Development of SNAP-tag based fusion proteins as novel auristatin F-containing immunoconjugates and photoimmunotheranostics in the detection and treatment of triple-negative breast cancer

Mungra, Neelakshi

Development of SNAP-tag based fusion proteins as novel auristatin F-containing immunoconjugates and photoimmunotheranostics in the detection and treatment of triple-negative breast cancer

Doctoral Thesis

2022

Abstract

Breast cancer represents one of the most common forms of female malignancy of the 21st century. Among the various breast cancer subtypes, triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). As an idiosyncratic disease, TNBC displays a conspicuously aggressive and invasive clinical course, with an unexplained partiality towards women of African ancestry. Its acute heterogeneity and complexity behave as mutually reinforcing negative factors, which further complicate prognosis, thereby increasing the burden of breast cancer-related mortality. With the absence of well-defined molecular targets in TNBC, there is a heightened reliance on tri-modality therapy (surgery, radiotherapy and chemotherapy), albeit with an increasing incidence of adverse effects and disease relapse. To this end, there is an urgent need to develop an arsenal of targeted diagnostics and therapeutics, which can be synergized to cover the vast majority of triple-negative breast tumors, paving the way towards the development of personalized regimens suitable for the particular needs and disease of each patient. As such, achieving selective cytotoxicity, with minimal or no collateral damage to healthy tissues, embodies the holy grail of targeted anti-cancer therapies. For instance, the high affinity and specificity of monoclonal antibodies (mAbs) and derivatives thereof, have cemented their application as revolutionary tools in the selective delivery of drugs to malignant cells. These therapeutic proteins, also known as antibody-drug conjugates (ADCs), might exhibit several advantages compared to their small-molecule counterparts, but their widespread clinical use is hampered by various developmental considerations. Traditional conjugation strategies employed to arm mAbs with cytotoxic warheads, usually give rise to heterogeneous mixtures of ADC species, bearing non-uniform drug-to-antibody ratios (DARs), pharmacologic characteristics and safety profiles. Fortunately, the implementation of self-labeling tags (such as SNAP-tag, CLIP-tag and Halo-tag) are providing renewed impetus to homogeneous recombinant immunotherapeutics development. More precisely, SNAP-tag is an engineered mutant of the human O(6)-alkylguanine-DNA alkyltransferase, endowed with the ability to specifically and irreversibly react with benzylguanine (BG) derivatives, forming a stable product. Based on the above premises, this research aims to use SNAP-tag technology as a cutting-edge site-specific conjugation method to: (1) develop a comprehensive antibody platform, consisting of single-chain antibody fragments (scFvs) genetically fused to SNAP-tag, to specifically screen and evaluate their predictive potential for chondroitin sulfate proteoglycan 4 (CSPG4), CD44 and aspartate (aspartyl/asparaginyl) β-hydroxylase (ASPH)-positive TNBC cells, (2) generate functional recombinant ADC formulations as robust delivery systems carrying the antimitotic drug monomethyl auristatin F (MMAF/AURIF), concurrently overcoming production constraints to yield therapeutically viable and homogeneous combination products, and (3) provide a fail-safe system that overcomes the lack of specificity of photodynamic therapy (PDT), by coupling scFv-SNAP-tag to the potent near-infrared (NIR) photosensitizer (PS) IRDye700DX (IR700) and to demonstrate its selective dose-dependent cytotoxic activities in vitro. Following in silico design of the open reading frames (ORFs) coding for each construct, standardized molecular cloning techniques were implemented to generate recombinant mammalian expression plasmids, encompassing Ig-Kappa leader as an efficient protein secretory system. After confirmation of the DNA integrity, protein expression was achieved through transient transfection into HEK293T cells. Thereafter, the resulting histidine-tagged fusion proteins (αCSPG4(scFv)-SNAP, αCD44(scFv)-SNAPf and αASPH(scFv)-SNAP) were harvested from the cell culture supernatant and subjected to immobilized metal affinity chromatography (IMAC). In order to evaluate the outcome of this protein expression and purification step, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were used to confirm the presence of full-length recombinant SNAP-tag based fusion proteins based on their molecular weights. Integration of the fluorescent dye Alexa Fluor 488 into the fusion proteins was carried out to investigate the self-labeling activity of the SNAP-tag moiety, as well as to provide qualitative and quantitative insights into the binding potential of the antibody fragments towards their cognate antigens. Subsequently, the AURIF and IR700-based immunoconjugates were generated by conjugating scFv-SNAP with their respective BG-modified substrates, in a defined 1:1 stoichiometric reaction. The specific and dose-dependent biological activities of the resulting bifunctional therapeutic proteins were then assessed on TNBC cells. In this study, pCB-αCD44(scFv)-SNAPf was successfully cloned and all 3 fusion proteins were effectively expressed, although with low yields and purity, yet adequate for downstream in vitro characterization. After showcasing the self-labeling potential of the SNAP-tag component, surface binding of the fluorescently labeled product was demonstrated on antigen-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell killing ability of the novel AURIF-based recombinant ADCs and IR700 photoimmunoconjugates, was illustrated by the induction of a 50% reduction in cell viability (IC50 value) at nanomolar to micromolar concentrations on target cell lines. This observable selective cytotoxicity revealed that conjugation of BG derivatives to SNAP-tag, did not affect the binding potential of the antibody fragment, nor abrogated the cytocidal activity of the payload. As a proof of concept, this research builds on existing work that promulgates the use of SNAP-tag as a state-of-the-art conjugation strategy that can circumvent the challenges associated with the use of antibodies as effective delivery systems for therapeutic molecules. By harnessing the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically acceptable immunoconjugates, the results herein presented, also highlight the prospects of such agents in disease-specific tumor suppression. While various architectural modifications could further improve cytotoxic activities of future combination products, this research underscores the duality of SNAP-tag in the development of immunodiagnostics and therapeutics, that could potentially be instrumental in instilling a shift towards a personalized medicine stratagem. In conclusion, the combination of such immunoconjugates with a robust companion diagnostic panel provided by SNAP-tag, represents a first step towards the effective management of TNBC, with potential impact on the economic, social and clinical settings.

Keywords

Chemical and Systems Biology

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PhD / Doctoral

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