University of Groningen
Production of novel protein therapeutics to improve targeted cancer therapy Al-Qahtani, Alanod
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Publication date: 2019
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Al-Qahtani, A. (2019). Production of novel protein therapeutics to improve targeted cancer therapy. University of Groningen.
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11
Chapter 1
General Introduction and Scope of
the Thesis
13
General Introduction and Scope of the Thesis
Cancer is one of the leading causes of death worldwide and one of the top ten causes of death in Qatar. Cancer research worldwide aims to enhance the understanding of cancer and develop a more effective treatments, which targets cancer cells only, with tolerable side effects than the current chemotherapy and radiotherapy. There are several pitfalls in the current cancer treatment that are either linked with the given drug or to the patient’s immune system. Regarding the drug, it lacks selectively, i.e. it damages the healthy tissues as well as the cancer cells. The lack of selectivity can cause severe side effects which in many cases terminate the treatment.1 The other pitfall is related to the fact that the drug should be given in
cycles to the patient. This leads the patient’s immune system to raise antibodies against the drug, which could hamper the efficacy and efficiency of the medicine. Due to the above weaknesses of these conventional treatments the attention has been given to the targeted cancer therapy. In this approach, the drug will be
directed mainly to the cancer tissue limiting its side effects and make it more tolerable to the patient.
In this thesis, we focus on two approaches on targeted cancer therapy, one is known as the Antibody Directed Enzyme Prodrug Therapy (ADEPT) and the other one is Targeted Tumor Superantigens (TTS).
In chapter 2, we covered the literature in relation to the targeted cancer therapy including our work as shown in the above chapters. The review chapter focused on PEGylation and Albumin fusion as the two strategies to produce long acting drugs. In chapter 3, 4 and 5, we successfully provided solutions to many limitations in the ADEPT, which will make it more effective and efficient. In chapter 6, we successfully managed to identify the part on the superantigen molecule which cause severe hypotension. This will lead to the production of novel superantigen variants to be used in TTS with less side effect.
Regarding the ADEPT, it is as a two-step approach in cancer treatment. The first step involves the administration of a cancer antibody-enzyme conjugate, which targets the tumor. Next, a prodrug is injected which will be activated by the enzyme at the site of the tumor, in an effort to circumvent the adverse impact on healthy tissues. The enzyme can activate many molecules of prodrug, which will result in a large amounts of drug being generated at the tumor vacinety.2,3 The activation of
the prodrug occurs extra cellular and can penetrate the cancer cell by diffusion causing cell death.2
One of the enzymes that is used in this technique is Carboxypeptidase G2 (CPG2) also known as glucarpidase. The enzyme CPG2 is a bacterial enzyme and a folate hydrolyzing enzyme.
On the other hand, the enzyme also can degrade the folate analogue, Methotrexate, which is used in chemotherapy treatment of cancer. CPG2, therefore, is not only
15 useful in targeted cancer therapy, but also in drug detoxification in case of a high doses.3
In Chapter 3, we successfully isolated a novel glucarpidase to be used in the detoxification of drugs and the ADEPT. We isolated novel glucarpidase producing bacteria from soil using folate as the only carbon or nitrogen source. We managed to isolate three novel enzyme producing bacterial. Two of the enzyme encoding genes, Xenophilus azovorans SN213 and Stenotrophomonas sp SA were cloned and molecularly characterized.
In chapter 4, we focused on the use of DNA shuffling to create enzyme variants with a new exerted feature, in our case, variants with higher enzyme activities.4
DNA shuffling is a practical process to induce directed molecular evolution in vitro by mimicking natural recombination. In addition to recombination, the technique also introduces point mutations at a controlled rate, which broadens the possibilities for evolving improved genes.5
In chapter 4, we successfully implemented the DNA shuffling techniques to produce novel CPG2 variants with higher enzyme activity than the wild type. We produced a DNA library using the DNA shuffling techniques and screening over four thousand variants on folate containing media plates, the variants were isolated depending on the desired phenotype. The best three novel variants with higher activity glucarpidase were analyzed and sequenced, to recognize the effector mutation, and the kinetics of each variant.
In Chapter 5, we have managed to implement two techniques to extend the serum half-life of CPG2 in the ADEPT technique and the detoxification of MTX. The first
approach is through PEGylation by the attachment of polyethylene glycol (PEG). PEG is a water-soluble and biocompatible polymer, which is used expensively in drug delivery. PEG conjugation increases the circulation half-life without affecting its activity.6 PEGylation prolongs the circulation time of the conjugated
therapeutics by increasing its hydrophilicity, reducing the rate of glomerular filtration, and masking the antigenic sites. PEG is a non-biodegradable polymer and it is primarily excreted through the renal system, whereas higher molecular weight PEG chains get eliminated by fecal excretion.6 Another approach that was
used in our work, is the fusion of the Human Serum Albumin (HSA) to CPG2, HSA is an excellent carrier and is responsible for transporting endogenous and exogenous compounds with the feature of providing a long average serum half-life. It also tends to accumulate around tumors and inflamed tissue sites, which makes the fused albumin aid in targeting the therapeutic site of interest. Both new variants produced in this chapter have been tested for their, solubility, stability in serum and immunogenicity in comparison to the free CPG2.
In Chapter 6, we focused on the other targeted therapy technique (TTS) as mentioned above. We studied the possible production of a new superantigen for tolerable cancer immunotherapy. Superantigens (SAGs) are a class of immunostimulatory proteins with the ability to activate large fractions of the T cell population. Activation requires simultaneous interaction of the SAG with the V beta domain of the T cell receptor (TCR) and with major histocompatibility complex (MHC) class II molecules on the surface of the antigen-presenting cell.7
The SAGs are able to non-specifically activate up to 20% of resting T-cells, whilst conventional antigen present results in the activation of only 0.001 - 0.0001% of
17 the T-cell population.8 This makes superantigens an excellent target for cancer
immunotherapy. The use of these molecules however, has a sever side effect such as sever vasodilation and hypotension. The purpose of this part of the work is to identify the amino acid sequence(s) which contributing to the sever hypotension the aim of which is to produce a novel variant of superantigens to be used in tolerable cancer immunotherapy.
Four super antigens (SEA, SEB, SPEA and TSST-1) were codon optimized and overexpressed in E .coli. We synthesized peptides to cover the whole molecule and we mapped the region which causes vasodilation and therefore, hypotension. Finally in chapter 7, 8 and 9, we provide in three different languages ( English, Dutch and Arabic) a comprehensive summery of the results and conclusion as well as perspective for future work to be developed on the new and conventional CPG2 in cancer therapy.
References:
1. He, H.; Liang, Q.; Shin, M. C.; Lee, K.; Gong, J.; Ye, J.; Liu, Q.; Wang, J.; Yang, V. Significance and strategies in developing delivery systems for bio-macromolecular drugs. Frontiers of Chemical Science and Engineering 2013, 7, (4), 496-507. 2. Francis, R. J.; Sharma, S. K.; Springer, C.; Green, A. J.; Hope-Stone, L. D.; Sena,
L.; Martin, J.; Adamson, K. L.; Robbins, A.; Gumbrell, L.; O'Malley, D.; Tsiompanou, E.; Shahbakhti, H.; Webley, S.; Hochhauser, D.; Hilson, A. J.; Blakey, D.; Begent, R. H. J. A phase I trial of antibody directed enzyme prodrug therapy (ADEPT) in patients with advanced colorectal carcinoma or other CEA producing tumours. British journal of cancer 2002, 87, 600.
3. Jeyaharan, D.; Brackstone, C.; Schouten, J.; Davis, P.; Dixon, A. M. Characterisation of the Carboxypeptidase G2 Catalytic Site and Design of New Inhibitors for Cancer Therapy. ChemBioChem 2018, 19, (18), 1959-1968.
4. 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, 508-513.
5. Marshall, S. H. DNA shuffling: induced molecular breeding to produce new generation long-lasting vaccines. Biotechnology Advances 2002, 20, (3), 229238.
6. Mishra, P.; Nayak, B.; Dey, R. K. PEGylation in anti-cancer therapy: An overview. Asian Journal of Pharmaceutical Sciences 2016, 11, (3), 337-348.
7. Li, H.; Llera, A.; Malchiodi, E. L.; Mariuzza, R. A. The structural basis of T cell activation by superantigens. Annual review of immunology 1999, 17, 435-66. 8. Papageorgiou, A. C.; Acharya, K. R. Superantigens as immunomodulators: recent
structural insights. Structure (London, England : 1993) 1997, 5, (8), 991-6.
