Cancer is one of the leading causes of death worldwide. Monoclonal antibodies (mAbs) have been proved effective for cancer therapy. MAbs possess advantages over chemical drugs and small molecular drugs in cancer treatment, such as high specificity, low toxicity, effector function, long half-life in circulation system and less side effects. There are eight FDA-approved anti-cancer antibody drugs now, and many more are under development. Antibodies have two functional domains, the Fab region that is responsible for antigen recognition, and the Fc region that couples the antibody to immune effector pathways. Fab-mediated interference with cancer cell signalling may lead to growth inhibition and direct cell death, while Fc-mediated effector function through interactions with Fc-gamma receptors (FcrRs) expressed in immune cells or through complement cascades may lead to target cell cytotoxicity. Antibody engineering to increase the binding affinity and effector function may improve antibody in vivo efficacy. Anti-Her2 mAb herceptin (trastuzumab) is effective in treatment of Her2-overexpressing breast cancer patients. However, only 25–30% of patients with Her2-overexpressing tumors respond to single agent trastuzumab, and drug resistance develops even in responding patients. Accumulating evidence showed that cross-talk between Her2 and the insulin-like growth factor receptor type I (IGF-IR), including receptor heterodimerization and transactivation, and elevated IGF-IR signalling have been associated with trastuzumab resistance. Therefore, we hypothesized that dual specific antibodies co-targeting both IGF-IR and Her2 may prevent or delay the emergence of resistance to mono-specific antibodies. Mouse monoclonal antibody, M590 showed very good binding activity to IGF-IR. By engineering the CH3 domain of human Fc in pDR12 plasmid, we developed a “knobs-into-holes” hybrid IgG expression system, and successfully produced M590-Herceptin bi-specific IgG, which showed high binding avidity for both antigens and preserved antibody-dependent cell-mediated cytotoxicity (ADCC), a main route of immune protections conferred by therapeutic antibodies in vivo. M590-Herceptin dual specific antibody inhibited breast cancer and ovarian cancer cell proliferation in vitro, and inhibited cancer growth in a SKOV-3 Her2- and IGF-IR-overexpressing ovarian cancer xenograft mouse model. M590-Herceptin hybrid showed better anti-cancer activity compared with M590 and Herceptin alone, or in combination. Meantime, I also constructed a phage display antibody Fab library using the mRNA of rabbits immunized by membrane proteins of SKOV-3 cells, and isolated a novel anti-Her2 mAb, designated as 1C6. Results from in vitro assays showed that 1C6 had anti-cancer activity which was comparable to that of herceptin. M590-1C6 hybrid IgG was also constructed, and the results from in vitro assays and mouse study showed that M590-1C6 hybrid IgG also possess better inhibitory activity of Her2 positive tumours compared with m590 or 1C6 alone. In summary, this study indicates that bi-specific antibodies co-targeting two elevated cancer receptors are more effective than mono-specific antibodies for cancer therapy. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Monoclonal antibodies (MAbs) have been raised against $ beta$-tubulin of B. pahangi and A. suum. Anti-B. pahangi MAbs were used to investigate the heterogeneity of tubulins from nematodes and mammals. One-dimensional SDS-PAGE showed that MAbs P3D and 1B6 react with $ beta$-tubulin from a number of filarial and intestinal nematodes, but not with tubulin from protozoan and mammalian cells. Two-dimensional SDS-PAGE demonstrated that MAb P3D recognizes two isoforms of $ beta$-tubulin and 1B6 recognizes one. Limited proteolysis showed that MAb 1B6 reacted with the amino-terminal fragments and MAb P3D with the carboxyl-terminal fragments of $ beta$-tubulin. The effect of anti-B. pahangi MAbs on the viability of adult B. pahangi was assessed using MTT assay. It was found that MAbs P3D and 1B6 caused an 80% and 40% reduction respectively, in worm viability, whereas anti-chick MAb 357 or mebendazole drug had no effect. Immunogold labelling of B. pahangi demonstrated the presence of tubulin in the median and basal layers of the cuticle, hypodermal layer and somatic muscle blocks, as well as the uterus of B. pahangi. The reduction in the viability of worms may, therefore, be due to the disruption of microtubules in the body wall muscle of B. pahangi. The total MBZ binding was highest in the intestine followed by the body wall muscle and in the reproductive tract extracts of A. suum. Electron microscopy of A. suum tissues demonstrated that the tubulin content decreased from the intestine through the body wall muscle to the reproductive tract. One dimensional SDS-PAGE revealed the presence of $ alpha,$ $ beta sb1$ and $ beta sb2$ tubulin subunits in all tissues of A. suum. This data confirmed the reduction of tubulin from the intestine through the body wall muscle to the reproductive tract. Two dimensional SDS-PAGE followed by Western blotting demonstrated that $ alpha$ and $ beta$ tubulin isoform patterns are dissimilar in different tissues of A. suum. Body wall muscle, inte

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published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

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Affinity molecules can serve as precision tools for selective recognition and measurement of specific biomolecules in the fields of therapeutic drug monitoring and quality control in recombinant protein production. In therapeutic drug monitoring, affinity molecules can enable the accurate quantification of drug concentrations within physiological fluids, enhancing both the safety and efficacy of clinical treatments. In the realm of recombinant protein production, these molecules can allow precise isolation and measurement of desired recombinant proteins from complex mixtures by selectively targeting specific protein tags or domains, ensuring the consistency and purity of protein products. Currently, antibodies are most commonly used affinity reagents in these fields but are limited by production complexity, batch variability, high cost, and low stability. Aptamers, known as ‘chemical antibodies’ but composed of nucleotides, are considered potential next-generation affinity reagents. Aptamers are obtained via a synthetic process, termed SELEX, of iterative affinity selection and polymerase chain reaction (PCR) amplification of target-binding members from a randomized oligonucleotide library. This process is traditionally labor and resource-intensive and time-consuming. In this thesis, microfluidic technology is employed to enable time-efficient and cost-effective generation of aptamers for monoclonal antibodies and recombinant proteins toward applications in therapeutic drug monitoring and quality control of recombinant protein production. This thesis starts with a comparative study of three SELEX strategies for aptamer isolation, including those using conventional agarose bead-based partitioning, microfluidic affinity selection (called “chip-selection SELEX”), and fully integrated microfluidic affinity selection and PCR amplification (termed “full-chip SELEX”). The comparison results indicate that chip-selection SELEX offers the lowest cost and highest efficiency in aptamer isolation. We then use chip-selection SELEX to streamline the process of isolating anti-idiotype aptamers targeting human monoclonal antibodies against spike protein of SARS-CoV-2 virus. The process is completed within only 5 rounds of SELEX within two days, which represented a significant improvement when compared to conventional methods whose completion generally requires more than 10 SELEX rounds in up to a month. These anti-idiotype aptamers are combined with a graphene-based affinity nanosensor to enable rapid antibody concentration measurements to inform therapeutic decisions in a timely manner. In addition, a microfluidic dual-aptamer sandwich assay with highly efficient isolation of aptamers is developed to enable rapid and cost-effective detection of tag-fused recombinant proteins. This approach addresses both the limitations of current dual-aptamer assays and commonly encountered difficulties in the lack of aptamers available for such assays, by first using chip-selection SELEX to generate aptamers and then employing these aptamers to implement a microfluidic dual-aptamer assay for quality control during recombinant protein production. Despite the high efficiency in aptamer isolation using chip-selection SELEX, the full-chip SELEX platform is still desired for minimal manual operation and reagent consumption. The current full-chip SELEX platform has low isolation efficiency and could not offer information of affinity selection process. Herein, by introducing asymmetric PCR into the full-chip SELEX process, we improve the efficiency in aptamer isolation and can successfully monitor the selection progress. This real-time monitoring capability allows us to identify the optimal point to terminate the SELEX process, preventing the potential loss of aptamer candidates and reducing the overall consumption of time and reagents. In addition, introducing solution phase-based asymmetric PCR addresses a notable technical challenge of on-chip PCR bead replenishment, toward complete automation of the full-chip SELEX platform. Furthermore, a holder equipped with connection pins is designed to enable the reversible connection between gold electrodes and electrical wires. This design promotes the reusability of gold electrode-deposited glass substrates, resulting in a substantial reduction in chip fabrication costs. In addition to the SELEX protocol development effort, we also present efficient and cost-effective microfluidic approaches for post-SELEX aptamer characterization, including aptamer identification and kinetic aptamer-target binding measurements. To mitigate the expensive and time-consuming nature of aptamer identification from SELEX-generated target-binding sequence pools, we present an approach that is based on a cost-effective and efficient procedure to generate modified single-stranded DNA copies of the aptamer candidates and then assess the affinity of the resulting modified ssDNA strands to target molecules. The approach is applied to identify aptamers from 12 candidates with consistent results, but at a cost three times lower than that of established methods. We also present a microfluidic fluorescence assay, which exploits a synergistic combination of microfluidic technology and fluorescence microscopy, to realize cost-effective and multiplexed measurement of kinetics of aptamer-target analyte binding without requiring special-purpose equipment.

Purpose: Control of micrometastatic ovarian cancer in the peritoneal cavity remains a major objective in post-surgical treatment. The purpose of this project was to investigate the efficacy and toxicity of targeted alpha therapy (TAT) for ovarian cancer in vitro and in vivo in animal models and to select the optimal targeting vector for an ovarian cancer clinical trial. Animal models of ovarian, breast and prostate cancer were developed and for further TAT; a phase I melanoma clinical trial was supported, paving the way for an ovarian cancer clinical trial. Methods: The expression of the turnor-associated antigens (Her2, MUC1, uPAfuPAR) on cancer cell line, animal model xenografts and human ovarian cancer tissue was tested by immunostaining. MTS and TUNEL assays were used to evaluate cell killing of alpha conjugates in monolayer and spheroids. Toxicity and maximum tolerance doses for different vectors were tested and determined in vivo. Pharmacokinetics was studied for different time points and different parameters. The antiproliferative effect of 213Bi-C595 and 213Bi-PAI2 was tested at 9 days post-peritoneal cell inoculation of the ovarian cancer cell line OVCAR3. The treatment efficacy of 213Bi-Herceptin was tested at a 2 days post-subcutaneous breast cancer cell BT474 inoculation. Mice were injected (i.p) with various concentrations of alpha conjugates (AC). Changes in cancer progression were assessed by girth size and tumor size. Results: uPA/uPAR and MUCI are expressed on ovarian cancer cell lines and more than 45% ovarian cancer tissue, while HER2 was only positive in one cell line and was positive in less than 15% of ovarian cancer tissues. The ACs can target and kill cancer cells in vitro in a dose dependent fashion. TUNEL positive cells were found after incubation with the different ACs. PAI2 and C595 vectors were selected for in vivo ascites model study of OVCARJ cell with high expression. Delayed and acute toxicity in animal models showed that radiation nephropathy was the cause of body weight loss. Biodistribution studies showed that kidney was the major uptake organ. L-lysine can reduce kidney uptake for 213Bi-PAI2, but no significant differences were found. A single ip injection of 213Bi-C595 or 213Bi-PAI2 can inhibit ascites growth, whereas, 213Bi-Herceptin can inhibit breast cancer growth in a nude mice model. Conclusion: 213Bi labelled targeting vectors can specifically target ovarian cancer cells in vitro and inhibit tumor growth in vivo. These ACs may be useful agents for the treatment of ovarian cancer at the minimum residual disease stage.

I established the phage antibody library platform for the identification of specific antibodies. In the first part of my study, I tried to identify antibody against SARS-CoV. Two fragments on the spike protein, which is responsible for inducing viral entry, was chosen as target for the selection of antibody. An antibody was identified which can selectively recognize the SARS-CoV infected cells, but not non-infected cells. Although this antibody was found to retain no neutralizing ability, this specific antibody may have potential to develop for diagnostic purpose. / I utilized the phage system-based cloning method as an attractive approach to screen and identify virus-specific antibodies that can be encoded by the human genome. Once a useful phage clone is identified, unlimited amounts of human monoclonal virus-specific antibodies can be manufactured, and potentially applied clinically for prophylactic and therapeutic uses. The study focuses on two of these new infections, both of which cause severe respiratory disease: SARS and avian influenza. / Identification of specific antibodies, either for diagnostic or therapeutic use, was successfully demonstrated in the two infectious disease models. The phage antibody platform offers a fast and cost-effective method to identify phage antibodies, which can easily be converted to human viral specific monoclonal antibodies for clinical use. / In the 21st century, a number of novel infectious diseases emerged suddenly and spread rapidly, endangering the lives and well-being of people around the world. Severe acute respiratory syndrome (SARS) is a life threatening form of atypical pneumonia that ravaged Hong Kong, Taiwan, China, Canada and many cities in 2003. In the same year, novel avian influenza viruses infected human beings on two continents. Both of these diseases originated in animals and crossed over into the human population. These emerging diseases pose significant public health threats while providing a chilling reminder that another influenza pandemic could occur at any time. Thus, the development of effective therapeutics to control the disease is of paramount importance. Although several vaccines against SARS and avian influenza are available nowadays, the poor clinical performance and frequent mutation of viral strains may limit the practical use and value of the vaccines. Moreover, there are no promising antiviral drugs available for the treatment. Therefore, I aimed to develop an immunotherapy as an alternative treatment option against these diseases. / In the second part of my study, the extracellular domain of matrix protein of avian influenza virus was chosen as target for the selection of antibody. I successfully identified an antibody which can neutralize the avian influenza virus infection. This promising result indicated this antibody has potential to develop for therapeutic use and these antibodies can be easily manufactured in unlimited amounts for clinical application. / Leung, Ka Man. / Adviser: Kwok Pui Fung. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0212. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 112-123). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.