Xu, Guolin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-128). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGEMENTS --- p.IV / LIST OF FIGURES --- p.VII / ABBBREVIATIONS --- p.X / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Antigens --- p.1 / Chapter 1.1.1 --- Preamble --- p.1 / Chapter 1.1.2 --- Types of antigen --- p.1 / Chapter 1.1.3 --- Autoantigens --- p.2 / Chapter 1.1.4 --- Telomerase is an important autoantigen --- p.6 / Chapter 1.2 --- Antibodies --- p.12 / Chapter 1.2.1 --- Preamble --- p.12 / Chapter 1.2.2 --- Ig structure --- p.12 / Chapter 1.2.3 --- Ig synthesis --- p.13 / Chapter 1.2.4 --- Immunoglobulin isotypes --- p.15 / Chapter 1.2.5 --- Monoclonal antibodies (mAb) --- p.17 / Chapter 1.2.6 --- Autoantibodies --- p.20 / Chapter 1.2.7 --- Telomerase detection and antibodies to telomerase --- p.22 / Chapter 1.3 --- Object and Scope of Study --- p.24 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Animals --- p.31 / Chapter 2.1.2 --- Antibodies --- p.31 / Chapter 2.1.3 --- Primers --- p.31 / Chapter 2.1.4 --- Culture media and reagents --- p.32 / Chapter 2.1.5 --- Chemicals and enzymes --- p.32 / Chapter 2.1.6 --- Miscellaneous chemicals --- p.33 / Chapter 2.1.7 --- Commercial kits --- p.33 / Chapter 2.1.8 --- Instruments --- p.33 / Chapter 2.1.9 --- Buffers --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Cells and cell culture --- p.35 / Chapter 2.2.2 --- Polymerase chain reaction (PCR) --- p.36 / Chapter 2.2.3 --- Cloning of C-terminal gene fragment to pGEX cloning vector --- p.36 / Chapter 2.2.4 --- Detection of antibody activity by ELISA --- p.37 / Chapter 2.2.5 --- Histochemical Staining --- p.38 / Chapter 2.2.6 --- Hybridoma production --- p.40 / Chapter 2.2.7 --- Protein analysis --- p.43 / Chapter 2.2.8 --- Flow cytometry --- p.46 / Chapter 2.2.9 --- Animal handling --- p.47 / Chapter 2.2.10 --- Statistical analysis --- p.48 / Chapter CHAPTER THREE --- PRELIMINARY STUDIES USING THE ANTI-N-TERT-TELOMERASE MAB DERIVED FROM HYBRIDOMA 476 --- p.49 / Chapter 3.1 --- Preamble --- p.49 / Chapter 3.2 --- Hybridoma 476 cells can be stained by the labeled recombinant N-TERT antigen --- p.50 / Chapter 3.3 --- Mouse spleen cells can also be stained by biotin-labeled N-TERT antigen --- p.52 / Chapter 3.4 --- Discussion --- p.53 / Chapter CHAPTER FOUR --- PRODUCTION OF MONOCLONAL ANTIBODIES TO C-TERT --- p.63 / Chapter 4.1 --- Preamble --- p.63 / Chapter 4.2 --- Construction of C-TERT expression vector --- p.63 / Chapter 4.3 --- Expression and purification of recombinant human C-terminal telomerase antigen --- p.64 / Chapter 4.4 --- Immunization of Balb/c mice with C-TERT-GST --- p.65 / Chapter 4.5 --- Generation of hybridomas to C-TERT --- p.65 / Chapter 4.6 --- Identification and selection of reactive clones --- p.65 / Chapter CHAPTER FIVE --- CHARACTERIZATION OF MAB A63 --- p.71 / Chapter 5.1 --- Preamble --- p.71 / Chapter 5.2 --- Characterization of mAb A63 by ELISA --- p.71 / Chapter 5.3 --- Characterization of mAb A63 by Western blotting analysis --- p.73 / Chapter 5.4 --- Characterization of mAb A63 by immuno-histochemical staining --- p.73 / Chapter 5.5 --- mAb A63 can also stain fish telomerase and human placenta --- p.75 / Chapter 5.6 --- mAb A63 can also stain telomerase in human tumors --- p.76 / Chapter 5.7 --- Hybridoma A63 can produce ascites fluid --- p.76 / Chapter 5.8 --- Discussion --- p.77 / Chapter CHAPTER SIX --- GENERAL DISCUSSION --- p.93 / Chapter 6.1 --- Why Enhancing buffer is required for the nuclear staining of hTERT when using mAb 476 or mAb A63 --- p.96 / Chapter 6.2 --- Why hybridoma 476 failed to form ascites while hybridoma A63 succeeded --- p.99 / Chapter 6.3 --- Can IL-6 be used to treat autoimmune diseases? --- p.102 / Chapter 6.4 --- Possible use of monoclonal antibodies in cancer therapy --- p.104 / Chapter 6.5 --- Prospects on study --- p.107 / REFERENCES --- p.111

The objective of this study was to produce a monoclonal antibody specific for the microgametocytes of Eimeria tenella, examine the site and stage specificity of the antibody, and investigate the immunopotency of the antibody. BALB/c mice were immunized with antigen containing Eimeria tenella microgametocytes isolated from in vitro systems. After three intraperitoneal immunizations with the antigen and one booster immunization administered by tail vein injection, the mice were sacrificed and their spleen cells fused with SP2/0 mouse myeloma cells using polyethylene glycol as a fusing agent. Resultant hybridomas were screened by immunoelectrophoresis, indirect immunofluorescent antibody assay, and immunoelectron microscopy to determine the isotype, subisotype, site and stages pecificity of the antibody. Of four 96 well plates seeded with fusion products, four hybridomas were found to be producing anti body specific for the target antigen. Only the most strongly positive of these hybridomas, clone T1A3B9, was used for the study. The antibody produced by this hybridoma was found to be of sub isotype IgG2b. T1A389 monoclonal antibody was introduced into Eimeria tenella infected cell cultures on days four, five, and six post-infection. At seven days post-infection, oocyst production was assayed by fixing, staining, and counting the resultant oocysts. Results of the in vitro experiments showed a greater than 50X reduction in oocyst product ion in experimental cultures over controls. Statistical significance of the data were confirmed by a Mann-Whitney U Test. These results indicate that the monoclonal a ntibod y was exert ing an inhibitory effect on the fertilization process. T1A3B9 monoclonal antibody was incubated with Eimeria tenella infected cecal scrapings and cell culture material, immunolabeled with colloidal gold conjugates, and observed by electron microscopy. Results showed that the antibody was binding to the microgametocytes and to no other life cycle stages of the parasite, nor was it binding to host tissue. This indicates that the antibody is stage specific. Additionally, the antibody was seen to bind only to areas in close proximity to the budding flagella of developing microgametes, thus indicating distinct site specificity.

Monoclonal antibodies were developed against USDA challenge strain of infectious laryngotracheitis virus (ILTV). Indirect immunofluorescence test was used to detect antibodies in supernatants of hybridomas. Hybridoma cells were developed by fusing Sp 2/0 myeloma cells with spleen cells obtained from mice immunized four times with partially purified USDA challenge strain of infectious laryngotracheitis virus. The supernatant of three hybridomas, designated as 2D1D8, 2E11G2, 2C6C7 were found positive for antibody activity against USDA challenge strain of ILTV. Hybridomas producing antibodies were cloned by the limiting dilution method. All three monoclonal antibodies reacted with USDA challenge strain of ILTV, S 88 00224 strain of ILTV, and 86 1169 strain of ILTV in an indirect immunofluorescence test. None of the monoclonal antibodies reacted with avian adenovirus 301 or parrot herpes virus in an indirect immunofluorescence test. The monoclonal antibodies were isotyped, and all three monoclonal antibodies were found to be IgM. / Graduation date: 1993

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Microbiology and Molecular Genetics." Includes bibliographical references (p. 55-60).

Thesis (Ph.D.)--Drexel University, 2010. / Includes abstract and vita. Includes bibliographical references (leaves 237-258).

abstract / Microbiology / Doctoral / Doctor of Philosophy

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

An effective HIV-1 vaccine will likely elicit broadly neutralizing antibodies (bnAbs). However, development of vaccine immunogens that induce bnAbs remains a challenging goal. Understanding the somatic maturation pathways of known broadly neutralizing HIV-1 human monoclonal antibodies (bnmAbs) may help vaccine immunogen design. All known HIV-1 bnmAbs are highly somatically matured, and the putative germline antibodies of the known HIV-1 bnmAbs lack measurable binding activity to HIV-1 envelope glycoprotein (Env). Based on these observations, we hypothesize that somatic maturation of known HIV-1 bnmAbs may be initiated by primary immunogens which may not be related to HIV-1 Env; such primary immunogens trigger the somatic maturation of the germline antibodies and induce intermediate antibodies that bind to HIV-1 Env and further mature to bnAbs upon HIV-1 infection or Env vaccination. The main objective of my study is to identify intermediate antibodies of bnmAb b12 in uninfected and infected human individuals, as well as in uninfected rhesus macaques, the model animals for vaccine development. I constructed two nonimmune cDNA antibody VH1 scFv libraries using the mRNAs isolated from pooled PBMCs of 200 uninfected healthy human individuals and one uninfected rhesus macaque, respectively, and identified 5 and 10 possible b12 intermediate immunoglobulin heavy chain V-segments (IGHVs) from the human and macaque nonimmune libraries, respectively. 454 deep sequencing of the VHs and VLs in the nonimmune and two immune human cDNA Fab libraries confirmed the existence of b12 intermediate IGHVs, but we did not find further maturation of the b12 intermediate IGHVs in HIV-1-infected human individuals. Further sequence analysis revealed the extremely low frequency of the VHs with exactly the same V(D)J recombination as b12, which may explain the lack of further maturation of the intermediate IGHVs of b12 in HIV-1-infected humans. Characterization of HIV-1 Env trimer may aid in Env-based vaccine immunogen design. Therefore, I investigated the importance of Env variable loops in Env-mediated viral function. A panel of gp160JRFL loop deletion/replacement mutants were constructed and tested. The results indicate that, besides the CD4 binding loop and V3, V1V2 and loop D are also critical for virus entry into permissive cells. Deletion of variable V4 or V5 loop or replacement of V4 or V5 loop with a flexible linker of the same length abolish Env cell surface display, which may result from the conformational changes of the V4 or V5 loop deletion or replacement Env proteins. V4 or V5 deletion or replacement knocks out the CD4 binding site and CD4-induced site on Env, but enhances the exposure of the membrane-proximal external region (MPER) and N-trimer structure. In summary, my study demonstrated the existence of intermediate b12 IGHVs in uninfected and HIV-1-infected humans and rhesus macaques. These intermediate antibody fragments may be used to identify primary immunogens that initiate b12 somatic maturation. My study also showed the importance of Env variable loops for Env structural integrity and Env-mediated viral function. The enhanced exposure of the MPER in gp160JRFL ΔV4 or ΔV5 may be further explored for vaccine development to induce MPER-specific bnAbs. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Two monoclonal antibodies (mabs), the newly generated mab anti-type I and the previously documented mab anti-zebrin II, were reacted with brainstem sections of two ostariophysan siluriforms, the gymnotoid Rhamphichthys rostratus and the siluroid Ictalurus punctatus. Mab anti-type I recognizes a 47 kD polypeptide present in the dendrites and soma of projection neurons. Mab anti-zebrin II recognizes a 36 kD polypeptide present throughout the neuronal cytoplasm, including the axon. Strongly type I immunopositive cells include all cerebellar Purkinje cells, pyramidal cells of the nucleus medialis, electrosensory lateral line lobe, and tectum, pacemaker relay cells, Mauthner neurons, lateral line ganglion cells, and cells of the reticular formation, lateral reticular nucleus, and inferior olive. Weakly reactive type I cells include neurons in the torus semicircularis, medial and efferent octavolateralis nuclei, magnocellular and lateral tegmentum, and motor neurons of the Vth, V I Ith, and Xth cranial nerves. All type I positive cells are projection neurons. Zebrin II expression is restricted to subsets of two cell types which also express the type I antigen -- Purkinje cells and developing acousticolateralis pyramidal cells. Both of these neurons develop from the region of the rhombic lip. Thus, the mutual expression of the type I antigen can be explained by the shared function of projection neurons, while the common expression of the zebrin II antigen may be due to a shared embryological lineage. / Department of Physiology and Health Science

Transfusion medicine is an important part of modern health care and the provision of reliably phenotyped red blood cells (RBC) is essential for safe and effective blood transfusions. For identification of many RBC antigens, monoclonal antibodies of either murine or human origin are available for use in agglutination assays, in which they perform as well as or better than the human polyclonal antibody preparations which they have replaced. However, the detection of some blood groups is still reliant on the use of human polyclonal antisera, which is a less reliable reagent source with respect to availability, batch to batch variation and bio-safety. The use of recombinant antibody and phage display technology for the discovery of new monoclonal antibodies with specificity for some of these RBC antigens has the potential to deliver an economical, unlimited supply of specific antibody reagents suitable for use in RBC phenotyping. Samples of human B cells from donors producing useful phenotyping antibodies were identified and transformed using Epstein Barr virus into lymphocyte cell lines. Antibody genes were obtained from the cell lines in the form ofRNA which was reverse transcribed, amplified by PCR and cloned into a phagemid vector system to generate several combinatorial antibody libraries. These antibody libraries were displayed on the surface of phage particles and subjected to antigen-driven selection by several rounds of phage display biopanning using soluble and cell based RBC antigens. In addition a large naIve library was biopanned against the same antigens in an attempt to isolate a wide range of antibodies suitable for blood typing. Several high quality combinatorial antibody libraries with respect to size (> 107 clones) and diversity were generated. Biopanning of recombinant libraries resulted in enrichment of phage antibodies specific for RBC antigens, and several clones were isolated which were shown to be specific for Duffy a antigen. The isolated antibodies would be ideal candidates for re-engineering into multivalent antibody molecules capable of direct agglutination of RBC and as such, have the potential to replace human polyclonal sera in the identification of Duffy a RBC antigen phenotyping.