This thesis, based on five research papers, presents results from development and evaluation ofmethods for identifying the interaction site of antibodies on their antigens and the functional investigation of these in different assays. As antibodies have proven to be invaluable tools in diagnostics, therapy and basic research, the demand of characterizing these binding molecules has increased. Techniques for epitope mapping in a streamlined manner are therefore needed, particularly in high throughput projects as the Human Protein Atlas that aims to systematically generate two antibodies with separate epitopes towards all human proteins.  In paper I we describe an approach to map the epitopes of polyclonal and monoclonal antibodies for the first time using staphylococcal display. This method was combined with peptide scanning and alanine scanning using suspension bead arrays, to create a streamlined approach of highresolution characterization of epitopes recognized by antibodies as demonstrated in paper II. Single epitopes were identified for the monoclonal antibodies and several (one to five) separate epitopes scattered throughout the antigen sequence were determined for each polyclonal antibody. Further, antibodies of different species origin showed overlapping binding epitopes. In paper III we studied the epitope patterns of polyclonal antibodies generated with the same antigen in different animals. Although common epitope regions could be identified the exact epitope pattern was not repeated, as some epitopes did not reoccur in the repeated immunizations. In paper IV, a potential biomarker for colon cancer, RBM3, was investigated using validated antibodies by epitope mapping and siRNA analysis. Finally, in paper V, a method for generating epitope-specific antibodies based on affinity purification of a polyclonal antibody is described. The generated antibodies were used in several immunoassays and showed a great difference in functionality. Paired antibodies with separate epitopes were successfully generated and could be used in a sandwich assay or to validate each other in immunohistochemistry. Taken together, in these studies we have demonstrated valuable concepts for the characterization of antibody epitopes. / QC 20120111

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept. 9, 2009). Includes bibliographical references.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Weiping Zhang / Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea to pigs at all ages, resulting in high mortality rate of 80-100% in piglets less than one week old. Within one year after the outbreak in April 2013, PEDV has rapidly spread in the US and causes the loss of over 10% of the US pig population. Monoclonal antibody (mAb) is a key reagent for rapid diagnosis of PEDV infection. In this study, we produced a panel of mAbs against nonstructural protein 8 (nsp8), spike(S) protein, and nucleocapsid (N) protein of PEDV. Four mAbs were selected, which can be used in various diagnostic assays, including indirect immunofluorescence assay (IFA), enzyme-linked immunoabsorbent assay (ELISA), Western Blot, immunoprecipitation (IP), immunohistochemistry (IHC) test and fluorescence in situ hybridization (FISH). The mAb 51-79 recognizes amino acid (aa) 33-60 of nsp8, mAb 70-100 recognizes aa1371-1377 of S2 protein, and mAb 66-155 recognizes aa 241-360 of N protein, while mAb 13-519 is conformational. Using the mAb70-100, the immunoprecipitated S2 fragment was examined by protein N-terminal sequencing, and cleavage sites between S1 and S2 was identified. In addition, this panel of mAbs was further applied to determine the infection site of PEDV in the pig intestine. IHC test result showed that PEDV mainly located at the mid jejunum, distal jejunum and ileum. Results from this study demonstrated that this panel of mAbs provides a useful tool for PEDV diagnostics and pathogenesis studies.

Radioimmunotherapy (RIT) and radioimmunolocalisation (RIL) are developing and promising technologies to diagnose and treat tumours by use of radiolabelled antibodies targeting tumour specific antigens. The major reason why RIL and RIT not are efficient enough, is the comparatively low accumulation of radiolabelled antibodies in the tumours. Irrespective of the antigen - antibody system used, the maximal tumour uptake in humans is often limited to below 0.1 % of the total injected dose, with significant radionuclide remaining in the blood pool and extravascular fluid. In the present thesis, the following putative improvement techniques for radioimmunotargeting have been evaluated in an experimental model using HeLa cell-xenografted nude mice: 1) Repetitive, simultaneous targeting of different antigens, 2) Removal of non-targeting antibodies using secondary antiidiotypic antibodies, 3) Preinjection of unlabelled antibody to remove shedded antigen and 4) Use of fractionated antibody administration. By use of multiple injections of mixtures of two different 131I-labelled monoclonal antibodies targeting placental alkaline phosphatase (H7) and cytokeratin 8 (TS1), respectively, a significant tumour growth inhibition compared to controls, was obtained. In the treated group, a negligible increase in tumour volume was seen compared to the control group, in which a 20-fold increase was observed. Quantitative determinations of volume densities of viable tumour cells, necrotic cells and connective tissue demonstrated no significant differences in the relative proportions between the groups, indicating that the irradiation caused decelerated growth. Using hybridoma technology, monoclonal antiidiotypic antibodies were generated against both TS1 and H7. The in vitro and in vivo effects of these antibodies, aH7 and aTSl, were investigated. Both these antiidiotypes were found to generate stable complexes with the radiolabelled idiotypic antibody, as revealed by gel-electrophoresis and autoradiography. Using biosensor technology (BIAcore, Pharmacia) the interactions were followed in real time and the association rate-, dissociation rate-, and affinity constants between the reactants were determined. In vivo, the antiidiotypes promoted a rapid dose dependent clearance of the 125I-labelled idiotypes with a decrease in total body radioactivity and concomitant dramatic increase in non-protein bound 125I excreted in the urine. The syngeneic monoclonal antiidiotypic antibody αTSl, was furthermore evaluated as a secondary clearing antibody at radioimmunolocalisation. Injection of αTSl in a molar ratio of 0.5-0.75:1 to TS1, 24 hours after the 125I-labelled TS1 improved the tumour to normal tissue ratio 2-3 fold. This was due to a decreased level of total body radioactivity as well as a slight decrease in tumour-radioactivity. A model describing the kinetics of the involved components, i.e. the antigen, the idiotype and the antiidiotype was presented. It is concluded that high affinity monoclonal antiidiotypes can be used as tools to regulate the levels of idiotypic antibodies in vivo. This strategy, combined with preinjection of non­labelled idiotypic antibodies, caused accumulated doses of 3 Gy to the tumour and 0.9 Gy to non tumour tissues as calculated for 125I-labelled antibodies (80 MBq/mg) by MIRD formalism based on repetitive quantitative radioimmunoscintigraphies. By approaching the maximal tolerated whole body radiation dose for mice (i.e. 6 Gy), it can be estimated that doses up to 20 Gy are possible to obtain following one single injection of labelled antibody. It was furthermore demonstrated that a single bolus injection of antibody is to be preferred, compared to exactly the same dose divided into three or ten fractions. Thus, not only the dose of radioactivity, but also the amount of antibody should be considered for fractionated RIT. In summary, the thesis demonstrates that several techniques can be used to improve radioimmunolocalisation and to approach the proposed 70 Gy required to sterilise tumours at radioimmunotherapy. / digitalisering@umu.se

Background: Both the innate and adaptive immune systems contribute to autoimmune injury in multiple sclerosis (MS). We have been particularly interested in elucidating the role of the innate γδ T-cell population in MS pathogenesis. In particular, some γδ T-cells that express Fc receptors (FcR), such as CD16, that bind antibody are more prominent with MS disease progression and have been shown to exert cytolysis via antibody-dependent cellular cytotoxicity (ADCC). We postulated that if there were also relevant and detectable antibodies in MS patients that might engage these FcR-bearing γδ T-cells then this might be a purported mechanism of neuro-axonal injury. A search for antibodies specific to axonal elements in MS revealed the presence of antibodies to neurofascin (Nfasc). Methods: Anti-Nfasc antibody titres, and concentrations of the light and heavy chains of neurofilament (NfL and NfH, respectively), markers of neuro-axonal injury, were measured in the sera and cerebrospinal fluid (CSF) of MS patients using enzyme-linked immunosorbent assays (ELISA), including those that underwent autologous hematopoietic stem cell transplantation (aHSCT), both prior to and yearly for 3 years thereafter. HeLa cells were transfected with the axonal variant of Nfasc, Nfasc-186, and were utilized as targets in ADCC assays involving γδ T-cells as the effectors, and anti-Nfasc antibodies that were enriched from MS patient sera. Results: Positive anti-Nfasc antibody titres were detected in of 22% and 25% of MS patient sera and CSF, respectively. The most elevated serum titres were in secondary progressive MS (SPMS), and highest CSF titres in relapsing-remitting MS (RRMS) (p<0.05 and p<0.0001, respectively, vs. other neurological disease [OND] controls). Patient serum and CSF antibody titres correlated and, in the CSF, the titres correlated positively with the concentration of NfL. Though NfL and NfH concentrations declined markedly following aHSCT in the CSF, anti-Nfasc antibody titres failed to decline. When co-cultured with CD16+ γδ T-cells in the presence of MS patient-derived anti-Nfasc antibodies, the percent specific cytolysis of the Nfasc-transfected HeLa cells was significantly greater than that of the non-transfected control HeLa cells, at 18% and 1%, respectively, indicating cytolytic kill via ADCC. Summary: Anti-Nfasc antibodies were detectable in the sera and CSF of MS patients, and rarely in OND controls, suggesting they are relevant to MS. Higher titres in the serum support peripheral synthesis, while higher CSF titres in the relapsing phase, that correlate with serum titres, imply that antibodies access the CNS during periods of active inflammation that are associated with disruption of the blood-CSF barrier. CSF anti-Nfasc antibody titres correlated strongly with the release of NfL, suggesting that axonal injury could be related to the presence of Nfasc-specific antibodies. Following aHSCT, CSF NfL and NfH release were reduced without concomitant CSF anti-Nfasc antibody reductions, suggesting that the presence alone of anti-Nfasc antibodies is not enough to cause axonal injury. Indeed, when co-cultured with CD16+ γδ T-cells in the presence of MS patient-derived anti-Nfasc antibodies, the percent specific cytolysis of the Nfasc-transfected HeLa cells was significantly greater than that of the non-transfected control HeLa cells, proving that FcR-bearing γδ T-cells can cause axonal damage by lysing axonal membranes via ADCC, when armed with axon-specific antibodies such as anti-Nfasc. This is the first report of γδ T-cell-mediated cytolysis by ADCC using both γδ T-cells and antibodies derived from MS patients.

Les cellules Natural Killer (NK) sont des lymphocytes capables de tuer les cellules tumorales avec une expression altérée des molécules du Complexe Majeur d’Histocompatibilité (CMH) de classe I . Chez l’homme, cette reconnaissance de “l’absence du soi” est relayée par l’absence d’engagement des antigènes des leucocytes humains (HLA) par les récepteurs inhibiteurs des cellules NK. Chez l’homme, ces récepteurs inhibiteurs incluent les récepteurs KIR qui ont comme analogues fonctionnels chez les rongeurs les lectines Ly49. Certaines tumeurs échappent à la surveillance des NK en augmentant leur expression des molécules HLA. Donc, bloquer les interactions entre les molécules KIR et HLA est une stratégie anti-tumorale intéressante qu’INNATE PHARMA a décidé d’explorer en développant l’anticorps thérapeutique anti-KIR 1-7F9. Mais, ces interactions sont nécessaires pour l’acquisition des fonctions des NK, i.e leur éducation. De plus, elles sont impliquées dans la tolérance aux cellules du soi par les cellules NK. Anticiper la toxicité de 1-7F9, évaluer ses propriétés anti-tumorales et son impact sur l’éducation des NK étaient les objectifs de notre travail. Deux modèles murins ont été développées à ces fins. Le premier, est basé sur les souris B6 : les récepteurs inhibiteurs Ly49 C/I ont pour ligand les molécules H-2b. Le second modèle est basé sur des souris transgéniques pour un unique récepteur KIR et son ligand, en l’absence des molécules endogènes de CMH de classe I murines. Ces deux modèles ont montré que le blocage des récepteurs inhibiteurs n’altère pas la tolérance au soi des NK et est une stratégie anti-tumorale efficace qui n’altère pas la fonctionnalité des NK. / Natural Killer cells (NK cells) are lymphocytes able to kill tumors with aletered expression of Major Histocompatibility Complex (MHC) class I molecules. This “missing self” recognition is mediated by the lack of engagement of Human Leukocytes Antigens (HLA) with NK inhibitory receptors that include Killer Immunoglobulin like Receptors (or KIR) in humans. In rodents, the functional analogues of KIR are Ly49 lectins. Some tumors escape NK cell immune surveillance by increasing the expression of HLA molecules on their surface. So, blocking interactions between KIR and HLA molecules constitutes an interesting therapeutic strategy that INNATE PHARMA decided to explore, developing the anti-KIR monoclonal antibody 1-7F9. Nevertheless, these interactions are necessary for the acquisition of NK functional properties, i.e for their “education”. They are also involved in self-tolerance on educated NK cells. For the pre-clinical development of 1-7F9, it was necessary to anticipate anti-KIR mAb safety and toxicity, evaluate its anti-cancer potential and its impact on NK education. A first part of our work was performed in a surrogate B6 mouse model:Ly49 C/I inhibitory receptors have H-2b molécules as endogenous ligand. Their interactions were blocked with anti-Ly49 C/I monoclonal antibody. In a second part, we report the generation of transgenic mice expressing a single inhibitory KIR in the context of its HLA ligand and in absence of endogenous mouse MHC class I molecules. Both models showed that the blockade of NK inhibitory receptors interactions with their endogenous ligands did not break self tolerance, had a strong anti-tumor effect and did not abrogate NK functionality.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21817号 / 農博第2330号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5189(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 間藤 徹, 教授 髙部 圭司, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Despite the progress made in cancer research, cancer remains one of the leading causes of death worldwide. Although the development of new cancer treatments has improved cancer patients' survival rate, a significant number of patients experience refractory and recurrence events with serious side effects. It is known that the immune system actively participates in eliminating cancer. However, cancer cells can develop mechanisms to evade the immune system resulting in immunotolerance. Immunotherapy aids the patient's immune system's ability to recognize and eliminate cancer cells. During the last three decades, immunotherapy has gradually emerged as an effective and more specific approach to treat cancer. Particularly monoclonal antibodies and adoptive cell therapies such as chimeric antigen receptor (CAR) T-cells have proven highly effective. Nevertheless, the success of these novel therapies depends on discovering suitable tumor targets. Recently, we reported localization of Thymidine Kinase 1 (TK1) to the plasma membrane of certain cancer cells but have not found such localization on normal cells. Similarly, another nucleotide salvage pathway enzyme Hypoxanthine Guanine Phosphoribosyltransferase (HPRT), has also been reported to be localized to the plasma membrane of certain cancer cells. Thus, TK1 and HPRT membrane-associated forms can be potential tumor targets for cancer immunotherapy. This dissertation describes the immunotargeting of TK1 for the selective elimination of tumor cells and the surface localization of HPRT on the plasma membrane of cancer cells. Using hybridoma and phage display technologies, we developed monoclonal antibodies (mAb) and isolated human single domain antibodies (sdAb) specific to human TK1. We confirmed that antibodies and sdAbs could target TK1 on the plasma membrane of lung, breast and colon cancer cells, but not on healthy cells. In addition, we demonstrated that cancer cells expressing membrane-associated TK1 (mTK1) co-cultured with human mononuclear cells (MNC) were selectively eliminated through antibody-dependent cell-mediated cytotoxicity (ADCC) when anti-TK1 mAbs were added. Furthermore, we designed novel TK1 specific tumor targeting receptors and expressed them in human T cells and human macrophages. Finally, we proposed using both TK1 and HPRT as biomarkers for the early detection and monitoring of follicular lymphoma (FL), a disease that is usually detected at advanced stages. The knowledge generated from the data presented in this dissertation indicates that TK1 and HPRT may be suitable immunotherapeutic targets for antibody-based and adoptive cell-based therapies against both liquid and solid malignancies. It also proposes the incorporation of TK1 and HPRT as molecular biomarkers for the early detection and monitoring of FL.

Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.