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.
University of Ph.D. dissertation. October 2010. Major: Chemistry. Advisor: Wagner, Carston R. 1 computer file (PDF); xiii, 156 pages. Ill. (some col.)
Chemically self-assembled antibody nanorings (CSANS): Design and characterization of an anti-CD3 IGM biomimetic..
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