Selective IgA immune unresponsiveness to Proteus mirabilis fumarate reductase A-chain in rheumatoid arthritis.

OBJECTIVE: To determine if selective immune unresponsiveness to microbial antigens is associated with predisposition to rheumatoid arthritis (RA). METHODS: Proteins from Proteus mirabilis lysate were isolated by SDS-PAGE and examined by Western blotting for antibody responses in sera from patients with RA compared to healthy subjects and patients with psoriatic arthritis (PsA). RESULTS: Although RA patients had marked IgA immune responses to many P. mirabilis proteins compared to healthy subjects, selective unresponsiveness was found in RA to a 66 kDa protein identified as fumarate reductase A-chain (FRD-A) by mass spectroscopy. This was confirmed in Western blots with recombinant FRD-A from P. mirabilis. IgA unresponsiveness to FRD-A was found in 21/59 (35.6%) RA patients compared to 7/63 (11.1%) healthy individuals (p < 0.01) and 6/52 (11.5%) patients with PsA (p < 0.01). IgA unresponsiveness to FRD-A was present in 20/46 (43.5%) RA patients with IgA rheumatoid factors (RF) compared to 1/13 (7.7%) without RF (p < 0.025). CONCLUSION: Our results identify a selective hole in the IgA immune repertoire for P. mirabilis FRD-A in a subset of IgA RF-positive patients with RA.

An intracellular delivery vehicle for protein transduction of micro-dystrophin.

The Fv fragment of an antibody that selectively targets and penetrates skeletal muscle in vivo was produced as a fusion protein with a micro-dystrophin for use as a delivery vehicle to transport micro-dystrophin into muscle cells. Fv-micro-dystrophin was produced as a secreted protein by transient transfection of Fv-micro-dystrophin cDNA in COS-7 cells and as a non-secreted protein by permanent transfection in Pichia pastoris. Isolated Fv-micro-dystrophin was shown to be full-length by Western blot analysis. Fv-micro-dystrophin penetrated multiple cell lines in vitro, and it localized to the plasma membrane of a cell line with membrane beta-dystroglycan. In the absence of membrane beta-dystroglycan, it localized to the cytoplasm. Antibody-mediated transduction of micro-dystrophin into muscle cells is a potential therapy for dystrophin-deficient muscular dystrophies.

Antibody-mediated transduction of p53 selectively kills cancer cells.

Some human cancers are caused by functional defects in p53 that are restored by gene therapy with wild-type p53. To circumvent the use of viral vectors, we reconstituted cancer cell lines with p53 by protein transduction. A fusion protein was produced from cDNA constructed from the Fv fragment of an antibody that penetrates living cells and wild-type p53 (Fv-p53). Fv-p53 penetrated and killed cancer cells that do not express p53. Additionally, Fv-p53 killed cancer cells that were malignant as a result of mutations within p53, nuclear exclusion of p53 and over-expression of MDM2. Non-specific toxicity was excluded by showing that Fv-p53 penetrated but did not kill primary cells and cancer cells unresponsive to p53. Fv fragments alone were not cytotoxic, indicating that killing was due to transduction of p53. Fv-p53 was shown to penetrate cancer cells engrafted in vivo. These results support continued efforts to evaluate the potential efficacy of Fv-p53 for the treatment of certain cancers in vivo.

Construction and expression of a bispecific single-chain antibody that penetrates mutant p53 colon cancer cells and binds p53.

A bispecific, single-chain antibody Fv fragment (Bs-scFv) was constructed from a single-chain Fv fragment of mAb 3E10 that penetrates living cells and localizes in the nucleus, and a single-chain Fv fragment of a non-penetrating antibody, mAb PAb421 that binds the C-terminal of p53. PAb421 binding restores wild-type functions of some p53 mutants, including those of SW480 human colon cancer cells. The Bs-scFv penetrated SW480 cells and was cytotoxic, suggesting an ability to restore activity to mutant p53. COS-7 cells (monkey kidney cells with wild-type p53) served as a control since they are unresponsive to PAb421 due to the presence of SV40 large T antigen that inhibits binding of PAb421 to p53. Bs-scFv penetrated COS-7 cells but was not cytotoxic, thereby eliminating non-specific toxicity of Bs-scFv unrelated to binding p53. A single mutation in CDR1 of PAb421 VH eliminated binding of the Bs-scFv to p53 and abrogated cytotoxicity for SW480 cells without altering cellular penetration, further supporting the requirement of PAb421 binding to p53 for cytotoxicity. Our study demonstrates the use of an antibody that penetrates living cells in the design of a bispecific single chain antibody to target and restore the function of an intracellular protein.

Nuclear delivery of p53 C-terminal peptides into cancer cells using scFv fragments of a monoclonal antibody that penetrates living cells.

scFv fragments of a monoclonal antibody that penetrates living cells and localizes in nuclei were designed as fusion proteins with C-terminal p53 peptides and tested for restoring p53 function in p53 mutant cancer cells. scFv fragments transported a 30-mer C-terminal peptide of p53 into cancer cells and induced cellular cytotoxicity in contrast to scFv fragments alone and other scFv-p53 fusion peptides. Cellular toxicity was not observed with scFv fragments containing a single mutation in VH that prevented antibody penetration. Our results demonstrate the potential efficacy of antibody scFv fragments as a nuclear delivery system in cancer cells.

Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb.

Methods for cell type specific targeted intracellular delivery of proteins in vivo remain limited. A murine monoclonal anti-dsDNA antibody, mAb 3E10, was selectively transported into skeletal muscle cells in vivo. The antibody bound a 200 kDa protein only found in lysates of skeletal muscle by Western blotting. The 200 kDa protein was purified from muscle lysate by antibody affinity chromatography and identified as the skeletal muscle specific heavy chain of myosin IIb by electrospray mass spectrometry. Antibody binding specificity for myosin IIb was demonstrated in Western blots by binding myosin in skeletal muscle lysates from mice null for myosin IId but not in mice null for myosin IIb. Myosin IIb is implicated in the specific targeting of mAb 3E10 to skeletal muscle.