Browsing by Subject "Antibodies"

Now showing 1 - 3 of 3
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    Chemically self-assembled antibody nanorings (CSANS): Design and characterization of an anti-CD3 IGM biomimetic.
    (2010-10) Li, Qing
    Based on our development of a highly efficient protocol for the chemically controlled self-assembling of protein nanorings, we have sought to exploit our methodology for engineering multivalent chemically self-assembled antibody-nanorings (CSANs) for tissue imaging and drug delivery. Two novel DHFR-DHFR-anti-CD3 scFv fusion proteins were constructed (13DDantiCD3 and 1DDantiCD3). In addition, the two DHFR cysteines were mutated to either alanine or serine to enhance correct folding. The protein was expressed in BL21 (DE3) cells, renatured with the SLS-based refolding protocol and purified by methotrexate affinity chromatography. Incubation of 13DDantiCD3 with the chemical dimerizer, bisMTX, resulted in almost exclusive formation of the bivalent CSANs, while incubation with 1DDantiCD3 resulted in formation of octavalent CSANs. Both antibody nanorings selectively blocked the killing of the CD3+ human T-leukemia HPB-MLT by a diphtheria-anti-CD3 immunotoxin. FACS analysis revealed nearly identical dissociation constants for both the selfassembled and parental monoclonal antibody and a 3-fold lower K d for the octavalent species. The chemically dimerized scFv's were shown to be stable in cell culture at 37°C and the dimerization was shown to be reversible by the addition of excess amounts of the non-toxic FDA approved DHFR antagonist trimethoprim. We also demonstrate that, similar to the parental bivalent anti-CD3 monoclonal antibody (MAB), anti-CD3 CSANs selectively bind to CD3+ leukemia cells, and undergo rapid internalization through a caveolin-independent pathway that requires cholesterol, actin polymerization and protein tyrosine kinase activation. While treatment with the monoclonal antibody leads to T-cell activation and nearly complete loss (i.e. 90%) of surface displayed T-cell receptor (TCR), only 25-30% of the TCR down regulate and no significant T-cell proliferation is observed after treatment of peripheral blood mononuclear cells (PBMCs) with anti-CD3 CSANs. Consistent with the proliferation findings, 15-25% less CD25 (IL-2 receptor) was found on the surface of PBMCs treated with either the polyvalent or bivalent anti-CD3 CSANs, respectively, than on PBMCs treated with the parental MAB. Comparative experiments with F(ab')2 derived from the MAB confirm that the activation of the T-cells by the MAB is dependent on the Fc domain, and thus interactions of the PBMC T-cells with accessory cells, such as macrophages. Taken together, our results demonstrate that anti-CD3 CSANs with valencies ranging from 2 to 8 could be employed for radionuclide, drug or potentially oligonucleotide delivery to T-cells without, as has been observed for other antibody conjugated nanoparticles, the deleterious affects of activation observed for MAB. Further the CSAN construct may be adapted for the preparation of other multivalent scFvs.
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    Evaluation of Actinobacillus pleuropneumoniae diagnostic tests using samples from experimentally infected pigs.
    (2009-12) Costa, Giordana Souza
    The early detection of pigs subclinically infected with A. pleuropneumoniae is a critical step in avoiding the introduction of this agent into naïve populations. Negative herds are usually monitored for the presence of A. pleuropneumoniae using serology. At least 3 different tests are available for detection of antibodies against A. pleuropneumoniae and results obtained with each of these tests often disagree. Unexpected or discordant serological results are usually further investigated by direct detection of A. pleuropneumoniae in the tonsils by isolation and/or PCR. At this time, there is no information available on how these tests compare regarding detection of A. pleuropneumoniae infection or which combination of tests provides a better definition of true infection status. In order to address these questions, we have evaluated the performance of different serological tests, isolation, and PCR for detection of A. pleuropneumoniae infection using samples from pigs experimentally infected with 7 clinically relevant serotypes. This thesis offers a comprehensive assessment of the diagnostic tools available for characterization of A. pleuropneumoniae subclinical infection.
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    Immunological benefits of a novel polycaprolactone-polyorthoester-based therapeutic vaccine in a mouse model of glioma
    (2014-08) Grinnen, Karen Lynn
    Cancer immunotherapy has led to significant improvement in the survival of patients with previously untreatable malignancies. The use of therapeutic vaccines is a promising form of immunotherapy, but their efficacy remains ambiguous. Much of the difficulty in identifying the optimal formulation and delivery is related to the complicated nature of the immune response, where it is uncertain which aspects would be most effective in destroying cancer cells. In this thesis, a novel polymeric delivery system, involving poly (caprolactone)-co-poly (ortho ester) [PCL-POE], was used to deliver tumor antigens and adjuvants in a controlled manner. We hypothesized that persistent release of tumor antigens from the biodegradable polymer would result in an increase in the number and persistence of anti-tumor lymphocytes in the effector state. To test this hypothesis, vaccines were administered to mice and the time dependent immunological response was evaluated. The polymeric delivery system resulted in an in vitro release profile characterized by a burst release of both antigen and adjuvant followed, in both cases, by a much slower phase of release. We also observed that the slow release provided by the PCL-POE polymer stimulated prolonged maturation of dendritic cells, activation and persistence of anti-OVA antibodies and antigen-specific T cells following a single vaccination. The vaccine system was also tested in a mouse model of glioblastoma multiforme (GBM). We observed a significant, potentially translatable increase in overall survival.