Graduation Date

Fall 12-18-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Maneesh Jain

Second Advisor

Surinder Batra

Abstract

The deregulation of cell surface glycoproteins, such as mucins, is a hallmark of many tumors of epithelial origin. The transmembrane mucin MUC4, is differentially overexpressed in several malignancies, including pancreatic, breast, ovarian, lung, cervical, and head and neck cancers. Of all the aforementioned cancers, the role of MUC4 has been the most thoroughly in pancreatic cancer (PC), which is the 4th leading cause of cancer-related deaths in the United States. While MUC4 is undetectable in the normal or inflamed pancreas (pancreatitis), its expression progressively increases during PC progression and its higher expression correlates with poor survival. The role of MUC4 in neoplastic transformation, enhanced motility, invasiveness, and drug resistance of cancer cells in vitro, and in tumorigenicity and metastasis in vivo has been conclusively established. Due to the differential overexpression of MUC4 in cancer cells and its functional involvement in disease pathobiology, it is an attractive therapeutic target. Of specific interest is the MUC4β-subunit of the MUC4 glycoprotein. The α-domain of MUC4 can be shed from the surface of the cell thus rendering it a feckless target. Thus, we propose to target the MUC4β-domain, a growth factor-like subunit that remains attached to the cell surface. To date no MUC4-targeted therapeutics have been developed. In the first part of the dissertation research, I sought to develop monoclonal antibodies (mAb) against the MUC4β subunit. MAb-based therapeutics have emerged as a promising cancer treatment modality due to their low toxicity and high specificity. Antibodies used in the treatment of solid tumors may inhibit oncogenic signaling, block cell-cell interactions, or engage immune effector cells to attack the tumor by antibody-dependent cellular cytotoxicity (ADCC) in a target expression-specific manner. Alternatively, mAbs can serve as vectors to deliver cytotoxic cargo (drugs, radionuclides, or toxins) to cancer cells in antigen-specific manner. The development of MUC4 specific mAbs was achieved through the use of hybridoma technology. The immunization of BALB/c mice with recombinant MUC4β protein purified from E.coli allowed for the development of antibodies specific to our protein of interest. Through the fusion of mouse splenocytes and myeloma cells we were able to create hybridoma cells that had the properties a B-cell allowing for the production and secretion antibodies of and had the immortality conferred by the myeloma cell partner.

The antibodies produced from this hybridoma fusion were screened for reactivity against MUCβ recombinant protein and against cell lysates from MUC4-expressing cells to identify antibody clones with high binding affinity. These clones were further purified and taken through specificity testing. We began testing for specificity by Western blotting cell lysates from MUC4-expressing and non-expressing cells lines. Determination of MUC4 molecular weights occurred through the use of both horizontal agarose gels for the detection of full length MUC4 and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) to detect the smaller MUC4β fragment. The specificity testing further extended to testing on the native conformation of the MUC4 protein in flow cytometry and confocal microscopy. The specific detection of MUC4 in PC patient samples was also performed using immunohistochemistry (IHC). The binding affinity of the mAbs was determined in surface plasmon resonance (SPR), which demonstrate binding with nanomolar affinity. Overall, the mAbs were validated for MUC4β specificity and could be used for further cell-based functional testing.

To determine the therapeutic potential of MUC4β antibodies, we propose to study their applications for inhibiting MUC4-mediated cell-based functions. Cell-based growth and motility assays were studied as part of the functional application of the MUC4β mAbs. Each assay was performed across several MUC4-expressing and non-expressing cell lines. Growth inhibition was only found to occur to a significant degree for the MUC4-expressing cell line CD18. The inhibition of growth was not found to occur through the induction of apoptosis. The inhibition of motility was only observed to occur to a significant degree for the cell lines engineered to express MUC4. Additional testing included use of the anti-MUC4β mAbs in combination with the chemotherapy gemcitabine against the cell line CD18. This combination demonstrated and additive effect at least equal to each of the therapeutics alone. Together these results demonstrate that the anti-MUC4β mAbs may have the capacity to be used as therapeutic agents and warrant further study in vivo.

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