Cellular adhesion research has recently focused on the small scale at the level of individual receptor-ligand bonds. This trend in research is primarily due to experimental advances which allow such individual bond force measurements. Here, one of these techniques, micromanipulation, has been extended to not only determine the bond force of individual receptor-ligand pairs, but also the intrinsic kinetic rates of the interaction. Using transmembrane (TM ) Fc gamma receptor III (CD16a-TM) and human IgG (hIgG), the dependence of adhesion probability on receptor-ligand expression densities, contract duration and contact area was quantitated. A probabilistic based theoretical formulation was developed and validated that relates the intrinsic molecular kinetic rates of the receptorVligand interaction to the experimentally determined adhesion probability. This theoretical formulation describing individual receptor-ligand kinetics has also allowed direct evaluation of existing biophysical bond strength/kinetics paradigms at the extreme condition of single bonds. A force-displacement model was also developed to quantitate the force exerted on the RBC membrane transducer during the micropipette retraction process and found to be in agreement with previous work. In addition to CD16a-TM, the kinetic rates of CD16a anchored via a glycosyl phosphatidylinositol (GPI) moiety (CD16a-GPI) and the two alleles of CD16b (NA1 and NA2) were determined for human, rabbit, and mouse IgG species. The binding affinity of these CD16 interactions to soluble IgG was also measured by traditional bulk chemistry approaches and compared to those measured via the micromanipulation protocol in which the IgG ligand is membrane bound in the solid phase. These data suggest that the membrane anchor itself can alter CD16 binding properties. This represents the first reported effect of the anchor on an intrinsic receptor property, its kinetic rates and binding affinity. This thesis presents two specific aims or goals. These goals were achieved and reported in this thesis. During the course of this research, I also explored other directions and gathered initial data. These directions were further explored by other researchers but the initial data is also presented here.

<p>Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. Howev-er, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific. Both clones were shown to have overlapping binding epitopes with their polyclonal rabbit antibody counterpart (rabbit anti-Sox11/KIAA0882) through competitive ELISA. The anti-Sox11 IgG was able to detect two bands in cell lysate in Western blot, of which one probably is Sox11 while the other band possibly could be Sox4. However, this needs to be confirmed in future experiments. The affinity of the anti-Sox11 IgG was measured in Biacore and compared to the affinity of its original scFv. This gave a rough estimation of the affinities, but the values are unreliable and the measurements need to be redone. Although more work has to be put into evaluating the potential of the produced IgGs, they compose a promising starting point to an improved understanding and improved diagnosis of MCL.</p>

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 164-204.

Animals deficient in complement components C1q, C4, C3, and CR1/2 have severely impaired antibody responses. C1q is primarily activated by antibody-antigen complexes. Antigen-specific IgM in complex with an antigen is able to enhance the antibody response against that antigen. This is dependent on the ability of IgM to activate complement. Naïve mice have very low amounts of specific antibodies and therefore it is surprising that classical pathway activation plays a role for primary antibody responses. It was hypothesized that natural IgM, present in naïve mice, would bind an antigen with enough affinity to activate C1q. To test this, a knock-in mouse strain, Cm13, with a point mutation in m heavy chain, making its IgM unable to activate complement was constructed. Surprisingly, the antibody responses in Cm13 were normal. Puzzled by the finding that the ability of IgM to activate complement was required only for some effects, the immunization protocol was changed to mimic an infectious scenario. With this regime, Cm13 mice had an impaired antibody response compared to wildtype (WT) mice. The antibody response in WT mice to these repeated low-dose immunizations was also enhanced. These observations suggest that IgM-mediated enhancement indeed plays a physiological role in initiation of early antibody responses. IgM-mediated enhancement cannot however compensate for the dependecy of T-cell help. Although IgM from WT mice enhanced the antibody response, the T-cell response was not enhanced. The connection between classical pathway activation and CR1/2 is thought to be generation of ligands for CR1/2. In mice, CR1/2 are expressed on B cells and follicular dendritic cells (FDC). Although CR1/2 are crucial for a normal antibody response, the molecular mechanism(s) are not understood. To investigate whether CR1/2 must be expressed on B-cells or FDC to generate a normal antibody response, chimeric mice between WT and CR1/2-deficient mice were constructed. The results show that CR1/2+ FDC were crucial for the generation of antibody responses. In the presence of CR1/2+ FDC, both CR1/2+ and CR1/2- B cells were equally good antibody producers. However, for an optimally enhanced antibody response against IgM-antigen complexes, both B cells and FDC needed to express CR1/2.

With the studies performed in this theses the local drug delivery technique FibMat developed by the biotech company AddBIO, was shown to be applicable to other plasma proteins and drugs than the fibrinogen-bisphosphonate combination that is today being commercialized. Hence the potential for a broader field of application was demonstrated. The application targeted today is as a surface modification giving improved strength to bone around screws used in bone implants. The effect of changing protein and manufacturing conditions was studied with null ellipsometry. It was demonstrated that with changes in incubation temperature, pH and salinity the fibrinogen could be successfully exchanged for the plasma proteins human serum albumin and immunoglobulin G. With liquid scintillation counting it was shown that the developed protein multilayers were able to absorb and release the bone strengthening drug alendronic acid in levels comparable to that of the fibrinogen based ditto. Disk susceptibility tests with the bacteria S. Aureus showed a potential for antibacterial functionalization with gentamicin. The release was, in the case of the fibrinogen multilayer, detectable up to 48 hours. Similar test revealed an inability of silver nanoparticle incorporated protein multilayers to achieve inhibitory levels.

The thesis is focused on site-specific labeling of affinity molecules for different applications where two types of binding proteins, Affibody molecules and antibodies, have been used. For the purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study, two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395). In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the 111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-MMA-ZHER2:2395 counterpart,resulting in improved tumor-to-organ ratios. In a second study the in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant. In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1, was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for 111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation resulted in the highest contrast. Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-binding regions might influence the affinity. A method for site-specific labeling of antibodies using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody by photoconjugation was optimized. By investigation of positions most suitable for incorporation of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses important for both diagnostic and therapeutic applications. In addition, optimized variants were used in combination with an incorporated click-reactive handle for selective labeling of the antibody with a detection molecule. / <p>QC 20140929</p>

This thesis focuses on surface science and bioengineering investigations, first for the development of an improved biomaterial for orthopaedic implant applications, and second, for the development of a biosensor device for biomedical diagnostics. A key component considered in this thesis was the covalent linkage of proteins to the material’s surface for retaining the protein’s immunological and biological activities and for generating a functional interface. Part 1 of this thesis investigated surface modification procedures for improving the bioactivity of titanium substrates. Titanium is first coated with a bioactive silica film grown by plasma enhanced chemical vapour deposition (PECVD), referred to as PECVD-Si-Ti. In previous studies, the bone-implant integration process was enhanced 1.6-fold for titanium implants coated with PECVD-Si films compared to uncoated titanium implants in vivo. However, in vitro studies carried out in this thesis showed that the growth of MG63 osteoblast-like cells was 7-fold higher on uncoated titanium compared to PECVD-Si coated titanium. Therefore, to improve cell growth on the surface and, by inference, the integration of PECVD-Si-Ti implants into bone tissue, the implant’s surface was functionalised with a mitogenic factor, insulin-like growth factor-1 (IGF-1). This was accomplished by modifying the PECVD-Si-Ti surface with an alkoxysilane, 3-isocyanatopropyl triethoxysilane (IPTES), and then by covalent bioconjugation of IGF-1 through isocyanate-amino chemistry. After 72 h of in vitro cell culture in serum-free medium, the growth of MG63 cells was enhanced 1.9-fold on IPTES functionalised PECVD-Si-Ti, which was loaded with covalently immobilised IGF-1 compared to IPTES functionalised PECVD-Si-Ti without IGF-1 (isocyanate reactive groups were quenched with ethanolamine hydrochloride). The attachment and adhesion of MG63 cells were also enhanced on PECVD-Si-Ti by the covalently immobilised IGF-1 in serum-free cell culture conditions. Therefore, the bioactivity of PECVD-Si-Ti was improved by covalently linking IGF-1 to the substrate surface through isocyanate-amino chemistry. Part 2 of this thesis involved the development of a new optical interferometric biosensor. The biosensor platform was constructed from electrochemically-prepared thin films of porous silicon that acted as a sensing matrix and transducer element. By reflective interferometry using white light, an enzyme-catalysed reaction was discovered (horseradish peroxidase (HRP) mediated oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB)), which led to an acceleration in the rate of porous silicon corrosion and represented the biosensor’s readout signal. We discovered that another substrate, which is also oxidised by HRP, OPD, produces an even more pronounced readout signal. The HRP-OPD system was used in an immunoassay for detecting human IgG from an Intragam solution. An important part in the design of the biosensor was the surface functionalisation approach where anti-human IgG, referred to as the capture antibody, is immobilised on the porous silicon surface. The readout signal (produced from the capture of human IgG) was enhanced 4-fold on the porous silicon biosensing platform functionalised with covalently linked anti-human IgG through isocyanate-amino chemistry compared to the porous silicon biosensing platform functionalised with adsorbed anti-human IgG. The optimised biosensor was used to detect IgG from a total human protein concentration of Intragam to a sensitivity of 100 ng/ml. In summary, isocyanate-amino bioconjugate chemistry was used to covalently link either IGF-1 to PECVD-Si-Ti for improving the biological activity of the orthopaedic implant and to covalently link IgG to porous silicon for developing a sensitive biosensor for the detection of proteins. This surface chemistry approach is very useful for biomaterial and biosensing applications.

Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 158-168.