Browsing by Subject "Cancer Therapy"
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Item Open Access Bench to bedside: A Bispecific Antibody for treating Brain Tumors(2019) Schaller, Teilo HMalignant gliomas are the most common primary brain tumor in adults, with an incidence of five cases per 100,000 persons per year. Grade IV glioblastoma is the most aggressive form and prognosis remains poor despite the current gold-standard first-line treatment – maximal safe resection and combination of radiotherapy with temozolomide chemotherapy – resulting in a median survival of approximately 20 months. Tumor recurrence occurs in virtually all glioblastoma patients, and there currently exists no accepted treatment for these patients. Recent advances in novel directed therapeutics are showing efficacy and have entered clinical trials. This work spans the pre-clinical and clinical development of a bispecific antibody – EGFRvIII:CD3 bi-scFv – for the treatment of malignant gliomas.
Chapter 1 reviews current front-line immunotherapy research in the fields of antibodies, including BiTEs and checkpoint inhibitors, and tumor vaccinations, including peptide and dendritic cell vaccinations. Furthermore, challenges specific to high-grade gliomas as well as opportunities for combination therapies are discussed. Chapter 2 introduces the architecture of the novel bispecific antibody EGFRvIII:CD3 bi-scFv and provides an overview of the molecule’s efficacy in various models. EGFRvIII:CD3 bi-scFv is a truncated antibody with dual specificity. One arm targets the epidermal growth factor receptor mutation variant III (EGFRvIII), a tumor-specific antigen found on glioblastoma. The other arm targets the human CD3 receptor on T cells. As an obligate bispecific antibody, simultaneous binding of both receptors by multiple EGFRvIII:CD3 bi-scFv’s results in the crosslinking of CD3 receptor, activation of T cells, and release of perforin/granzyme which lyses the proximal EGFRvIII-expressing tumor cells. EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma.
Chapter 3 outlines the in-house development of a scalable clinical production process using a WAVE (GE) bioreactor and describes the cGMP-compliant clinical production of EGFRvIII:CD3 bi-scFv. The 250-liter cGMP-production run yielded more than four grams of clinical drug material.
Chapter 4 demonstrates that EGFRvIII:CD3 bi-scFv produced using the cGMP development process is efficacious in both in vitro and in vivo models of glioblastoma. The chapter also describes the approach used to calculate the starting dose for the upcoming first-in-human clinical trial. First-in-human clinical trials require careful selection of a safe yet biologically relevant starting dose. Typically, such starting doses are selected based on toxicity studies in a pharmacologically relevant animal model. However, with the advent of target-specific and highly active immunotherapeutics, both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have provided guidance that recommend determining a safe starting dose based on a minimum anticipated biological effect level (MABEL) approach. In order to establish a first-in-human dose, as advised by the FDA for bispecific antibodies, this work uses a MABEL approach to select a safe starting dose for EGFRvIII:CD3 bi-scFv, based on a combination of in vitro data, in vivo animal studies, and theoretical human receptor occupancy modeling. Using the most conservative approach to the MABEL assessment, a dose of 57.4 ng EGFRvIII:CD3 bi-scFv/kg body weight was selected as a safe starting dose for a first-in-human clinical study.
Chapter 5 describes the pharmacokinetic properties of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, the chapter first describes the development of a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, was used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ~8 minutes and a terminal half-life of ~2.5 hours. These results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and Phase I studies of EGFRvIII:CD3 bi-scFv.
Finally, Chapter 6 provides an outlook of the future development of cancer therapeutics for treating malignant gliomas.
Item Open Access Structure-Guided Design of Novel Therapeutics Targeting Translesion DNA Synthesis and Lipid A Biosynthesis(2019) Najeeb, JavariaCancer is one of the most devastating diseases in modern society, with over 1.6 million new cancer cases occurring in the US alone each year. DNA-damaging agents are often the first line of defense against rapidly dividing cancer cells. However, cancer cells can become resistant to chemotherapy by up-regulating an error-prone DNA-repair process called translesion DNA synthesis (TLS). The Rev1 polymerase orchestrates this pathway by recruiting one of three inserter polymerases and the extender polymerase (Pol ζ) to bypass the lesion. Here we report the discovery and characterization of an inhibitor of the protein-protein interaction between Rev1 and Rev7, a subunit of Pol ζ, using biochemical and biophysical techniques. Our X-ray crystallographic structural analysis of the Rev1 and the inhibitor (JH-RE-06) complex reveals that the inhibitor blocks Rev7 binding by inducing Rev1 dimerization. Such an unexpected observation is confirmed by an in vitro crosslinking assay. In vitro cell-killing assays show that JH-RE-06 enhances sensitivity of a variety of cancer cell lines to a wide range of chemotherapeutic agents; furthermore, co-administration of JH-RE-06 with cisplatin significantly suppresses melanoma growth in mice and prolongs the survival time of tumor bearing mice, highlighting the therapeutic potential of translesion synthesis inhibitors as a novel class of cancer adjuvant therapeutics to enhance the outcome of chemotherapy currently available to cancer patients.
Due to their compromised immune systems, cancer patients are particularly susceptible to opportunistic bacterial infections, many of which are becoming rapidly resistant to current antibiotic therapies. We describe the combined use of X-ray crystallography and NMR spectroscopy to delineate a cryptic inhibitor envelope for optimization of a small molecule inhibitor of LpxC, an enzyme essential to the survival of Gram-negative bacteria. The resulting inhibitor shows vast improvement over its parent compound over a wide range of bacterial orthologs.
In summary, we demonstrate successful structural characterization and structure-guided design and optimization of lead compounds in two different systems. These studies have profound implications for drug discovery and lead optimization in other disease-relevant systems as well.