle, R. J. The Spatial Distribution of
Immunotoxins in Solid Tumors: Assessment by Quantitative Autoradiography; 1993; Vol. 53.
(45) Yap, W. Y.; Hwang, J. S. Response of Cellular Innate Immunity to Cnidarian Pore-Forming Toxins.
Molecules. MDPI AG October 4, 2018. https://doi.org/10.3390/molecules23102537.
(46) Narat, M.; Maček, P.; Kotnik, V.; Sedmak, B. The Humoral and Cellular Immune Response to a Lipid Attenuated Pore-Forming Toxin from the Sea Anemone Actinia Equina L. Toxicon 1994, 32 (1), 65–71. https://doi.org/10.1016/0041-0101(94)90022-1.
(47) Siegall, C. B.; Haggerty, H. G.; Warner, G. L.; Chace, D.; Mixan, B.; Linsley, P. S.; Davidson, T. Prevention of Immunotoxin-Induced Immunogenicity by Coadministration with CTLA4Ig Enhances Antitumor Efficacy. J. Immunol. 1997, 159 (10), 5168–5173.
(48) Tsutsumi, Y.; Onda, M.; Nagata, S.; Lee, B.; Kreitman, R. J.; Pastan, I. Site-Specific Chemical Modification with Polyethylene Glycol of Recombinant Immunotoxin Anti-Tac(Fv)-PE38 (LMB-2) Improves Antitumor Activity and Reduces Animal Toxicity and Immunogenicity. Proc. Natl. Acad.
Sci. U. S. A. 2000, 97 (15), 8548–8553. https://doi.org/10.1073/pnas.140210597.
(49) Harding, F. A.; Stickler, M. M.; Razo, J.; DuBridge, R. B. The Immunogenicity of Humanized and Fully Human Antibodies: Residual Immunogenicity Resides in the CDR Regions. MAbs 2010, 2 (3), 256–265. https://doi.org/10.4161/mabs.2.3.11641.
(50) Mathew, M.; Verma, R. S. Humanized Immunotoxins: A New Generation of Immunotoxins for Targeted Cancer Therapy. Cancer Sci. 2009, 100 (8), 1359–1365. https://doi.org/10.1111/j.1349-7006.2009.01192.x.
(51) Mazor, R.; Kaplan, G.; Park, D.; Jang, Y.; Lee, F.; Kreitman, R.; Pastan, I. Rational Design of Low Immunogenic Anti CD25 Recombinant Immunotoxin for T Cell Malignancies by Elimination of T Cell Epitopes in PE38. Cell. Immunol. 2017, 313, 59–66. https://doi.org/10.1016/j.cellimm.2017.01.003. (52) Onda, M.; Beers, R.; Xiang, L.; Lee, B.; Weldon, J. E.; Kreitman, R. J.; Pastan, I. Recombinant
Immunotoxin against B-Cell Malignancies with No Immunogenicity in Mice by Removal of B-Cell Epitopes. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (14), 5742–5747. https://doi.org/10.1073/
(53) Mazor, R.; Onda, M.; Park, D.; Addissie, S.; Xiang, L.; Zhang, J.; Hassan, R.; Pastan, I. Dual B- and T-Cell de-Immunization of Recombinant Immunotoxin Targeting Mesothelin with High Cytotoxic Activity. Oncotarget 2016, 7 (21), 29916–29926. https://doi.org/10.18632/oncotarget.9171.
SAMENVATTING
Kanker is wereldwijd een van de belangrijkste doodsoorzaken.1 De meest gebruikte
behandelmethoden zijn momenteel chirurgie, bestraling en chemotherapie. Deze veel voorkomende kankerbehandelingen hebben enkele ernstige nadelen, zoals ineffectiviteit of ernstige bijwerkingen veroorzaakt door off-target toxiciteit.2,3
Hoofdstuk 1 geeft een algemene inleiding tot de huidige benaderingen om de nadelen van de
meest voorkomende kankerbehandelingen te overwinnen. De laatste tijd hebben onderzoekers zich gericht op het ontwikkelen van gerichte kankertherapieën om de effectiviteit van de huidige behandelingen te overtreffen en om beschadiging van gezonde cellen te voorkomen.4,5 Gerichte
kankertherapieën zijn geneesmiddelen die zijn ontwikkeld om te werken tegen kankerspecifieke
moleculen die betrokken zijn bij de ontwikkeling van kanker, tumorgroei of metastase.6
Vooral op proteïne gebaseerde medicijnen worden onderzocht als een nieuwe veelbelovende groep van potentiële actieve werkzame stoffen. Hun therapeutisch potentieel werd mogelijk gemaakt door vooruitgang op het gebied van genetische manipulatie en eiwit-engineering en door hun technische vermogen om de eigenschappen van het eiwit te verbeteren.7,8 Een van de
zwaartepunten van geneesmiddelenonderzoek tegen kanker is de ontwikkeling van monoklonale antistoffen die tumorantigenen met hoge selectiviteit binden, waardoor hun biologische functie
wordt geblokkeerd en de cellen voor het lichaamseigene immuunsysteem worden gemarkeerd.9,10
Ze hebben echter een beperkte anti-tumoractiviteit en werden daarom geconjugeerd aan medicijnen11, toxines12, cytokines13 of radioactieve deeltjes14 om ze naar de juiste doelcel te leiden.
Op deze manier werd de selectiviteit van de celdodende middelen voor de doelcellen vergroot.6
Een voorbeeld ter verbetering van de selectiviteit zijn immunotoxines, die bestaan uit een element dat selectief aan de doelcel bindt en een sterk toxine.15
In Hoofdstuk 2 beschrijven we de productie van immunotoxines, die bestaan uit porievormende toxines (Engels pore-forming toxins PFT’s). Immunotoxinen waren oorspronkelijk gebaseerd
op plantentoxines zoals ricine16 of bacteriële toxines zoals Pseudomonas exotoxine A17
en difterietoxine18, die cellen lyseren door de proteïnesynthese te remmen. De efficiëntie
van deze sterke toxines hangt af van het vermogen in tumorweefsel door te dringen en de internalisatiepercentage van de doelreceptor, omdat het nodig is om voldoende toxine te verrijken om de celdood te veroorzaken.19,20 Als alternatief kunnen immunotoxinen geproduceerd worden
uit PFT’s, die oligomere poriën vormen in het doelcelmembraan, wat op zijn beurt leidt tot celdood.21 Daarom hebben we cytolysine A (ClyA) van Salmonella typhi chemisch geconjugeerd
tot overexpressie brengen. Bio-orthogonale chemische bindingen kunnen instabiel zijn en tot heterogene monsters leiden. Daarom hebben we ClyA ook genetisch gefuseerd met een nanobody, het variabele domein van een antistof, tegen de EGF-receptor (Engels epidermal growth factor
receptor). De cytotoxiciteit van PFT’s hangt echter af van de membraansamenstelling.22 Om onze
aanpak te veralgemenen, hebben we ClyA ingeruild voor de Actinoporin Fragaceatoxin C (FraC) van Actina fragacea. Conjugatie aan de celbindende component leidde tot een dubbele toename van de cytotoxiciteit tegen cellijnen die de overeenkomstige receptor tot overexpressie brengen. Een belangrijke beperking van PFT’s voor farmacologische toepassingen is hun toxiciteit voor alle cellen. We hebben daarom de natuur geïmiteerd en off-target toxiciteit voorkomen door FraC te produceren als protoxine. De activiteit werd volledig opgeheven door een polypeptidesegment aan het transmembraan N-terminus toe te voegen via een linker die een furin splitsingsplaats bevat. Kankercellen brengen proteasen zoals furin of cathepsine B vaak tot overexpressie. Deze bevorderen invasie en metastase door afbreken van de extracellulaire matrix en de penetratie van de basale lamina.23,24 Dit maakt in situ proteïnesplitsing door het kanker-geassocieerde
protease furin mogelijk en leidt tot de activering van FraC. We hebben de twee benaderingen, bevestiging van een nanobody en toevoeging van een polypeptidesegment aan het N-terminus, gecombineerd om een PFT te creëren waarvan de activiteit op twee manieren wordt gecontroleerd. Daarnaast hebben we om de eigenschappen van ons op proteïne gebaseerde actieve ingrediënt te verfijnen directionele evolutie gebruikt, waardoor natuurlijke selectie kan worden nagebootst en proteïne-eigenschappen kunnen worden geoptimaliseerd in herhalende cycli. Het gen dat voor het betreffende proteïne codeert, wordt door mutagenese gediversifieerd en tot expressie gebracht in een geschikte gastheer. Daarna worden proteïne met de gewenste eigenschappen geïdentificeerd met behulp van geschikte screening- of selectiemethoden. Uiteindelijk worden geselecteerde mutanten gedupliceerd en dienen ze als template voor daaropvolgende cycli van gerichte evolutie.25 We gebruikten gerichte evolutie om de oplosbare expressie te verbeteren, de
affiniteit van het doel te verhogen en de off-target affiniteit van ons immunotoxine te verlagen. Ten slotte bestaat ons gerichte kankermedicijn uit drie bouwstenen, een celbindend element, een porievormend toxine en een protease-trigger, welke gemakkelijk kunnen worden uitgewisseld. Het toevoegen van een externe trigger kan de controle over PFT-activiteit verbeteren. Een bijzonder aantrekkelijke trigger is licht, omdat het niet-invasief, bioorthogonaal is en ruimtelijke en temporele controle mogelijk maakt.26
In Hoofdstuk 3 beschrijven we het creëren van een fotogestuurde nanopore. Hiervoor is FraC uitgerust met fotowisselbare azobenzenen om de vorming van nanoporiën door licht te beheersen.
met licht wordt omgezet in de metastabiele cis-vorm. We hebben azobenzenen geselecteerd op basis van hun gunstige fotofysische eigenschappen, zoals hun hoge fotostationaire toestand en goede kwantumopbrengst, evenals hun snelle fotoisomerisatie bij lage fotoblekende snelheden.27
Verschillende azobenzeenverbindingen waren covalent gekoppeld aan cysteïne-gemodificeerde
FraC. De porievorming van FraC hangt af van de aandeel sfingomyeline in het membraan,28
dus introduceerden we cysteïnen op verschillende posities in de buurt van de sfingomyeline-bindingsplaats in de hoop de rol van sfingomyelinen als membraanankers te vervangen. We verwachtten dat de verandering tussen de twee isomeren de affiniteit van de FraC-monomeren voor het biomembraan zou veranderen. De cytolytische activiteit van de verschillende met azobenzeen gemodificeerde FraC varianten werd bepaald met behulp van een test voor hemolyseactiviteit. We waren in staat om enkele constructen te vinden die inactief waren in de thermische rusttoestand (trans-isomeer), maar actief waren in de door licht stimuleerde toestand (cis-isomeer). In het bijzonder toonde FraC gemodificeerd op positie Y138 en geassocieerd met het para-sulfoneerde azobenzeen (FraC-Y138C-C) een vijfvoudig hogere hemolyseactiviteit in cis vergeleken met trans. Het is essentieel dat trans-FraC-Y138C-C volledig inactief was in geselecteerde concentraties, terwijl dezelfde concentratie cis-FraC-Y138C-C complete lysis van rode bloedcellen veroorzaakte. Ten slotte zou de cytotoxiciteit van FraC-Y138C-C volledig kunnen worden gestopt door bestraling met wit licht. De omkeerbare controle van nanoporievorming biedt mogelijkheden voor gebruik in klinische toepassingen. Het inactieve toxine kan worden geactiveerd door bestraling direct op de tumorplaats en schade aan het aangrenzende gezonde weefsel kan voorkomen worden doordat diffuse actieve monomeren worden geïnactiveerd met wit licht. Naast het potentiële gebruik als medicijn, kan lichtgestuurde FraC ook voordelig zijn in biosensor toepassingen. FraC nanoporiën kunnen worden gebruikt om individuele moleculen, zoals peptiden en proteïnen, te herkennen en te detecteren.29 In het bijzonder zijn nu duizenden
individuele nanoporiën ingebouwd in draagbare en goedkope apparaten om DNA te sequencen.30
De mogelijkheid om de vorming van nanoporiën met licht te regelen, zou de productie van arrays met individuele nanoporiën vergemakkelijken en dus de kosten voor multiplexing verlagen. Een voortdurende uitdaging bij de ontwikkeling van kankergeneesmiddelen is het gebrek aan selectiviteit en de daaruit voortvloeiende ernstige bijwerkingen. In de Hoofdstukken 2 en
3 presenteerden we twee verschillende benaderingen om de off-target toxiciteit van PFT’s te
verminderen. De volgende stap is het combineren van de concepten en het introduceren van een dubbel activeringsmechanisme om ons op proteïne gebaseerde actieve werkzame stoffen verder te verbeteren. Daarom beschrijven we in Hoofdstuk 4 de productie van een genetisch gecodeerde PFT die zowel door proteasen als door licht met verschillende golflengten wordt geactiveerd. Het lichtgevoelige light-oxygen-voltage domein van Avena sativa (AsLOV2) werd gebruikt om de
functie van FraC op een lichtafhankelijke manier te reguleren. In het donker bindt het AsLOV2 domein niet-covalent zijn flavine-mononucleotide (FMN) cofactor. Bestraling met blauw licht leidt tot de vorming van een covalente binding tussen de FMN en een geconserveerde cysteïne
van het AsLOV2 domein.31 Deze adductvorming bevordert het ontvouwen en loslaten van de
C-terminale α-helix van de kern van β-sheets van het AsLOV2 domein.32 Daarom hebben we
het C-terminus van het AsLOV2 domein gekoppeld aan het N-terminus van FraC met behulp van een linker van 10 aminozuren die een proteasesplitsingsplaats bevat. In het donker is het fusieproteïne inactief omdat het AsLOV2 domein de proteasesplitsingsplaats verbergt. Bestraling met blauw licht leidt tot structurele veranderingen in het AsLOV2 domein en de openleggen van de proteasesplitsingsplaats. Bestraling met licht en gelijktijdige in situ proteolytische splitsing leidt dus tot de activering van FraC en daarmee tot cytotoxiciteit. Blauw licht heeft echter het nadeel dat het cytotoxisch is en slechts in beperkte mate door het weefsel dringt.33,34 AsLOV2 domeinen,
die worden geactiveerd met licht met een hogere golflengte, zouden verdere toepassingen in de optogenetica mogelijk kunnen maken, zoals de parallelle selectieve controle van verschillende celfuncties met licht van verschillende kleuren. Het is opmerkelijk dat ons fusieproteïne kan worden gebruikt als een screeningplatform om AsLOV2 domeinen te ontwikkelen met verbeterde fotoactiverende eigenschappen. Sommige rondes van gerichte evolutie verminderden de activiteit van AsLOV2-FraC in het donker. Extra willekeurige mutagenese en DNA-shuffling met daaropvolgende screening op rode bloedcellen kunnen mutanten identificeren die worden geactiveerd met groen of zelfs rood licht. Interessant is dat onze resultaten suggereren dat bestraling met een hogere golflengte de verandering in conformatie veroorzaakt door een parallel mechanisme dat onafhankelijk is van de vorming van de FMN-cysteïnebinding. AsLOV2-FraC varianten die reageren op blauw en groen licht zijn met succes gebruikt om specifiek kankercellen te lyseren die het overeenkomstige protease tot expressie brengen en zouden daarom potentiële kandidaten kunnen zijn voor gerichte kankertherapie.
Samenvattend beschrijft dit proefschrift een methode om gerichte kankergeneesmiddelen te ontwikkelen op basis van porievormende toxines. We presenteren een modulaire benadering om porievormende toxinen uit te rusten met verschillende targeting- en trigger-mechanismen. We gebruikten ook directionele evolutie om de eigenschappen van onze op proteïne gebaseerde actieve werkzame stoffen te verfijnen.
REFERENTIES
(1) Dagenais, G. R.; Leong, D. P.; Rangarajan, S.; Lanas, F.; Lopez-Jaramillo, P.; Gupta, R.; Diaz, R.; Avezum, A.; Oliveira, G. B. F.; Wielgosz, A.; et al. Variations in Common Diseases, Hospital Admissions, and Deaths in Middle-Aged Adults in 21 Countries from Five Continents (PURE): A Prospective Cohort Study. Lancet 2019.
(2) Sereno, M.; Brunello, A.; Chiappori, A.; Barriuso, J.; Casado, E.; Belda, C.; de Castro, J.; Feliu, J.; González-Barón, M. Cardiac Toxicity: Old and New Issues in Anti-Cancer Drugs. Clin. Transl. Oncol.
2008, 10 (1), 35–46.
(3) Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P. G. Cancer Drug Resistance: An Evolving Paradigm. Nature Reviews Cancer. 2013, 714–726.
(4) Aggarwal, S. Targeted Cancer Therapies. Nature Reviews Drug Discovery. 2010, 427–428.
(5) Chari, R. V. J. Targeted Cancer Therapy: Conferring Specificity to Cytotoxic Drugs. Acc. Chem. Res.
2008, 41 (1), 98–107.
(6) Baudino, T. Targeted Cancer Therapy: The Next Generation of Cancer Treatment. Curr. Drug Discov.
Technol. 2015, 12 (1), 3–20.
(7) Vasserot, A. P.; Dickinson, C. D.; Tang, Y.; Huse, W. D.; Manchester, K. S.; Watkins, J. D. Optimization of Protein Therapeutics by Directed Evolution. Drug Discovery Today. 2003, 118–126.
(8) Stryjewska, A.; Kiepura, K.; Librowski, T.; Lochyñski, S. Biotechnology and Genetic Engineering in the New Drug Development. Part I. DNA Technology and Recombinant Proteins. Pharmacol. Reports
2013, 65 (5), 1075–1085.
(9) Sela-Culang, I.; Kunik, V.; Ofran, Y. The Structural Basis of Antibody-Antigen Recognition. Front.
Immunol. 2013, 4.
(10) Scott, A. M.; Wolchok, J. D.; Old, L. J. Antibody Therapy of Cancer. Nature Reviews Cancer.2012, 278–287.
(11) Lambert, J. M.; Berkenblit, A. Antibody-Drug Conjugates for Cancer Treatment. Annu. Rev. Med.
2018, 69 (1), 191–207.
(12) Alewine, C.; Hassan, R.; Pastan, I. Advances in Anticancer Immunotoxin Therapy. Oncologist 2015,
20 (2), 176–185.
(13) Pasche, N.; Neri, D. Immunocytokines: A Novel Class of Potent Armed Antibodies. Drug Discovery
Today. 2012, 583–590.
(14) Kitson, S. L.; Cuccurullo, V.; Moody, T. S.; Mansi, L. Radionuclide Antibody-Conjugates, a Targeted Therapy towards Cancer. Curr. Radiopharm. 2013, 6 (2), 57–71.
(15) Pastan, I.; Hassan, R.; Fitzgerald, D. J.; Kreitman, R. J. Immunotoxin Therapy of Cancer. Nat. Rev.
Cancer 2006, 6 (7), 559–565.
(16) Krolick, K. A.; Uhr, J. W.; Slavin, S.; Vitetta, E. S. In Vivo Therapy of a Murine B Cell Tumor (BCL1) Using Antibody-Ricin A Chain Immunotoxins. J. Exp. Med. 1982, 155 (6), 1797–1809.
(17) Mansfield, E.; Amlot, P.; Pastan, I.; FitzGerald, D. J. Recombinant RFB4 Immunotoxins Exhibit Potent Cytotoxic Activity for CD22-Bearing Cells and Tumors. Blood 1997, 90 (5), 2020–2026. (18) Williams, D. P.; Parker, K.; Bacha, P.; Bishai, W.; Borowski, M.; Genbauffe, F.; Strom, T. B.; Murphy, J.
R. Diphtheria Toxin Receptor Binding Domain Substitution with Interleukin-2: Genetic Construction and Properties of a Diphtheria Toxin-Related Interleukin-2 Fusion Protein. Protein Eng 1987, 1 (6), 493–498.
(19) Polito, L.; Mercatelli, D.; Bortolotti, M.; Maiello, S.; Djemil, A.; Battelli, M.; Bolognesi, A. Two Saporin-Containing Immunotoxins Specific for CD20 and CD22 Show Different Behavior in Killing Lymphoma Cells. Toxins (Basel). 2017, 9 (6), 182.
(20) Shan, L.; Liu, Y.; Wang, P. Recombinant Immunotoxin Therapy of Solid Tumors: Challenges and Strategies. J. Basic Clin. Med. 2013, 2 (2), 1–6.
(21) Cosentino, K.; Ros, U.; García-Sáez, A. J. Assembling the Puzzle: Oligomerization of α-Pore Forming Proteins in Membranes. Biochim. Biophys. Acta - Biomembr. 2016, 1858 (3), 457–466.
(22) Rojko, N.; Anderluh, G. How Lipid Membranes Affect Pore Forming Toxin Activity. Acc. Chem. Res.
2015, 48 (12), 3073–3079.
(23) Mohamed, M. M.; Sloane, B. F. Cysteine Cathepsins: Multifunctional Enzymes in Cancer. In Nature
Reviews Cancer; 2006, 6, 764–775.
(24) Jaaks, P.; Bernasconi, M. The Proprotein Convertase Furin in Tumour Progression. Int. J. Cancer
2017, 141 (4), 654–663.
(25) Packer, M. S.; Liu, D. R. Methods for the Directed Evolution of Proteins. Nat. Rev. Genet. 2015, 16 (7), 379–394.
(26) Hüll, K.; Morstein, J.; Trauner, D. In Vivo Photopharmacology. Chemical Reviews. 2018, 10710–10747. (27) Beharry, A. A.; Woolley, G. A. Azobenzene Photoswitches for Biomolecules. Chem. Soc. Rev. 2011,
40 (8), 4422–4437.
(28) Tanaka, K.; Caaveiro, J. M. M.; Morante, K.; González-Manãs, J. M.; Tsumoto, K. Structural Basis for Self-Assembly of a Cytolytic Pore Lined by Protein and Lipid. Nat. Commun. 2015, 6, 6337. (29) Huang, G.; Willems, K.; Soskine, M.; Wloka, C.; Maglia, G. Electro-Osmotic Capture and Ionic
Discrimination of Peptide and Protein Biomarkers with FraC Nanopores. Nat. Commun. 2017, 8 (1), 935.
(30) Bayley, H. Nanopore Sequencing: From Imagination to Reality. Clin. Chem. 2015, 61 (1), 25–31. (31) Salomon, M.; Eisenreich, W.; Dürr, H.; Schleicher, E.; Knieb, E.; Massey, V.; Rüdiger, W.; Müller, F.;
Bacher, A.; Richter, G. An Optomechanical Transducer in the Blue Light Receptor Phototropin from Avena Sativa. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (22), 12357–12361.
(32) Harper, S. M.; Neil, L. C.; Gardner, K. H. Structural Basis of a Phototropin Light Switch. Science (80-.
). 2003, 301 (5639), 1541–1544.
(33) Yoshida, A.; Shiotsu-Ogura, Y.; Wada-Takahashi, S.; Takahashi, S.; Toyama, T.; Yoshino, F. Blue Light Irradiation-Induced Oxidative Stress in Vivo via ROS Generation in Rat Gingival Tissue. J.
Photochem. Photobiol. B. 2015, 151, 48–53.
(34) Ash, C.; Dubec, M.; Donne, K.; Bashford, T. Effect of Wavelength and Beam Width on Penetration in Light-Tissue Interaction Using Computational Methods. Lasers Med. Sci. 2017, 32 (8), 1909–1918.
ZUSAMMENFASSUNG
Krebs ist weltweit eine der häufigsten Todesursachen.1 Die derzeit am häufigsten genutzten
Behandlungsmethoden sind operative Eingriffe, Strahlen- und Chemotherapie. Diese üblichen Krebsbehandlungen haben einige schwerwiegende Nachteile wie zum Beispiel mangelnde
Wirksamkeit oder starke Nebenwirkungen verursacht durch Off-Target Toxizität.2,3
Kapitel 1 enthält eine allgemeine Einführung gegenwärtiger Vorgehensweisen, um die Nachteile
der gebräuchlichsten Krebsbehandlungen zu überwinden. In letzter Zeit konzentrierten sich Forscher auf die Entwicklung gezielter Krebstherapien um den Wirkungsgrad derzeitiger
Behandlungen zu übertreffen und um Schädigungen gesunder Zellen zu vermeiden.4,5 Gezielte
Krebstherapien sind Medikamente, die entwickelt wurden um gegen bestimmte Krebs spezifische Moleküle vorzugehen, die an der Krebsentstehung, am Tumorwachstum oder
an der Metastasierung beteiligt sind.6 Insbesondere Medikamente auf Proteinbasis werden
als neue vielversprechende Gruppe potentieller Wirkstoffe erforscht. Ihr therapeutisches Potenzial wurde möglich durch Fortschritte in der Gentechnik und im Protein-Engineering sowie durch die technische Fähigkeit Proteineigenschaften zu verbessern.7,8 Ein Schwerpunkt
in der Krebsmedikamentenforschung ist die Entwicklung monoklonaler Antikörper die mit hoher Selektivität Tumorantigene binden, und dadurch ihre biologische Funktion blockieren
sowie die Zellen für das körpereigene Immunsystem kennzeichnen.9,10 Jedoch haben sie nur
eine eingeschränkte Antitumoraktivität, und wurden deshalb mit Arzneistoffen11, Toxinen12,
Cytokinen13 oder radioaktiven Teilchen14 konjugiert, um diese zur richtigen Zielzelle zu leiten.
Auf diese Weise wurde die Selektivität der zelltötenden Wirkstoffe für die Zielzellen erhöht.6 Ein
Beispiel sind Immuntoxine, diese bestehen aus einem Element das selektiv an die Zielzelle bindet und einem starken Toxin.15
In Kapitel 2 beschreiben wir die Herstellung von Immuntoxinen, die aus porenbildenden Toxinen (englisch pore-forming toxins PFTs) bestehen. Ursprünglich basierten Immuntoxine
auf Pflanzentoxinen wie Rizin16 oder bakteriellen Toxinen wie Pseudomonas Exotoxin A17
und Diphtherietoxin18, diese lysieren Zellen in dem sie die Proteinsynthese hemmen. Die
Effizienz dieser starken Toxine hängt von der Fähigkeit ab in Tumorgewebe einzudringen und den Internalisierungsraten des Zielrezeptors, denn es ist notwendig ausreichend Toxin
anzureichern um den Zelltod auszulösen.19,20 Alternativ können Immuntoxine aus PFTs
hergestellt werden, diese bilden oligomere Poren in der Zielzellmembran die wiederum zum
Zelltod führen.21 Daher konjugierten wir Cytolysin A (ClyA) von Salmonella typhi chemisch
zu lysieren. Bioorthogonale chemische Bindungen können instabil sein und zu heterogenen Proben führen. Deshalb fusionierten wir ClyA auch genetisch mit einem Nanobody, der variablen Domäne eines Einzeldomänenantikörpers, gegen den EGF-Rezeptor (englisch epidermal growth
factor receptor). Die Zytotoxizität von PFTs hängt jedoch von der Membranzusammensetzung
ab22. Um unseren Ansatz zu verallgemeinern, tauschten wir daher ClyA gegen das Actinoporin
Fragaceatoxin C (FraC) von Actina fragacea aus. Konjugation an die zellbindende Komponente führte zu einer zweifach erhöhten Zytotoxizität gegen Zelllinien die den entsprechenden Rezeptor überexpremieren. Eine Haupteinschränkung von PFTs für die pharmakologische Anwendungen ist deren Toxizität für sämtliche Zellen. Daher haben wir von der Natur abgeschaut und unterbinden Off-Target Toxizität, indem wir FraC als Protoxin herstellen. Durch Hinzufügen eines Polypeptidsegments an den Transmembran N-Terminus über einen Linker, der eine Furin Spaltstelle enthält konnte die Aktivität vollständig aufgehoben werden. Krebszellen überexpremieren oft Proteasen wie zum Beispiel Furin oder Cathepsin B. Diese fördern die Invasion und Metastasierung durch Aufspalten der extrazellulären Matrix und vordringen in die Basallamina.23,24 Dies ermöglicht die in situ Proteinspaltung durch die krebsassoziierte
Protease Furin und führt zur Aktivierung von FraC. Wir kombinierten die beiden Ansätze, um ein PFT herzustellen dessen Aktivität auf zwei Arten kontrolliert wird. Zusätzlich verwendeten wir für die Feinabstimmung der Eigenschaften unseres proteinbasierenden Wirkstoffs gerichtete Evolution, dabei wird die natürliche Selektion nachgeahmt und Proteineigenschaften können in sich wiederholenden Zyklen optimiert werden. Das Gen, dass das betreffende Protein kodiert wird diversifiziert durch Mutagenese und in einem geeigneten Wirt exprimiert. Dann werden Proteine mit den gewünschten Eigenschaften durch geeignete Screenings- oder Selektionsmethoden identifiziert. Letztendlich werden ausgewählte Mutanten vervielfältigt und dienen als Template für nachfolgende Zyklen der gerichteten Evolution.25 Wir verwendeten die
gerichtete Evolution zur Verbesserung der löslichen Expression, der Erhöhung der Zielaffinität, und der Verringerung der Off-Target Affinität unseres Immuntoxins. Schlussendlich besteht unser gezieltes Krebsmedikament aus drei Bausteinen, einem zellbindenden Element, einem porenbildenden Toxin und einem Protease Trigger, diese können einfach ausgetauscht werden. Durch Hinzufügen eines externen Triggers könnte eine verbesserte Kontrolle über die PFT Aktivität erreicht werden. Ein besonders attraktiver Trigger ist Licht, denn es ist nichtinvasiv, bioorthogonal, und ermöglicht eine räumlich-zeitliche Kontrolle.26
In Kapitel 3 beschreiben wir die Erstellung einer photokontrollierten Nanopore. Dafür wurde FraC mit photoschaltbaren Azobenzolen ausgestattet, um die Nanoporenbildung durch Licht
welche durch Bestrahlung mit Licht in die metastabile cis Form umgewandelt wird. Wir wählten Azobenzole aufgrund ihrer vorteilhaften photophysikalischen Eigenschaften aus, wie zum Beispiel ihres hohen photostationären Zustands und einer guten Quantenausbeute, sowie ihrer schnellen
Photoisomerisierung bei niedrigen Photobleichraten.27 Verschiedene Azobenzolverbindungen
wurden kovalent mit Cystein-modifizierten FraC verbunden. Porenbildung von FraC hängt vom Anteil Sphingomyeline in der Membran ab,28 daher führten wir Cysteine an verschiedenen
Positionen in der Nähe der Sphingomyelin-Bindungstelle ein, in der Hoffnung, die Rolle von Sphingomyelinen als Membrananker zu ersetzten. Wir erwarteten, dass der Wechsel zwischen den zwei Isomeren die Affinität der FraC-Monomere für die Biomembran verändert. Die zytolytische Aktivität der verschiedenen Azobenzol-modifizierten FraC Varianten wurde mittels Hämolyseaktivitätsassay bestimmt. Wir konnten einige Konstrukte finden die im thermischen Ruhezustand (trans Isomer) inaktiv, aber im durch Licht angeregten Zustand (cis Isomer) aktiv waren. Insbesondere zeigte FraC modifiziert an Position Y138 und verbunden mit dem para-sulfonierten Azobenzol (FraC-Y138C-C) eine fünffach höhere Hämolyseaktivität in cis verglichen mit trans. Wesentlich ist, dass trans-FraC-Y138C-C in ausgewählten Konzentrationen vollständig inaktiv war, während die gleiche Konzentration cis-FraC-Y138C-C eine vollständige Lyse der roten Blutkörperchen verursachte. Schlussendlich konnte die Zytotoxizität von FraC-Y138C-C vollständig gestoppt werden durch Bestrahlung mit weißem Licht. Die reversible Kontrolle der Nanoporenbildung bietet Potential für die Nutzung in klinischen Anwendungen. Das inaktive Toxin kann durch Bestrahlung direkt an der Tumorstelle aktiviert werden und die Schädigung des benachbarten gesunden Gewebes kann verhindert werden, indem diffundierende aktive Monomere mit weißem Licht inaktiviert werden. Abgesehen von ihrer Verwendungsmöglichkeit als Medikament, könnte lichtgesteuertes FraC auch in der nanobiotechnischen Anwendung von Vorteil sein. FraC Nanoporen können zum Erkennen und Nachweisen von einzelnen Molekülen,
wie zum Beispiel Peptiden und Proteinen verwendet werden.29 Insbesondere sind heutzutage
tausende einzelne Nanoporen in tragbaren und preiswerten Geräten zum DNA sequenzieren eingebaut.30 Die Möglichkeit Nanoporenbildung mit Licht zu kontrollieren würde die Herstellung
von Arrays mit einzelnen Nanoporen erleichtern und somit die Kosten für das Multiplexen senken.
Eine bleibende Herausforderung bei der Entwicklung von Krebsmedikamenten ist die mangelnde Selektivität und den daraus resultierenden schweren Nebenwirkungen. In Kapitel
2 und 3 stellten wir zwei verschiedene Vorgehensweisen vor, um die Off-Target Toxizität von
PFTs zu reduzieren. Der nächste Schritt besteht darin die Konzepte zu kombinieren und einen doppelten Aktivierungsmechanismus einzuführen, um unseren proteinbasierten Wirkstoff weiter zu verbessern. Demzufolge beschreiben wir in Kapitel 4 die Herstellung eines genetisch
kodierten PFT, das sowohl durch Proteasen als auch durch Licht unterschiedlicher Wellenlänge aktiviert wird. Die photoempfindliche light-oxygen-voltage Domäne von Avena sativa (AsLOV2) wurde verwendet, um die Funktion von FraC lichtabhängig zu regulieren. Im Dunkeln bindet die AsLOV2 Domäne nichtkovalent ihren Flavinmononukleotid (FMN) Cofaktor. Bestrahlung mit blauem Licht führt zur Bildung einer kovalenten Bindung zwischen dem FMN und einem
konservierten Cystein der AsLOV2 Domäne.31 Diese Adduktbildung fördert die Entfaltung und
die Ablösung des C-terminalen α-Helix vom Kern aus β-Faltblättern der AsLOV2 Domäne.32
Deshalb verbanden wir den C-Terminus der AsLOV2 Domäne mit dem N-Terminus von FraC mittels eines 10 Aminosäure langen Linkers, der eine Proteasespaltstelle enthält. Im Dunkeln ist das Fusionsprotein inaktiv, weil die AsLOV2 Domäne die Proteasespaltstelle verbirgt. Bestrahlung mit blauem Licht führt zu Strukturänderungen der AsLOV2 Domäne und zur Aufdeckung der Proteasespaltstelle. Die Bestrahlung mit Licht und gleichzeitiger in
situ proteolytischer Spaltung führt somit zur Aktivierung von FraC und dementsprechend zu
Zytotoxizität. Blaues Licht hat jedoch den Nachteil das es zytotoxisch ist und nur eingeschränkt
Gewebe durchdringt.33,34 AsLOV2 Domänen die mit Licht höherer Wellenlänge aktiviert
werden, könnten weitere Anwendungen in der Optogenetik ermöglichen, so wie zum Beispiel die parallele selektive Kontrolle unterschiedlicher Zellfunktionen mit verschiedenfarbigen Licht. Bemerkenswerterweise konnte unser Fusionsprotein als Screening-Plattform verwendet werden, um AsLOV2 Domänen mit verbesserten Photoaktivierungseigenschaften zu entwickeln. Einige Runden der gerichteten Evolution konnten die Aktivität von AsLOV2-FraC im Dunkeln verringern. Zusätzliche Zufallsmutagenese und DNA-Shuffling mit anschließendem Screening auf roten Blutkörperchen konnte Mutanten identifizieren, die mit grünem oder sogar rotem Licht aktiviert werden. Interessanterweise deuten unsere Ergebnisse darauf hin, dass die Bestrahlung mit Licht höherer Wellenlänge die Konformationsänderung durch einen parallelen Mechanismus auslöst, der unabhängig von der Bildung der FMN-Cystein Bindung ist. AsLOV2-FraC Varianten, die auf blaues und grünes Licht reagieren wurden erfolgreich eingesetzt, um gezielt Krebszellen zu lysieren, die die entsprechende Protease exprimieren, und könnten daher mögliche Kandidaten für eine gezielte Krebstherapie sein.
Zusammenfassend beschreibt diese These eine Methode, um auf der Grundlage von porenbildenden Toxinen gezielte Krebsmedikamente zu entwickeln. Wir präsentieren hier einen modularen Ansatz um porenbildende Toxine mit unterschiedlichen Targeting und Trigger Mechanismen auszustatten. Zusätzlich nutzten wir gerichtete Evolution, um die Eigenschaften unserer proteinbasierenden Wirkstoffe zu optimieren.
LITERATUR
(1) Dagenais, G. R.; Leong, D. P.; Rangarajan, S.; Lanas, F.; Lopez-Jaramillo, P.; Gupta, R.; Diaz, R.; Avezum, A.; Oliveira, G. B. F.; Wielgosz, A.; et al. Variations in Common Diseases, Hospital Admissions, and Deaths in Middle-Aged Adults in 21 Countries from Five Continents (PURE): A Prospective Cohort Study. Lancet 2019.
(2) Sereno, M.; Brunello, A.; Chiappori, A.; Barriuso, J.; Casado, E.; Belda, C.; de Castro, J.; Feliu, J.; González-Barón, M. Cardiac Toxicity: Old and New Issues in Anti-Cancer Drugs. Clin. Transl. Oncol.
2008, 10 (1), 35–46.
(3) Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P. G. Cancer Drug Resistance: An Evolving Paradigm. Nature Reviews Cancer. October 2013, pp 714–726.
(4) Aggarwal, S. Targeted Cancer Therapies. Nature Reviews Drug Discovery. June 2010, pp 427–428. (5) Chari, R. V. J. Targeted Cancer Therapy: Conferring Specificity to Cytotoxic Drugs. Acc. Chem. Res.
2008, 41 (1), 98–107.
(6) Baudino, T. Targeted Cancer Therapy: The Next Generation of Cancer Treatment. Curr. Drug Discov.
Technol. 2015, 12 (1), 3–20.
(7) Vasserot, A. P.; Dickinson, C. D.; Tang, Y.; Huse, W. D.; Manchester, K. S.; Watkins, J. D. Optimization of Protein Therapeutics by Directed Evolution. Drug Discovery Today. January 21, 2003, pp 118–126. (8) Stryjewska, A.; Kiepura, K.; Librowski, T.; Lochyñski, S. Biotechnology and Genetic Engineering in the New Drug Development. Part I. DNA Technology and Recombinant Proteins. Pharmacol. Reports
2013, 65 (5), 1075–1085.
(9) Sela-Culang, I.; Kunik, V.; Ofran, Y. The Structural Basis of Antibody-Antigen Recognition. Front.
Immunol. 2013, 4 (OCT).
(10) Scott, A. M.; Wolchok, J. D.; Old, L. J. Antibody Therapy of Cancer. Nature Reviews Cancer. March 22, 2012, pp 278–287.
(11) Lambert, J. M.; Berkenblit, A. Antibody-Drug Conjugates for Cancer Treatment. Annu. Rev. Med.
2018, 69 (1), 191–207.
(12) Alewine, C.; Hassan, R.; Pastan, I. Advances in Anticancer Immunotoxin Therapy. Oncologist 2015,
20 (2), 176–185.
(13) Pasche, N.; Neri, D. Immunocytokines: A Novel Class of Potent Armed Antibodies. Drug Discovery
Today. June 2012, pp 583–590.
(14) Kitson, S. L.; Cuccurullo, V.; Moody, T. S.; Mansi, L. Radionuclide Antibody-Conjugates, a Targeted Therapy towards Cancer. Curr. Radiopharm. 2013, 6 (2), 57–71.
(15) Pastan, I.; Hassan, R.; Fitzgerald, D. J.; Kreitman, R. J. Immunotoxin Therapy of Cancer. Nat. Rev.
Cancer 2006, 6 (7), 559–565.
(16) Krolick, K. A.; Uhr, J. W.; Slavin, S.; Vitetta, E. S. In Vivo Therapy of a Murine B Cell Tumor (BCL1) Using Antibody-Ricin A Chain Immunotoxins. J. Exp. Med. 1982, 155 (6), 1797–1809.
(17) Mansfield, E.; Amlot, P.; Pastan, I.; FitzGerald, D. J. Recombinant RFB4 Immunotoxins Exhibit Potent Cytotoxic Activity for CD22-Bearing Cells and Tumors. Blood 1997, 90 (5), 2020–2026. (18) Williams, D. P.; Parker, K.; Bacha, P.; Bishai, W.; Borowski, M.; Genbauffe, F.; Strom, T. B.; Murphy, J.
R. Diphtheria Toxin Receptor Binding Domain Substitution with Interleukin-2: Genetic Construction and Properties of a Diphtheria Toxin-Related Interleukin-2 Fusion Protein. Protein Eng 1987, 1 (6), 493–498.
(19) Polito, L.; Mercatelli, D.; Bortolotti, M.; Maiello, S.; Djemil, A.; Battelli, M.; Bolognesi, A. Two Saporin-Containing Immunotoxins Specific for CD20 and CD22 Show Different Behavior in Killing Lymphoma Cells. Toxins (Basel). 2017, 9 (6), 182.
(20) Shan, L.; Liu, Y.; Wang, P. Recombinant Immunotoxin Therapy of Solid Tumors: Challenges and Strategies. J. Basic Clin. Med. 2013, 2 (2), 1–6.
(21) Cosentino, K.; Ros, U.; García-Sáez, A. J. Assembling the Puzzle: Oligomerization of α-Pore Forming Proteins in Membranes. Biochim. Biophys. Acta - Biomembr. 2016, 1858 (3), 457–466.
(22) Rojko, N.; Anderluh, G. How Lipid Membranes Affect Pore Forming Toxin Activity. Acc. Chem. Res.
2015, 48 (12), 3073–3079.
(23) Mohamed, M. M.; Sloane, B. F. Cysteine Cathepsins: Multifunctional Enzymes in Cancer. In Nature
Reviews Cancer; 2006; Vol. 6, pp 764–775.
(24) Jaaks, P.; Bernasconi, M. The Proprotein Convertase Furin in Tumour Progression. Int. J. Cancer
2017, 141 (4), 654–663.
(25) Packer, M. S.; Liu, D. R. Methods for the Directed Evolution of Proteins. Nat. Rev. Genet. 2015, 16 (7), 379–394.
(26) Hüll, K.; Morstein, J.; Trauner, D. In Vivo Photopharmacology. Chemical Reviews. November 2018, pp 10710–10747.
(27) Beharry, A. A.; Woolley, G. A. Azobenzene Photoswitches for Biomolecules. Chem. Soc. Rev. 2011,
40 (8), 4422–4437.
(28) Tanaka, K.; Caaveiro, J. M. M.; Morante, K.; González-Manãs, J. M.; Tsumoto, K. Structural Basis for Self-Assembly of a Cytolytic Pore Lined by Protein and Lipid. Nat. Commun. 2015, 6, 6337. (29) Huang, G.; Willems, K.; Soskine, M.; Wloka, C.; Maglia, G. Electro-Osmotic Capture and Ionic
Discrimination of Peptide and Protein Biomarkers with FraC Nanopores. Nat. Commun. 2017, 8 (1), 935.
(30) Bayley, H. Nanopore Sequencing: From Imagination to Reality. Clin. Chem. 2015, 61 (1), 25–31. (31) Salomon, M.; Eisenreich, W.; Dürr, H.; Schleicher, E.; Knieb, E.; Massey, V.; Rüdiger, W.; Müller, F.;
Bacher, A.; Richter, G. An Optomechanical Transducer in the Blue Light Receptor Phototropin from Avena Sativa. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (22), 12357–12361.
(32) Harper, S. M.; Neil, L. C.; Gardner, K. H. Structural Basis of a Phototropin Light Switch. Science (80-.
). 2003, 301 (5639), 1541–1544.
(33) Yoshida, A.; Shiotsu-Ogura, Y.; Wada-Takahashi, S.; Takahashi, S.; Toyama, T.; Yoshino, F. Blue Light Irradiation-Induced Oxidative Stress in Vivo via ROS Generation in Rat Gingival Tissue. J.
Photochem. Photobiol. B. 2015, 151, 48–53.
(34) Ash, C.; Dubec, M.; Donne, K.; Bashford, T. Effect of Wavelength and Beam Width on Penetration in Light-Tissue Interaction Using Computational Methods. Lasers Med. Sci. 2017, 32 (8), 1909–1918.
