Browsing by Subject "Antibody"

Now showing 1 - 6 of 6
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    Antibodies In Cancer Therapy: New Targets, Applications And Combination Strategies
    (2019-07) Khanna, Vidhi Devendra
    Over the last decade, antibodies have become an important component in the arsenal of cancer therapeutics. Their high-specificity, low-off target effects, desirable pharmacokinetics and high success rate are a few of the many attributes that make them amenable for development as drugs. The work presented here explores the targeting, mechanisms and use of antibody-based cancer therapy. In the first chapter, we used a phage display-based cell panning procedure to develop two fully humanized antibodies, Tw1S4_6 and Tw1S4_AM6, that bind specifically to HSPG2/perlecan, a protein found to be overexpressed on tumor cells. Immunohistochemistry studies revealed high HSPG2 expression across various tumor sub-types including melanoma, bladder cancer, glioblastoma and ovarian cancer. There was significant correlation between high HSPG2 expression and poor survival in triple negative breast cancer, bladder and ovarian cancers. The data presented here points towards the relevance of HSPG2 as a novel target for not only triple negative breast cancer but other malignancies as well. Based on its over-expression in different solid tumors, we evaluated HSPG2 as a therapeutic target in the second chapter. We observed significant tumor growth inhibition with Tw1S4_AM6 in the triple negative MDA-MB-231-LM2 breast cancer xenograft model. This efficacy was reduced in NSG mice, suggesting NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) as a potential mechanism of action. In vitro studies using human PBMCs confirmed induction of ADCC with anti-HSPG2 antibodies. In addition, conjugation of Tw1S4_AM6 on the surface of polymeric nanoparticles enabled increased tumor cell uptake of nanoparticles, suggesting Tw1S4_AM6 could be valuable as a targeting ligand for drug delivery systems. There is a significant interest in designing therapeutic agents that can enhance ADCC and thereby improve clinical responses with approved antibodies. We have developed a suite of highly substituted imidazoquinolines, which activate TLR 7 and/or 8 and induce significantly higher levels of cytokines compared to the FDA-approved TLR7 agonist, imiquimod. In the third chapter, we evaluated our series of TLR7/8 agonists for their ability to improve ADCC. Our studies show that the second generation TLR 7/8 agonists induce robust pro-inflammatory cytokine secretion and activate NK cells. These agonists also enhanced ADCC in vitro. Finally, we found that these agonists significantly improved the anticancer efficacy of two monoclonal antibodies in vivo. Thus, the work presented here encompasses the three critical aspects of antibody therapeutics: identifying the target, understanding their mechanisms, and leveraging these mechanisms to improve their efficacy.
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    Antibody Conjugated Nanoparticles for Targeting Metastatic Triple Negative Breast Cancer
    (2016-09) Khanna, Vidhi Devendra
    Early detection and the availability of new treatments have improved the survival rates of patients presenting with local or regional breast cancer to as high as 99% and 85%, respectively. On the contrary, patients with metastatic disease have a dismal 5-year survival rate of 17%.1 Thus, there is an urgent need for treatment strategies directed towards metastasis. Our lab has developed antibodies (Clone 6 and AM6) capable of recognizing tumor cells that have undergone epithelial-to-mesenchymal transition (EMT), a key step in the generation of circulating tumor cells and metastasis. The goal of the current study was to determine whether we use these antibodies as targeting ligands for directing anticancer drug-loaded polymeric nanoparticles to metastatic triple negative breast cancer cells as a novel therapeutic option. Polymeric PLGA nanoparticles loaded with paclitaxel, a chemotherapeutic agent, were functionalized with the antibodies using thiol-maleimide chemistry. We optimized the conjugation reaction in order to achieve maximal cell uptake of nanoparticles without compromising antibody binding. In vitro studies were carried out in an MDA-MB-231 derivative cell line with enhanced lung metastatic potential as well as a melanoma metastatic cell line M12. Clone 6 nanoparticles and AM6 nanoparticles showed significant improvement in cellular uptake as well as retention. A competition experiment confirmed target-mediated uptake of nanoparticles. Cytotoxicity studies showed improved cell kill using Clone 6 nanoparticles and AM6 nanoparticles. Based on these promising in vitro results, we are currently carrying out in vivo studies in mice. The development of a targeted drug delivery system for the treatment of metastatic triple negative breast cancer can significantly enhance the survival rate for patients who often times have a life-expectancy of less than one year.2
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    Antibody glycoengineering for drug delivery applications
    (2019-12) Sehgal, Drishti
    Monoclonal antibodies (mAbs) are frontline drugs for the treatment of many diseases including cancer 1 and rheumatoid arthritis 2 . In addition to their natural role as neutralizers of pathogens and toxins as well as in the recruitment of immune elements (complement, improving phagocytosis, antibody dependent cytotoxicity), they can be used as carriers for tumor-targeted delivery of therapeutic and diagnostic agents 3 . However, conjugation of drug or drug-encapsulated nanoparticles to antibodies can often result in reduced affinity of the antibody towards the target antigen. The overall objective of this thesis is to advance a new antibody glycoengineering technology that will allow for facile synthesis of antibody-based drug delivery systems. Most therapeutic mAbs are of the IgG class, which contains a glycosylation site in the Fc region at position 297 4 . In chapter 2, we investigated a glycoengineering strategy that enables the introduction of artificial azide groups at this glycosylation site without affecting their antigen affinity. This is based on the observation that glycosyltransferases present in mammalian cells can incorporate non-natural sugars (e.g., azido mannose) at glycosylation sites on an IgG molecule during the post translational modification. The azide groups in these artificial sugars are then available to react with alkynes through copper-catalyzed ‘click’ chemistry or with strained alkynes such as dibenzyl cyclooctyne (DBCO) allowing for biorthogonal, copper-free ‘click’ chemistry. Because the sugars are added reproducibly and at a site that does not affect antigen binding, the glycoengineering technology would overcome problems associated with traditional conjugation strategies. Using this approach, azide groups were introduced in anti-CD133 and anti-perlecan (AM6) antibodies. Further, the azide groups were available to react with various DBCO conjugates including fluorophores, drug molecules and nanoparticles. Importantly, the addition of artificial sugar and subsequent azide-alkyne reaction did not affect the affinity of the antibody for the target antigen. Antibody–drug conjugates (ADCs) have emerged as the next generation anticancer therapeutic agents. In chapter 3, glycoengineered AM6 antibody was used to generate an ADC with monomethyl auristatin E (MMAE) as the cytotoxic drug. The glycoengineering approach resulted in an ADC with a DAR of 2-3 drug molecules per antibody. The AM6- MMAE conjugate demonstrated enhanced cell kill in vitro and significantly improved anticancer efficacy in vivo compared to free MMAE. Similarly, in chapter 4, glycoengineered AM6 antibody was used to generate antibody conjugated polymeric nanoparticles loaded with paclitaxel. These perlecantargeted nanoparticles showed enhanced antitumor efficacy in vitro and in vivo in TNBC tumor models. Similarly, antibody conjugated nanoparticles showed enhanced antitumor efficacy in vitro and complete tumor growth inhibition in vivo in a non-muscle invasive bladder cancer model. We expect that this glycoengineering strategy will prove to be a unique platform technology that will have a significant impact on antibody-based therapeutics.
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    Antibody repertoire dynamics in the changing landscape of infection
    (2013-05) Schwartz, John Charles
    Antibody responses are fundamentally important to effector and memory mechanisms of disease resistance. In order to respond to a nearly infinite array of possible antigens, the antibody repertoire must be suitably diverse. To achieve this necessary high level of diversity, the antibody repertoire has evolved a unique recombinatorial system consisting of a large number of gene segments that can recombine in different combinations to yield an astronomical array of potential antigen-binding structures. Understanding the antibody repertoire of swine (Sus scrofa) can inform about host genetic differences that may affect disease susceptibility and resistance. Also, it may allow identification of antibody molecules that are important in the host immune response against specific pathogens. Such knowledge could potentially be used in the future to develop selective breeding programs for animals that possess desirable immunological traits, and to screen for specific antibody molecules that are of either therapeutic or diagnostic importance. Knowledge of antibody repertoire diversity in swine has heretofore been lacking. While most previous studies have focused heavily on understanding the heavy chain repertoire by analyzing hundreds of cDNA clones, there have been few investigations of the porcine light chain repertoire. This study was designed to characterize the organization and complexity of both the kappa and lambda light chain loci in the pig genome. Findings revealed extensive allelic variation between both homologous pairs of chromosomes in a single sow and suggested non-crossover homologous recombination (i.e. gene conversion) as a potential evolutionary mechanism to explain at least part of that variation. Armed with this new information, and with that from the previously characterized heavy chain locus, antibody variable region amplicon libraries were generated from lymphoid tissues of pigs either infected (n=2) or mock-infected (heavy chain, n=2; light chain, n=3) with the major swine pathogen, porcine reproductive and respiratory syndrome virus (PRRSV). It is hypothesized that the major anti-PRRSV antibody responses would be detectible in infected animals compared to their control counterparts. Approximately a half-million reads for each heavy and light chain library were generated. From this data, diversity of the expressed antibody repertoire was assessed, including gene segment usage and allelic variability, and anti-PRRSV responses. As predicted, due to biological necessity, the heavy and light chain repertoires possessed a rich array of putatively functional antibody transcripts (heavy chain richness estimate, 3x105 molecules; kappa light chain, 1.5x105; lambda light chain, 2.3 x 105), despite being restricted in their germline to a small number of functional D and J gene segments, a single heavy chain V gene segment family and four light chain V gene families. Interestingly, a power-law distribution of antibody abundances was detected similar to what has previously been reported in zebrafish (Danio rerio), whereby a small number of antibody sequences are exceptionally common and the vast majority are exceptionally rare. Substantial allelic variation was also detected, most notably in the lambda locus. Four out of 5 pigs possessed a functional copy of a previously undescribed V gene segment (IGLV3-1-1) which substantially contributed to the expressed repertoire of the animals that possessed a copy. Importantly, a small number of antibody sequences were detected which were incredibly abundant (>1% of the entire repertoire) in PRRSV-infected pigs and rare in uninfected pigs. It is hypothesized that these highly abundant antibody molecules are PRRSV-specific. Using the knowledge obtained from these studies, future investigations will examine the repertoire for specific heavy and light chain pairs from PRRSV-infected pigs that can neutralize PRRSV using an antibody yeast-display system. In addition, specific heavy and light chain pairs identified in our expression analysis and deemed putatively PRRSV-specific are to be tested for epitopic specificity against labeled PRRSV as well as individual PRRSV recombinant antigens. This last method represents a potential novel and non-lethal means of generating antigen-specific recombinant antibodies derived from lymphoid tissue of immunized animals.
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    Detection, diagnostics, and characterization of virus-like organisms and conformational disease-like proteins in plants
    (2016-10) Bratsch, Sara Ann
    The projects in this thesis all investigated virus-like organisms in agronomically important plants. Chapter one consists of reports describing viruses identified in new hosts or locations. Tobacco rattle virus (TRV) was identified in symptomatic Phryma leptostachya L, a native perennial, from plants in an uncultivated habitat and suggests that TRV may be endemic to North America. Since TRV is the causal organism of corky ringspot of potato this study raises the possibility that native perennial plants could serve as a potential reservoir to cause disease in potato. Canna yellow mottle virus was isolated for the first time from symptomatic Canna indica in Kenya. Cut flowers are a major agronomic crop of Kenya and growers should plant only virus indexed plants to limit losses from virus infection. Orchid fleck virus was confirmed by microscopy and sequence analysis for the first time in the United States in Phalaenopsis hybrida. Asymptomatic P. hybrida tested by one step reverse transcription polymerase chain reaction (RT-PCR) did not yield the expected product while a two step RT-PCR, creating cDNA first, yielded the expected product. This indicates the one step RT-PCR diagnostic test can yield false negatives for asymptomatic plants. Chapter two describes the production of polyclonal antibodies for the detection of Orchid fleck virus (OFV). OFV is a mite transmitted virus and has been reported word wide. The previous project identified a need for a reliable, inexpensive method to detect OFV in plants use for propagation, breeding, conservatories, or virus indexing projects. Polyclonal antibodies were produced in rabbits against Escherichia coli expressed OFV phosphoprotein and matrix protein. The resulting antiserums were assayed in PTA-ELISA and DAS-ELISA. OFV phosphoprotein antisera in PTA-ELISA readily differentiated between healthy and OFV infected orchid (Phalaenopsis hybrida) tissue. OFV matrix antisera in PTA-ELISA detected bacterially-expressed protein but did not differentiate between healthy or OFV infected tissue. The OFV phosphoprotein antiserum can be used by in PTA-ELISA to reliably detect OFV. Chapter three describes the molecular and biological characterization of a new Nepovirus causing a leaf mottling disease in Petunia hybrida. The sequence of the majority of the genome was determined by next generation sequencing and the sequence of remainder of the genome was obtained using a 5’ RACE amplification and RT-PCR using poly-A tail and virus specific primers. Due to phylogenetic relationship and sufficient genome dissimilarity to characterized viruses I propose the name of Petunia Chlorotic Mottle Virus for a new Nepovirus. The fourth chapter describes characterization of filamentous virus-like particles in members of the Asteraceae plant family including sunflower, chrysanthemum, coneflower, gerbera daisy, and zinnia. The filaments were 7-10 nanometers in diameter and could exceed 3,000 nm in length. The N-terminal sequences of the major proteins associated with purified filaments from several species were nearly identical and shared homology with the kunitz soybean trypsin inhibitor (KTI) family of proteins. CID MS/MS sequencing of the major proteins of purified sunflower filaments also shared homology with a KTI sequence. A Western blot using antiserum prepared against recombinant sunflower KTI protein labeled the observable protein bands from sunflower filaments. Filaments composed of a major protein of KTI have been found across the Asteraceae family but have not been observed in dandelion, thistle, or lettuce.
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    Humanized antibody development using phage display: applications to solid tumor metastasis
    (2016-07) Kalscheuer, Stephen
    The outlook for cancer patients who present with evidence of metastasis is best characterized as a precipitous decline in prognosis and overall survival. This dramatic reduction in survival suggests a need to focus on the development of therapeutic and diagnostic reagents that are tailored to cancer in its disseminated form. In pursuit of this goal, a phage display based phenotype screening platform was developed to generate humanized antibodies for use in both circulating tumor cell detection, and therapeutic intervention, using in vivo models of breast cancer metastasis. A broader perspective on this work is that it highlights methods that focus on biologic drug development based on disease phenotype, as opposed to conventional target-based methods. In the context of metastasis, the present work focused on the relevant cancer cell phenotype, termed epithelial to mesenchymal transition, which is believed to be the driver phenotype of cancer dissemination. Phenotype screening approaches do not require prior knowledge of potential targets, and are thus amenable to cancer biomarker discovery, which in turn can lead to innovative, first-in-class approaches to cancer management. A broadly applicable method for deriving humanized antibodies from cell based phenotype screening was developed. Target deconvolution approaches to identify the binding partner of candidate antibodies were also explored. Finally, the fine tuning of antibody binding affinity via affinity maturation methods was also explored through physiologically based pharmacokinetic modelling, in an attempt to establish optimal targeting affinities for both solid tumors and metastases. The work concludes with in vitro and in vivo characterization of two candidate antibodies as therapeutic agents.