University of Groningen
Studies on Superantigens and Antibody Directed Enzyme Prodrug Therapy for Tolerable Targeted Cancer Treatment
Bashraheel, Sara
DOI:
10.33612/diss.96169444
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Publication date: 2019
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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
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Chapter 1: Introduction to the Thesis
Chapter 1: Introduction to the Thesis
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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
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.
Chapter 1: Introduction to the Thesis
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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
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.
Chapter 1: Introduction to the Thesis
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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).
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