Covalent antibody display--an in vitro antibody-DNA library selection system.

The endonuclease P2A initiates the DNA replication of the bacteriophage P2 by making a covalent bond with its own phosphate backbone. This enzyme has now been exploited as a new in vitro display tool for antibody fragments. We have constructed genetic fusions of P2A with single-chain antibodies (scFvs). Linear DNA of these fusion proteins were processed in an in vitro coupled transcription-translation mixture of Escherichia coli S30 lysate. Complexes of scFv-P2A fusion proteins covalently bound to their own DNA were isolated after panning on immobilized antigen, and the enriched DNAs were recovered by PCR and prepared for the subsequent cycles of panning. We have demonstrated the enrichment of scFvs from spiked libraries and the specific selection of different anti-tetanus toxoid scFvs from a V-gene library with 50 million different members prepared from human lymphocytes. This covalent antibody display technology offers a complete in vitro selection system based exclusively on DNA-protein complexes.

A novel angiopoietin-2 selective fully human antibody with potent anti-tumoral and anti-angiogenic efficacy and superior side effect profile compared to Pan-Angiopoietin-1/-2 inhibitors.

There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.

Immunosuppressive anti-immunoglobulin autoantibodies: specificity, gene structure and function in health and disease.

Anti-immunoglobulin autoantibodies (anti-Ig auto-Abs) are part of the physiological immune repertoire. Herein we focus on Abs directed against the F(ab')2 region of the Ig molecule. The vast majority of previously described anti-F(ab')2 auto-Abs were antiidiotypes. The antibodies we studied recognize epitopes located in the hinge region of IgG and in other conserved domains of F(ab')2. Gene structure analyses revealed germline gene identity of VL chains and 88% homology with the closest germline gene of VH chains. We present evidence for a B cell suppressive role of anti-Ig Abs and discuss the mechanism of suppression. Moreover, the role of anti-Ig auto-Abs in immunoregulation as well as in the pathogenesis of autoimmune and other diseases is discussed. There is substantial evidence indicating that anti-Ig auto-Abs are important immunoregulatory molecules.

Human IgG monoclonal anti-alpha(IIb)beta(3)-binding fragments derived from immunized donors using phage display.

Previous studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have relied on serum analysis and have shown the frequent development of Abs directed against the alpha(IIb)beta(3) integrin. However, little is known about the molecular diversity of the humoral immune response to alpha(IIb)beta(3) due to the paucity of mAbs issuing from these pathologies. We have isolated human IgG anti-alpha(IIb)beta(3) binding fragments using combinatorial libraries of single-chain IgG created from the B cells of a GT and an AITP patient, both with serum Abs. Ab screening was performed using activated platelets or activated alpha(IIb)beta(3)-expressing Chinese hamster ovary cells. Sequencing of selected phage Abs showed that a broad selection of genes from virtually all V gene families had been used, indicating the diversity of the immune response. About one-half of the V(H) and V(L) segments of our IgG anti-alpha(IIb)beta(3) fragments displayed extensive hypermutations in the complementarity-determining region, supporting the idea that an Ag-driven immune response was occurring in both patients. The H chain complementarity-determining region 3 analysis of phage Abs revealed motifs other than the well-known RGD and KQAGDV integrin-binding sequences. To our knowledge, our study is the first to illustrate multiple human IgG anti-alpha(IIb)beta(3) reactivities and structural variations linked to the anti-platelet human immune response. Human alpha(IIb)beta(3) Abs preferentially directed against the activated form of the integrin were further characterized because platelet alpha(IIb)beta(3) inhibitors are potential therapeutic reagents for treating acute coronary syndromes. Currently available alpha(IIb)beta(3) antagonists do not specifically recognize the activated form of the integrin.