Production of novel protein therapeutics to improve targeted cancer therapy Al-Qahtani, Alanod
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):
Al-Qahtani, A. (2019). Production of novel protein therapeutics to improve targeted cancer therapy. University of Groningen.
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.
211
Chapter 7
213
Summary and Future Prospects:
Targeted cancer therapies are currently the focus of many anticancer drug treatments with the ability to interfere with cancer cell growth or survival. Many targeted cancer therapies have been approved by the Food and Drug Administration (FDA) to treat specific types of cancer. Others are being studied in clinical trials, and many more are in preclinical testing. Targeted therapies on cancer cells may have limitations, possibly involving drug resistance, or lack of dug specificity. Each chapter below focuses on a possible limitation of the treatment, and provides a new approaches that can be applied to overcome these limitations.
In Chapter 2, we carried out a review of different strategies to produce long acting therapeutic drugs in cancer, and their benefits for drug delivery. PEGylation and albumin fusion have provided a particular focus and represent the most widely used and discussed. The application of the two forms in therapeutic proteins and their use in cancer treatment and improving drug delivery are crucial and are the future for therapeutic proteins. The modified therapeutic agents have been demonstrated to have increased serum half-life and solubility, and an improved potential for drug delivery while maintaining their activity. Our studies suggest roles in immunotherapy as well in targeted cancer therapy and in a manner that protects the protein/enzyme from the immune system. In chapter 3, we discuss how we managed to isolate a novel CPG2 variant. Despite the close similarity between the new and the conventional enzyme, the polyclonal antibody raised against our new form of CPG2 did not react with the CPG2 from Pseudomonas strain RS-16 that is currently in clinical use. This indicates that the two enzymes are antigenically distinct. This feature may have advantages in that administration of the two drugs consecutively in the ADEPT protocol could minimize the antibody responses that hampers repetitive administration of the current treatment with Ps CPG2. The availability of the new glucarpidase could be of great importance in improving
its therapeutic usefulness in cancer therapy and will provide the opportunity for dose studies that, in turn, might lead to the escalation of methotrexate doses for more efficient treatment. Future studies should carry out in vivo studies to investigate if the patient’s immune system will react favorably, if the two enzymes are given consecutively comparing to one enzyme only.
In Chapter 4, we discuss how with DNA shuffling we created new CPG2 variants with mutations that produced 110-200% more activity than the wild type glucarpidase. Analysis of the DNA sequences indicated that single point mutation occurred in each variant causing amino acid substitutions I100T, T239A, and G123S. CD study of the shuffled CPG2 showed a higher alpha helix content than the wild type. This work indicates a basis for economical production of a new recombinant glucarpidase with enhanced enzyme activities for the potential use in ADEPT and/or the detoxification of drugs. The application of the newly generated glucarpidase could be extended to clinical applications other than cancer. The new glucarpidase with higher activity that we have produced could be of value in detoxification, in cases of methotrexate overdose. Modified drugs (as discussed below in chapter 5) could be applied with the new shuffled variants to produce a longer active drug with even higher activity using the PEGylation techniques and also the genetic fusion of HSA.
In Chapter 5, the aim was to produce a long-acting glucarpidase. We created two longacting forms of CPG2: a mono-PEGylated glucarpidase, and a HSA-fused glucarpidase.
Biochemical and bioactivity analyses indicated that each form had an improved half-life, and the functional activities of the glucarpidase conjugates were maintained. They also exhibited high stability in human serum and were more resistant to key human proteases than the native glucarpidase. To our knowledge, this study is the first to report stable and less immunogenic glucarpidase variants produced by PEGylation and fusion with HSA. Our findings suggest that they
215
might have greater efficacy in drug detoxification and ADEPT. The two forms can be given to patients consecutively to prolong the cycle administration, thereby, significantly improving the cancer treatment strategy. Our work paves the way for clinical investigation, and clinical trials using our novel modified forms of CPG2 for cancer treatment and drug detoxification. Production of different modifiers to glucarpidase (CPG2) and its new variants should allow novel CPG2 forms to be produced for more cycle of administration of the enzyme. Animal testing will be a crucial bridge to clinical studies, and permit an initial assessment of the half-lives of the different forms of CPG2,
in vivo, their immunogenicity and stability.
In Chapter 6, we used different molecules for cancer treatment, superantigens. The aim is to identify the region(s) on these molecules, which cause the severe hypotension if the host was injected by superantigen. The identification of the region causing this hypotension will then lead to the production of modified superantigen by the removal of these regions. The novel superantigen molecules will then be used for targeted cancer treatment with less side effects. We first overexpressed four codon optimized superantigens, SEA, SEB, TSST-1 and SPEA. These had the ability to activate the T cells to an extent at least 66% greater than that with the available SEB. The recombinant superantigens had the ability to kill DLD1 cancer cells when mixed with PBMCs which activated T cells and triggered the production of cytokines. We showed, using two of the superantigens (SEA and SPEA), that they cause vasodilation which could lead to hypotension, and creating an unwanted side effect. We then designed and synthesized nineteen overlapping peptides from SPEA, and were able to identify peptides which contribute to the superantigen-induced hypotension. Removing the amino acids identified should pave the way for production of superantigen variants with reduced potential for the side effect of vasodilation.
