Immune response with long-term memory triggered by amorphous aggregates of misfolded anti-EGFR VHH-7D12 is directed against the native VHH-7D12 as well as the framework of the analogous VHH-9G8.

We previously demonstrated that amorphous aggregates of misfolded VHH-7D12 antibodies (VHH-Mis), a potential anti-EGFR drug, can generate a robust serum IgG response. Here we investigate the immunogenic nature, especially the specificity of the immune response induced by VHH-Mis. To this end, we used two natively folded and 77% identical anti-EGFR VHHs (VHH-7D12 and VHH-9G8) that possess a common framework but distinct complementarity determining regions (CDRs). In 60% of mice immunized with VHH-Mis, the anti-VHH-7D12 IgG titer was stronger than the anti-VHH-9G8 titer (Group-1). In the remaining mice (40%; Group-2), the anti-VHH-7D12 and anti-VHH-9G8 titer were almost identical. We rationalized these results by hypothesizing that mice in Group-1 produced IgG mostly against the VHH-7D12's CDRs, whereas in Group-2 mice, they targeted the VHH's framework. The IgG specificity against VHH-7D12 and VHH-9G8 was essentially unchanged over 17 weeks in both groups. Further, in all mice (Group-1&2) re-immunized with native VHH-7D12, the IgG titer against VHH-7D12 increased sharply but not against VHH-9G8. On the other hand, none of the three Group-1 mice re-immunized with native VHH-9G8 showed immunogenicity against VHH-7D12 nor VHH-9G8. Whereas, in Group-2 mice (three/three) re-immunized with VHH-9G8, the IgG titers against both VHHs increased but slowly. Flow-cytometric studies showed that VHH-Mis immunized mice generated a higher number of effector and central memory T-cells. Overall, these observations indicate that amorphous aggregates made of a misfolded VHH can induce serum IgG against its natively folded self and analogous VHHs having a similar framework but distinct CDRs. Furthermore, a robust long-term immune response with memory was established against its natively folded self but with a nil-to-moderate immune response against natively folded VHH analogs.

A new model of induced experimental systemic lupus erythematosus (SLE) in pigs and its amelioration by treatment with a tolerogenic peptide.

INTRODUCTION: Systemic lupus erythematosus (SLE) is characterized by a variety of autoantibodies and systemic clinical manifestations. A tolerogenic peptide, hCDR1, ameliorated lupus manifestations in mice models. The objectives of this study were to induce experimental SLE in pigs and to determine the ability of hCDR1 to immunomodulate the disease manifestations. RESULTS AND DISCUSSION: We report here the successful induction, by a monoclonal anti-DNA antibody, of an SLE-like disease in pigs, manifested by autoantibody production and glomerular immune complex deposits. Treatment of pigs with hCDR1 ameliorated the lupus-related manifestations. Furthermore, the treatment downregulated the gene expression of the pathogenic cytokines, interleukin (IL)-1beta, tumor necrosis factor alpha, interferon gamma, and IL-10, and upregulated the expression of the immunosuppressive cytokine transforming growth factor beta, the antiapoptotic molecule Bcl-xL, and the suppressive master gene, Foxp3, hence restoring the expression of the latter to normal levels. Thus, hCDR1 is capable of ameliorating lupus in large animals and is a potential candidate for the treatment of SLE patients.

Structural analysis and in vivo administration of an anti-idiotypic antibody against mAb 2F5.

Anti-idiotypic antibody (Ab2) 3H6 is directed against the human monoclonal antibody 2F5, which is one of a few neutralising antibodies against HIV-1. Since the binding epitope of 2F5 is cryptic and no neutralising immune response could be elicited by several potential vaccines comprising this region, Ab2/3H6 represents a potent vaccine candidate for active immunisation. Here we describe the molecular features of Ab2/3H6 after changing the antigen binding specificity by single point mutations in the complementarity-determining region 3 of the Ab2/3H6 heavy chain. The resulting Ab2/3H6 mutants were compared in several experimental settings to the wild type Ab2/3H6 Fab fragment. Moreover, we report about an immunisation study with Ab2/3H6 Fab variants, which elicited a specific 2F5-like humoral immune response in BALB/c mice.

Amelioration of lupus manifestations by a peptide based on the complementarity determining region 1 of an autoantibody in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes of systemic lupus erythematosus (SLE) patients.

A peptide based on the complementarity determining region (CDR)1 of a human monoclonal anti-DNA autoantibody (hCDR1) was shown to either prevent or treat an already established murine lupus in systemic lupus erythematosus (SLE)-prone mice or in mice with induced experimental SLE. The present study was undertaken to determine the therapeutic potential of hCDR1 in a model of lupus in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes (PBL) of patients with SLE. To this end, PBL obtained from lupus patients were injected intraperitoneally into two equal groups of SCID mice that were treated either with the hCDR1 (50 micro g/mouse) once a week for 8 weeks, or with a control peptide. Mice were tested for human IgG levels, anti-dsDNA autoantibodies, anti-tetanus toxoid antibodies and proteinuria. At sacrifice, the kidneys of the successfully engrafted mice were assessed for human IgG and murine complement C3 deposits. Of the 58 mice transplanted with PBL of SLE patients, 38 (66%) were engrafted successfully. The mice that were treated with the control peptide developed human dsDNA-specific antibodies. Treatment with hCDR1 down-regulated the latter significantly. No significant effect of the treatment on the levels of anti-tetanus toxoid antibodies could be observed. Treatment with hCDR1 resulted in a significant amelioration of the clinical features manifested by proteinuria, human IgG complex deposits as well as deposits of murine complement C3. Thus, the hCDR1 peptide is a potential candidate for a novel specific treatment of SLE patients.