• No results found

University of Groningen Studies on Superantigens and Antibody Directed Enzyme Prodrug Therapy for Tolerable Targeted Cancer Treatment Bashraheel, Sara

N/A
N/A
Protected

Academic year: 2021

Share "University of Groningen Studies on Superantigens and Antibody Directed Enzyme Prodrug Therapy for Tolerable Targeted Cancer Treatment Bashraheel, Sara"

Copied!
9
0
0

Bezig met laden.... (Bekijk nu de volledige tekst)

Hele tekst

(1)

University of Groningen

Studies on Superantigens and Antibody Directed Enzyme Prodrug Therapy for Tolerable Targeted Cancer Treatment

Bashraheel, Sara

DOI:

10.33612/diss.96169444

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.

Document Version

Publisher's PDF, also known as Version of record

Publication date: 2019

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):

Bashraheel, S. (2019). Studies on Superantigens and Antibody Directed Enzyme Prodrug Therapy for Tolerable Targeted Cancer Treatment. University of Groningen. https://doi.org/10.33612/diss.96169444

Copyright

Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

(2)

7

Chapter 1: Introduction to the Thesis

(3)

Chapter 1: Introduction to the Thesis

8

Introduction to the Thesis

Cancer is one of the leading causes of death worldwide and the second leading cause of death in Qatar, according to Qatar Population Health Report from the Ministry of Public Health [1]. Recent global cancer statistics suggest that the worldwide cancer burden has increased to 18.1 million cases and 9.6 million cancer deaths. In fact, the global incidence of cancer is around: 48.4% for Asia, 23.4% for Europe, 21% for the US, 5.8% for Africa and 1.4% for Oceania [2].

The available treatment protocols can achieve a survival rate of 98% in some types of cancers. The rate of survival, however, in some other types of cancer is only 1% [3]. It is, therefore, of paramount importance to continue to develop novel cancer treatments and to improve the current therapies.

It is essential to understand the biology of cancer to be able to develop more effective therapies. The therapies used for most types of advanced cancer are cytoreductive surgery, chemotherapy and radiation therapy, separately or in combination. Conventionally used chemotherapies are quite often associated with severe side effects due to their lack of specificity, which leads to the killing of healthy tissue as well as cancer cells [4]. Thus, much recent scientific research has focused on targeted cancer therapies to overcome this limitation [5].

The work in this thesis deals with two different approaches for targeted cancer therapy. One is the production of new superantigen variants for the development of a safer tumor targeted superantigen (TTS), and the other is improvements to antibody-directed enzyme prodrug therapy (ADEPT). Chapter 2 provides a full review of recent developments in targeted cancer therapies, and with TTS and ADEPT in particular.

Part 1: Tumor Targeted Superantigens for Cancer Immunotherapy

Superantigens are microbial toxins known for their ability to induce the immune system massively by the activation of unspecific T-cells and through the large-scale production of cytokines. To achieve these effects, superantigens crosslink the Vb

(4)

9 domain of the T-cell receptor (TCR), present on the surface of T-cells, with Major Histocompatibility Complex (MHC) class II molecules present on the surface of an Antigen Presenting Cell (APC). This sort of interaction activates up to 20% of resting T-cells, whereas a conventional antigen results in the activation of between 0.001 - 0.0001% of the T-cell population [6, 7]. Therefore, superantigens have been studied for cancer immunotherapy and targeted immunotherapy, where the superantigen is linked to a specific monoclonal antibody or to a tumor-specific ligand [8-11]. This approach is known as tumor-targeted superantigen therapy. TTS has been used in animal studies and for the treatment of breast cancer [8, 12, 13], bladder cancer [14], and melanoma [12, 13, 15]. All these treatments were achieved, but with many serious side effects, including severe vasodilation leading to severe, life-threatening hypotension.

Therefore, the purpose of the work in Chapter 3 was to study the vasodilation effect of superantigens, to investigate the mechanism by which superantigens cause vasodilation and to map the regions on the superantigen that contribute to vasodilation and hence possible severe hypotension. To achieve this goal, four superantigens (SEA, SEB, SPEA and TSST-1) were codon-optimized, cloned, overexpressed in E. coli and assessed for their superantigenicity and T-cell dependent tumor killing. We then investigated the direct effect of SAgs on vascular tone using two recombinant SAgs, SEA and SPEA. The roles of nitric oxide (NO) and potentially hyperpolarization, which is dependent on activation of the K+

channel, were also explored. To map the region on the superantigen that causes vasodilation and possibly hypotension, a series of 20 overlapping peptides, spanning the entire sequence of SPEA were synthesized. The vascular response of each peptide was measured, and peptides with vasodilation effect were identified. The successful identification of these regions pave the way for the construction and production of superantigen variants with reduced vasodilatory side effects, which could be used for tolerable targeted cancer immunotherapy.

(5)

Chapter 1: Introduction to the Thesis

10

Part 2: Antibody-Directed Enzyme Prodrug Therapy for improved cancer treatment

In this part we focused on Antibody-Directed Enzyme Prodrug Therapy. The ADEPT strategy uses an enzyme with no human homologue to convert a non-toxic substance to a toxic drug only at the tumor site, thereby restricting the cytotoxic activity to the tumor. [16, 17]. The most common enzyme used in this strategy is Carboxypeptidase G2, also known as glucarpidase. Carboxypeptidase G2 (CPG2) is a folate hydrolyzing bacterial enzyme that can also degrade methotrexate (MTX), a folate analogue drug used in chemotherapy of cancer treatment. CPG2 has also been used for methotrexate drug detoxification in cases of MTX overdose, in addition to its use in ADEPT for cancer treatment [18].

Repeated cycles of ADEPT and the use of glucarpidase in the detoxification of cytotoxic methotrexate (MTX) are highly desirable for cancer therapy but are hampered by the induced human antibody response to glucarpidase. In Chapters

4, 5 and 6 we successfully produced several CPG2 variants to overcome this

limitation of ADEPT. This work includes the isolation of a new CPG2 variant with a different major epitope, the production of a new variant with higher activity and the production of long-acting CPG2 enzyme.

In Chapter 4, we isolated a novel glucarpidase variant with epitopes that appear to differ from those associated with the CPG2 that is currently in use. We collected soil samples from farms where vegetables rich in folate grow. Screening for folate-degrading bacteria led to the isolation of three stains, Pseudomonas lubricans strain SF 168, Xenophilus azovorans SN213 and Stenotrophomonas sp SA. Isolation of the Xen CPG2 gene from Xenophilus azovorans SN213 was followed by cloning, protein overexpression and functional characterization of the soluble protein. Immunoblotting analysis established that anti-Xen CPG2 antibody does not bind to the conventional CPG2 protein from Pseudomonas, which suggests that the two enzymes have different epitopes. Alternating between the Ps CPG2

(6)

11 and Xen CPG2s in repetitive ADEPT cycles may lead to reduced immunogenicity and hence to more effective cancer treatment.

Chapter 5 describes the production of new variants of CPG2 with enhanced

enzyme activities by random mutagenesis and DNA shuffling. Error prone PCR was used to introduce random mutations into the gene, followed by DNA shuffling, which is an in vitro recombination process used to rapidly increase mutations and broaden the possibilities for evolving improved genes [19, 20]. A DNA library of four thousand variants was screened for folate hydrolyzing activity. Three novel variants with higher activity were further analyzed and sequenced. We propose that the novel CPG2 mutants would potentially enhance the efficiency of ADEPT by reducing the number of cycles that are required, thereby reducing the risk that patients will develop antibodies to glucarpidase. The new variants also could be useful in drug detoxification.

In Chapter 6, we successfully produced CPG2 variants with an extended half-life in serum for improved ADEPT and MTX detoxification. This was achieved using two techniques. The first used PEGylation, i.e. the attachment of polyethylene glycol (PEG) molecules to the protein, while the other fused the CPG2 protein to Human Serum Albumin (HSA). PEG is a water-soluble molecule and thus increases the hydrophilicity of the CPG2 enzyme. PEG works by masking the antigenic site without affecting the enzyme’s activity, thereby protecting the protein from an immune response [21]. HSA, on the other hand, is the most abundant protein in the human body. One of its advantages is that it accumulates around the tumor cell due to its specificity for the glycoprotein 60 (gp60) receptor found on the surface of many cancer cells [22]. Both approaches are commonly used to facilitate drug delivery, as both are known for their biocompatibility, biodegradability and non-immunogenicity. The CPG2 variants that were produced were both tested for their solubility, stability in serum and immunogenicity in comparison to the free CPG2.

(7)

Chapter 1: Introduction to the Thesis

12

Finally, in Chapters 7, 8 and 9, we provide a comprehensive summery of the results and conclusions, along with perspectives for future work on targeted cancer therapy in three languages (English, Dutch and Arabic).

(8)

13

References:

1. Cancer: Ministry of Public Health; 2017. Available from: https://phs.moph.gov.qa/data/cancer/.

2. New Global Cancer Data: GLOBOCAN 2018; 2018. Available from: https://www.uicc.org/new-global-cancer-data-globocan-2018.

3. UK CR. Cancer survival for common cancers. 2011.

4. He H, Liang Q, Shin MC, Lee K, Gong J, Ye J, et al. Significance and strategies in developing delivery systems for bio-macromolecular drugs. Frontiers of Chemical Science and Engineering. 2013;7(4):496-507. doi: 10.1007/s11705-013-1362-1.

5. Ayyar BV, Arora S, O'Kennedy R. Coming-of-Age of Antibodies in Cancer Therapeutics. Trends in pharmacological sciences. 2016;37(12):1009-28. Epub 2016/10/18. doi: 10.1016/j.tips.2016.09.005. PubMed PMID: 27745709.

6. Li H, Llera A, Malchiodi EL, Mariuzza RA. The structural basis of T cell activation by superantigens. Annual review of immunology. 1999;17:435-66. Epub 1999/06/08. doi: 10.1146/annurev.immunol.17.1.435. PubMed PMID: 10358765.

7. Papageorgiou AC, Acharya KR. Superantigens as immunomodulators: recent structural insights. Structure (London, England : 1993). 1997;5(8):991-6. Epub 1997/08/15. PubMed PMID: 9309216.

8. Yousefi F, Siadat SD, Saraji AA, Hesaraki S, Aslani MM, Mousavi SF, et al. Tagging staphylococcal enterotoxin B (SEB) with TGFaL3 for breast cancer therapy. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2016;37(4):5305-16. Epub 2015/11/13. doi: 10.1007/s13277-015-4334-x. PubMed PMID: 26561468.

9. Dohlsten M, Hansson J, Ohlsson L, Litton M, Kalland T. Antibody-targeted superantigens are potent inducers of tumor-infiltrating T lymphocytes in vivo. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(21):9791-5. Epub 1995/10/10. PubMed PMID: 7568219; PubMed Central PMCID: PMCPMC40888.

10. Liu X, Zeng L, Zhao Z, Xie Y, Wang S, Zhang J, et al. Construction, Expression, and Characterization of rSEA-EGF and In Vitro Evaluation of its Antitumor Activity Against Nasopharyngeal Cancer. Technology in cancer research & treatment. 2018;17:1533033818762910. Epub 2018/03/20. doi: 10.1177/1533033818762910. PubMed PMID: 29551087; PubMed Central PMCID: PMCPMC5862366.

11. Yousefi F, Mousavi SF, Siadat SD, Aslani MM, Amani J, Rad HS, et al. Preparation and In Vitro Evaluation of Antitumor Activity of TGFalphaL3-SEB as a Ligand-Targeted Superantigen. Technology in cancer research & treatment. 2016;15(2):215-26. Epub 2015/03/12. doi: 10.1177/1533034614568753. PubMed PMID: 25759426.

12. Yu J, Tian R, Xiu B, Yan J, Jia R, Zhang L, et al. Antitumor activity of T cells generated from lymph nodes draining the SEA-expressing murine B16 melanoma and secondarily activated with dendritic cells. International journal of biological sciences. 2009;5(2):135-46. Epub 2009/01/29. PubMed PMID: 19173035; PubMed Central PMCID: PMCPMC2631223.

13. Sundstedt A, Celander M, Hedlund G. Combining tumor-targeted superantigens with interferon-alpha results in synergistic anti-tumor effects. International immunopharmacology. 2008;8(3):442-52. Epub 2008/02/19. doi: 10.1016/j.intimp.2007.11.006. PubMed PMID: 18279798.

14. Han C, Hao L, Chen M, Hu J, Shi Z, Zhang Z, et al. Target expression of Staphylococcus enterotoxin A from an oncolytic adenovirus suppresses mouse bladder tumor growth and recruits

(9)

Chapter 1: Introduction to the Thesis

14

CD3+ T cell. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2013;34(5):2863-9. Epub 2013/05/21. doi: 10.1007/s13277-013-0847-3. PubMed PMID: 23686803.

15. Jeudy G, Salvadori F, Chauffert B, Solary E, Vabres P, Chluba J. Polyethylenimine-mediated in vivo gene transfer of a transmembrane superantigen fusion construct inhibits B16 murine melanoma growth. Cancer gene therapy. 2008;15(11):742-9. Epub 2008/07/12. doi: 10.1038/cgt.2008.42. PubMed PMID: 18617917.

16. Mishra AP, Chandra S, Tiwari R, Srivastava A, Tiwari G. Therapeutic Potential of Prodrugs Towards Targeted Drug Delivery. The open medicinal chemistry journal. 2018;12:111-23. Epub 2018/12/07. doi: 10.2174/1874104501812010111. PubMed PMID: 30505359; PubMed Central PMCID: PMCPMC6210501.

17. Bagshawe KD, Sharma SK, Begent RH. Antibody-directed enzyme prodrug therapy (ADEPT) for cancer. Expert opinion on biological therapy. 2004;4(11):1777-89. Epub 2004/10/27. doi: 10.1517/14712598.4.11.1777. PubMed PMID: 15500406.

18. Jeyaharan D, Brackstone C, Schouten J, Davis P, Dixon AM. Characterisation of the Carboxypeptidase G2 Catalytic Site and Design of New Inhibitors for Cancer Therapy. ChemBioChem. 2018;19(18):1959-68. doi: 10.1002/cbic.201800186.

19. Li H, Chu X, Peng B, Peng X-x. DNA shuffling approach for recombinant polyvalent OmpAs against V. alginolyticus and E. tarda infections. Fish & Shellfish Immunology. 2016;58:508-13. doi: https://doi.org/10.1016/j.fsi.2016.09.058.

20. Marshall SH. DNA shuffling: induced molecular breeding to produce new generation long-lasting vaccines. Biotechnology Advances. 2002;20(3):229-38. doi: https://doi.org/10.1016/S0734-9750(02)00015-0.

21. Mishra P, Nayak B, Dey RK. PEGylation in anti-cancer therapy: An overview. Asian Journal of Pharmaceutical Sciences. 2016;11(3):337-48. doi: https://doi.org/10.1016/j.ajps.2015.08.011. 22. Lomis N, Westfall S, Farahdel L, Malhotra M, Shum-Tim D, Prakash S. Human Serum Albumin Nanoparticles for Use in Cancer Drug Delivery: Process Optimization and In Vitro Characterization. Nanomaterials (Basel, Switzerland). 2016;6(6). Epub 2016/01/01. doi: 10.3390/nano6060116. PubMed PMID: 28335244; PubMed Central PMCID: PMCPMC5302621.

Referenties

GERELATEERDE DOCUMENTEN

Chapter (9) ﺔﻟﺎﺳرﻟا صﺧﻠﻣ 197 ﺺﺨﻠﻤﻟا : ﺔﯿﻟﺎﻌﻓ ﻦﯿﺴﺤﺘﻟ نﺎطﺮﺴﻠﻟ فﺪﮭﺘﺴﻤﻟا جﻼﻌﻟا لﺎﺠﻣ ﻲﻓ زرﺎﺑ مﺪﻘﺗ ﻆﺣﻮﻟ ،ﻦﯿﯿﺿﺎﻤﻟا ﻦﯾﺪﻘﻌﻟا ىﺪﻣ ﻰﻠﻋ ﻢﺗ ﻲﺘﻟا نﺎطﺮﺴﻠﻟ ةدﺎﻀﻤﻟا ﺔﯾودﻷا ﻢﻈﻌﻣ

This journey was very exceptional and long for me, many people came into my way throughout my study, and had a very supportive influence, I would love to express my gratitude to

Studies on Ligand Directed Enzyme Prodrug Therapy and Production of Long Acting Protein Therapeutics for Targeted Cancer Treatment. University

Chapter 6: Production of “biobetter” glucarpidase variants to improve Drug Detoxification and Antibody Directed Enzyme Prodrug Therapy for Cancer Treatment

In this review, we discuss several targeted cancer strategies that use antibodies, enzymes or small molecules, for example, the use of antibody-directed enzyme

Our study paves the way for in vivo investigation of the novel peptides as antihypertensive drugs and the possible production of superantigen variants with less or no

The isolated recombinant Xen CPG2 showed high glucarpidase activity toward folate degradation on agar plates (Fig 6) in comparison to the Ps CPG2 recombinant enzyme in

The three randomly produced glucarpidase mutation substitution, I100T, G123S and T329A, increased the enzyme activity in each case but are predicted to decrease the stability