Drug-specific Treg cells are induced during desensitization procedure for rituximab and tocilizumab in patients with anaphylaxis.

Biologic agents (BA) are able to induce an adaptive immune response in a proportion of exposed patients with the onset of anti-drug antibodies (ADA), which are usually responsible for hypersensitivity reactions (HR). Drug desensitization (DD) for BA allows transient clinical tolerance to the drug in reactive patients. The paper aimed to analyse the modification of drug-specific immune responses along DD in two patients with previous ADA-mediated HR (anaphylaxis) to rituximab and tocilizumab. The in vivo and in vitro assays of humoral and cellular response to drugs were carried out in a longitudinal manner throughout the DD cycles. We observed a progressive decrease of the pre-procedure ADA titer with negativization during the DD cycles in both patients. The monitoring of the drug-specific effector cell response showed the decrease in the BA-induced proliferation, while T cell response to unrelated antigens resulted unmodified along the DD cycles. Lastly, the increase of circulating drug-specific Treg cells mainly producing IL-35 were shown during the DD treatment. This study provides evidence that DD treatment to two BA inhibits humoral and cellular anti-drug response by increasing regulatory T cells and cytokines in an antigen-restricted manner. These modifications could contribute to the safety of the procedure.

The Role of IgM Antibodies in T Cell Lymphoma Protection in a Novel Model Resembling Anaplastic Large Cell Lymphoma.

MRL/lpr mice typically succumb to immune complex-mediated nephritis within the first year of life. However, MRL/lpr mice that only secrete IgM Abs because of activation-induced deaminase deficiency (AID-/-MRL/lpr mice) experienced a dramatic increase in survival. Further crossing of these mice to those incapable of making secretory IgM (µS mice) generated mice lacking any secreted Abs but with normal B cell receptors. Both strains revealed no kidney pathology, yet Ab-deficient mice still experienced high mortality. In this article, we report Ab-deficient MRL/lpr mice progressed to high-grade T cell lymphoma that can be reversed with injection of autoreactive IgM Abs or following adoptive transfer of IgM-secreting MRL/lpr B cells. Anti-nuclear Abs, particularly anti-dsDNA IgM Abs, exhibited tumor-killing activities against a murine T cell lymphoma cell line. Passive transfers of autoreactive IgM Abs into p53-deficient mice increased survival by delaying onset of T cell lymphoma. The lymphoma originated from a double-negative aberrant T cell population seen in MRL/lpr mice and most closely resembled human anaplastic large cell lymphoma. Combined, these results strongly implicate autoreactive IgM Abs in protection against T cell lymphoma.

Harnessing SLE Autoantibodies for Intracellular Delivery of Biologic Therapeutics.

Intracellular delivery of therapeutic antibodies is highly desirable but remains a challenge for biomedical research and the pharmaceutical industry. Approximately two-thirds of disease-associated targets are found inside the cell. Difficulty blocking these targets with available drugs creates a need for technology to deliver highly specific therapeutic antibodies intracellularly. Historically, antibodies have not been believed to traverse the cell membrane and neutralize intracellular targets. Emerging evidence has revealed that anti-DNA autoantibodies found in systemic lupus erythematosus (SLE) patients can penetrate inside the cell. Harnessing this technology has the potential to accelerate the development of drugs against intracellular targets. Here, we dissect the mechanisms of the intracellular localization of SLE antibodies and discuss how to apply these insights to engineer successful cell-penetrating antibody drugs.

Structural identifiability for mathematical pharmacology: models of myelosuppression.

Structural identifiability is an often overlooked, but essential, prerequisite to the experiment design stage. The application of structural identifiability analysis to models of myelosuppression is used to demonstrate the importance of its considerations. It is shown that, under certain assumptions, these models are structurally identifiable and so drug and system specific parameters can truly be separated. Further it is shown via a meta-analysis of the literature that because of this the reported system parameter estimates for the "Friberg" or "Uppsala" model are consistent in the literature.

Anti-IFN-α/ß receptor antibody treatment ameliorates disease in lupus-predisposed mice.

The demonstration in humans and mice that nucleic acid-sensing TLRs and type I IFNs are essential disease mediators is a milestone in delineating the mechanisms of lupus pathogenesis. In this study, we show that Ifnb gene deletion does not modify disease progression in NZB mice, thereby strongly implicating IFN-α subtypes as the principal pathogenic effectors. We further document that long-term treatment of male BXSB mice with an anti-IFN-α/ß receptor Ab of mouse origin reduced serologic, cellular, and histologic disease manifestations and extended survival, suggesting that disease acceleration by the Tlr7 gene duplication in this model is mediated by type I IFN signaling. The efficacy of this treatment in BXSB mice was clearly evident when applied early in the disease process, but only partial reductions in some disease characteristics were observed when treatment was initiated at later stages. A transient therapeutic effect was also noted in the MRL-Fas(lpr) model, although overall mortality was unaffected. The combined findings suggest that IFN-α/ß receptor blockade, particularly when started at early disease stages, may be a useful treatment approach for human systemic lupus erythematosus and other autoimmune syndromes.

The relevance of antigen binding to the pathogenicity of lupus autoantibodies.

Although lupus autoantibodies provide diagnostic value, discordance between serum levels and nephritis poses mechanistic questions. Krishnan and co-workers report that only those that crossreact with basement membrane components produce immune deposits. Thus, other glomerular binding properties probably define where deposits form. Thereafter, Fc- and complement-mediated events influence disease expression. Clearly other factors determine the ultimate phenotype; however, the findings provide insights into the variable disease patterns in lupus nephritis.

Anti-DNA autoantibodies initiate experimental lupus nephritis by binding directly to the glomerular basement membrane in mice.

The strongest serological correlate for lupus nephritis is antibody to double-stranded DNA, although the mechanism by which anti-DNA antibodies initiate lupus nephritis is unresolved. Most recent reports indicate that anti-DNA must bind chromatin in the glomerular basement membrane or mesangial matrix to form glomerular deposits. Here we determined whether direct binding of anti-DNA antibody to glomerular basement membrane is critical to initiate glomerular binding of anti-DNA in experimental lupus nephritis. Mice were co-injected with IgG monoclonal antibodies or hybridomas with similar specificity for DNA and chromatin but different IgG subclass and different relative affinity for basement membrane. Only anti-DNA antibodies that bound basement membrane bound to glomeruli, activated complement, and induced proteinuria whether injected alone or co-injected with a non-basement-membrane-binding anti-DNA antibody. Basement membrane-binding anti-DNA antibodies co-localized with heparan sulfate proteoglycan in glomerular basement membrane and mesangial matrix but not with chromatin. Thus, direct binding of anti-DNA antibody to antigens in the glomerular basement membrane or mesangial matrix may be critical to initiate glomerular inflammation. This may accelerate and exacerbate glomerular immune complex formation in human and murine lupus nephritis.

The endoplasmic reticulum stress-inducible protein, Herp, is a potential triggering antigen for anti-DNA response.

Anti-dsDNA Abs are highly specific indicators of systemic lupus erythematosus (SLE) and play a pathogenic role in lupus nephritis. Human anti-dsDNA Abs are most likely generated by an Ag-driven mechanism. However, the Ag responsible for triggering anti-dsDNA Ab production has not been identified. To search for proteins that are cross-reactive with anti-dsDNA Abs, we screened a cDNA library from a patient with SLE with single-chain Fv of O-81 human anti-ss/dsDNA mAb by using a two-hybrid system. Homocysteine-induced ER protein (Herp), an endoplasmic reticulum (ER) stress-inducible ER membrane protein, was identified and shown to bind to original O-81 Ab and human lupus anti-dsDNA Abs. Some IgG purified from patients with active SLE by Herp-immobilized affinity chromatography bound to dsDNA. BALB/c mice immunized with Herp showed IgG anti-dsDNA Abs, IgG anti-nucleosome Abs, and glomerular IgG deposition. Herp reactivity was strongly positive in a proportion of PBLs from patients with active SLE, but undetectable in those from healthy controls. Moreover, activation of caspases was observed in the Herp-positive cells, implying that ER stress-induced apoptosis likely occurs in patients with active SLE. Herp is exposed on blebs of ER stress-induced apoptotic cells, suggesting that Herp can be recognized by immune cells. These results indicate that Herp mimics structural determinants of DNA immunologically and can be immunogenic in vivo. Thus, Herp represents a candidate autoantigen for anti-DNA Abs. This study may help explain how common environmental factors induce the production of anti-DNA Abs and contribute the development of SLE.

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.

Antinucleosome antibody-modified liposomes and lipid-core micelles for tumor-targeted delivery of therapeutic and diagnostic agents.

Liposomes and lipid-core micelles prepared of polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugates have been modified with nucleosome-specific monoclonal antinuclear autoantibody (ANA) 2C5 (mAb 2C5) specifically recognizing a broad variety of cancer cells through the cancer cell surface-bound nucleosomes. mAb 2C5 preserves its specific properties upon the binding with the lipid-based pharmaceutical nanocarriers, and 2C5-modified immunoliposomes and immunomicelles demonstrate an enhanced binding with tumor cells both in vitro and in vivo. We have investigated the delivery of therapeutic and diagnostic agents with such tumor-targeted immunoliposomes and immunomicelles to various tumors in vivo and in vitro. Both lipid-based nanocarriers provided enhanced tumor delivery of imaging agents ((111)In) and antitumor drugs (doxorubicin and photodynamic therapy agents) to tumor cells under different experimental settings. Pharmaceutical lipid-based nanoparticular carriers modified with mAb 2C5 could represent universal systems for tumor-specific delivery of various soluble and insoluble pharmaceuticals.

Accelerated ischemia/reperfusion-induced injury in autoimmunity-prone mice.

Natural Abs have been implicated in initiating mesenteric ischemia/reperfusion (I/R)-induced tissue injury. Autoantibodies have affinity and self-Ag recognition patterns similar to natural Abs. We considered that autoimmunity-prone mice that express high titers of autoantibodies should have enhanced I/R-induced injury. Five-month-old B6.MRL/lpr mice displayed accelerated and enhanced intestinal I/R-induced damage compared with 2-mo-old B6.MRL/lpr and age-matched C57BL/6 mice. Similarly, older autoimmune mice had accelerated remote organ (lung) damage. Infusion of serum IgG derived from 5-mo-old but not 2-mo-old B6.MRL/lpr into I/R resistant Rag-1-/- mice rendered them susceptible to local and remote organ injury. Injection of monoclonal IgG anti-DNA and anti-histone Abs into Rag-1-/- mice effectively reconstituted tissue injury. These data show that like natural Abs, autoantibodies, such as anti-dsDNA and anti-histone Abs, can instigate I/R injury and suggest that they are involved in the development of tissue damage in patients with systemic lupus erythematosus.

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.

Anti-idiotype antibodies abrogate the tissue deposition of anti-RNP human autoantibodies injected into neonatal BALB/c mice.

OBJECTIVE: The goals of the current work are: 1) to examine the epidermal deposition of anti-RNP IgG human autoantibodies in neonatal BALB/c mice; 2) to look for immunoregulatory effects of anti-idiotypes allowing one to inhibit the epidermal deposition of anti-RNP antibodies; and 3) to elicit antinuclear antibodies in adult BALB/c mice by internal images of anti-idiotypes. MATERIALS AND METHODS: Anti-idiotype antibodies were produced with human anti-RNP IgG obtained by ion exchange chromatography; F(ab')2 fragments were recovered from pepsin digestion and were purified using Sephacryl S-300. F(ab')2 fragments were then used to immunize New Zealand rabbits. RESULTS: The anti-RNP IgG recognized the 70 kDa protein and the A (31 kDa) and C (19 kDa) proteins, while the anti-idiotype antibody specifically recognized the light or heavy chain of the anti-RNP (Fab')2 fragments. Additionally, anti-idiotypes recognized the anti-RNP IgG from some sera, but not the IgG from other specificities or from normal IgG. When anti-RNP IgG was injected intraperitoneally into BALB/c mice it induced immune complex deposition in the epidermis and at the dermal-epidermal junction. Previous injection of anti-idiotype antibodies abrogated the anti-RNP IgG deposits. Vaccination with anti-idiotypes elicit antinuclear antibodies in adult BALB/c mice. CONCLUSIONS: Anti-idiotype antibodies abrogate in vitro the antinuclear antibody deposition in neonatal BALB/c mice. Anti-idiotype antibodies elicit antinuclear antibodies in adult BALB/c mice.

Induction of anti-DNA antibodies by immunization with anti-DNA antibodies: mechanism and characterization.

Two well-characterized IgG monoclonal antibodies, reactive with double-stranded (ds) DNA and nucleosomes, were administered to normal BALB/c mice to examine the reproducibility and the biology of a previously reported model of anti-DNA antibody induction by immunization with anti-DNA antibodies. The monoclonal antibodies were purified either with or without a high-salt wash to remove nucleosomal antigens bound to them during the cell culture. Both monoclonal antibodies, but not normal IgG, induced significant IgG anti-dsDNA antibody production from 1 week to 25 weeks after the last immunization. The antibodies produced in this manner possess different binding preferences to ds synthetic polynucleotides than the antibodies used for the immunization, and they did not react with nucleosomes. The monoclonal antibodies purified with the high-salt wash were more effective in anti-DNA antibody induction than those purified without the high-salt wash. Even when bound to these monoclonal antibodies, neither dsDNA, nucleosomes, or ds synthetic polynucleotides exert significant antigenicity. For example, anti-DNA antibodies produced by mice immunized with an immune complex formed by poly(dA-dT) and one of the monoclonal antibodies that has a high affinity to this polynucleotide did not show an increased affinity to poly(dA-dT). Together, these results suggest that anti-DNA antibody molecules or processed antibody peptides, and not DNA/nucleosomes carried by anti-DNA antibodies, play a role in this model of anti-DNA antibody production.

An autoantibody is modified for use as a delivery system to target the cell nucleus: therapeutic implications.

A murine monoclonal anti-dsDNA antibody was found to penetrate living cells and localize in the nucleus without pathologic effects. A single mutation in VH markedly enhanced cellular penetration. The mutant antibody was produced as recombinant Fab and single chain antibody fragments to investigate its use as a delivery system to target the cell nucleus. Complexes were made containing Fab fragments and alkaline phosphatase conjugated goat antibodies to mouse |gk chains. Fab fragments transported 305 kDa goat antibody-enzyme complexes into the nucleus in COS-7 and CHO cells. A single chain antibody cDNA was constructed by splice overlap extension PCR and expressed in COS-7 cells. Binding of the single chain antibody to dsDNA was shown by ELISA, and cellular penetration and nuclear localization were demonstrated in COS-7 and CHO cells. The single chain antibody cDNA was ligated into the expression vector, pEGFP, to produce a fusion protein with green fluorescent protein. The fusion protein penetrated COS-7 cells and localized in the cell nucleus. The single chain antibody produced during sustained expression in CHO cells re-entered antibody-producing cells and localized in the nucleus without affecting cell viability. Our results demonstrate the potential use of a modified autoantibody as a delivery system to target the cell nucleus.

Genetic analysis of experimentally induced lupus in mice.

The DBA/2 and C57BL/6 mouse strains, as well as the BXD RI lines derived from these strains, were used to map the genes controlling experimentally induced systemic lupus erythematosus (SLE). SLE was induced using two immunologic approaches: (1) immunization with the human monoclonal anti-DNA antibody expressing the 16/6Id, to which the DBA/2 strain is susceptible (responder) and the C57BL/6 strain is resistant (nonresponder); and (2) induction of autoimmune GVHD in B6D2F1 hosts by inoculation of parental DBA/2 (induces SLE) or C57BL/6 (does not induce SLE) T cells. By both approaches the BXD RI lines could be divided into distinct DBA/2-like and C57BL/6-like categories. Concordance of SLE induced by both methods was observed for susceptibility and resistance in 13/15 BXD lines (P < 0.005). The results suggest that at least two non-H-2 genes control susceptibility and resistance to experimentally induced SLE, one mapping to chromosome 7 and the other mapping to chromosome 14.

Mechanisms of cellular penetration and nuclear localization of an anti-double strand DNA autoantibody.

An anti-dsDNA Ab, mAb 3E10, was identified that bound membranes of fixed human renal tubular cells, penetrated live murine renal tubular cells in vivo, and localized in the cell nucleus. mAb 3E10 binds both dsDNA and an extracellular matrix protein, HP8/HEVIN, expressed in high endothelial venules. Previous studies showed both shared and distinct binding determinants of mAb 3E10 VH for DNA and HP8/HEVIN. To independently assess the requirement of DNA and HP8/HEVIN in cellular penetration, site-directed mutants of mAb 3E10 VH and V kappa were studied for penetrating kidney cell lines. The results showed that residues required for binding DNA, but not HP8/HEVIN, were necessary for Ab penetration, indicating that cellular penetration required the presence of DNA or binding of Ab to a membrane determinant precisely resembling DNA. Ab Fab penetrated cells, indicating that neither the Fc nor multivalent Ab binding is necessary for cellular penetration. Ab synthesized in the cytoplasm as a result of deleting heavy and light chain signal peptides was not translocated to the nucleus, indicating a mechanism distinct from the usual protein nuclear localization signals and suggesting the need for a membrane-mediated pathway or for post-translational modification of the Ab.

Anticardiolipin, but not the 16/6 Id anti-DNA antibody induces pregnancy failure.

The primary antiphospholipid syndrome or the antiphospholipid syndrome in systemic lupus erythematosus patients (defined as secondary antiphospholipid syndrome) are characterized by the presence of thrombosis, thrombocytopenia and recurrent fetal loss in association with anticardiolipin antibodies. To determine the causal role of these antibodies in the pathogenesis of pregnancy failure we studied the effects of immunization with monoclonal anti-DNA antibody (designated 16/6 Id; no cardiolipin reactivity) and anticardiolipin monoclonal antibody (designated 2C4C2; binds DNA as well) on the outcome of allogeneic pregnancies in BALB/c mice. Mating of BALB/c females 4 weeks after active immunization with the 16/6 Id, anti-DNA monoclonal antibody resulted in normal pregnancy outcome, similar to control mouse groups. In contrast to that, immunization with the 2C4C2 anticardiolipin antibodies resulted in severe gestational failure with low pregnancy rate, low numbers of fetuses and high rates of resorptions. The fertility index of those mice was extremely low as compared to the 16/6 Id-immunized mice or the control groups. Furthermore, a correlation was shown between the presence of anticardiolipin antibody levels in the sera of the mice at the time of gestation and the pregnancy fate. The 2C4C2-immunized mice which produced high levels of anticardiolipin antibodies demonstrated severe pregnancy failure, whereas normal gestations were observed in the 16/6 Id primed or the control mouse groups that did not produce measurable amounts of the latter antibodies. Thus, our studies demonstrate that anticardiolipin but not the 16/6 Id anti-DNA antibodies can induce severe gestational impairment.

The pathogenic human monoclonal anti-DNA that induces experimental systemic lupus erythematosus in mice is encoded by a VH4 gene segment.

Systemic lupus erythematosus (SLE) can be induced in mice by immunization with a human anti-DNA IgM mAb that was derived from a patient with cold agglutinin disease. The latter anti-DNA mAb expresses the common idiotype (Id) designated 16/6 Id. The original human hybridoma 16/6 that secreted an IgM antibody that bound ssDNA and carried the 16/6 Id had switched in culture to secrete an IgG molecule. Herein we show that the IgG 16/6 antibody contains the previously reported characteristics of the original IgM 16/6 mAb: it expresses the 16/6 Id and is capable of inducing experimental SLE in susceptible mouse strains. The identify of the IgG 16/6 anti-DNA mAb to the original IgM mAb was shown both by serological techniques and at the T cell level. The human IgG 16/6 mAb was found to be encoded by a germline gene from the human VH4 gene family, with high similarity to the germline gene VH4.21 that was previously shown to code for anti-DNA antibodies isolated from SLE patients. The VH4.21 germline gene was found to also code for most antibodies with cold agglutinin activity that were isolated from patients with cold agglutinin disease.

Injections of complexes made of dsDNA and specific polyclonal antibodies extend MRL lpr mouse survival: a pilot study.

Antibodies towards double-strain (ds) DNA are responsible for the development of lupus nephritis both in human and animal models. A method by which one would suppress the production of pathogenic idiotypes could therefore prevent the development of nephritis. To this end, we prepared polyclonal anti-dsDNA antibodies by immunoaffinity from a serum pool of MRL/MpJ-lpr mice, a strain that develops an early form of nephritis identical to its human counterpart. Antigen-antibody complexes were prepared by addition of dsDNA. Such complexes have the potential of altering the anti-DNA antibody response and boosting the production of specific anti-idiotypic antibodies. Two groups of 14 MRL lpr mice were treated by regular intraperitoneal injections of 10 micrograms dsDNA-anti-dsDNA complexes or carrier buffer, starting at the age of 4 weeks, namely, prior to the appearance of nephritogenic anti-dsDNA IgG antibodies. We show here that such a treatment significantly extended the survival of treated mice compared with the control group. Five treated mice were still alive at month 11 compared with two in the control group. In addition, microscopic kidney examination at the time of death showed less lesions in the treated group compared with controls. This study indicates that complexes made of dsDNA and anti-dsDNA can delay the development of nephritis in the MRL lpr mouse strain.