Shlomo Dagan

Monoclonal antibody HCV-AbXTL68 in patients undergoing liver transplantation for HCV: results of a phase 2 randomized study.

A randomized, double‐blind, dose‐escalation study evaluated the safety and efficacy of hepatitis C virus (HCV)‐AbXTL68, a neutralizing, high‐affinity, fully human, anti‐E2 monoclonal antibody, in 24 HCV‐positive patients undergoing liver transplantation. HCV‐AbXTL68 or placebo was administered at doses from 20‐240 mg as 2‐4 infusions during the first 24 hours after transplantation, followed by daily infusions for 6 days, weekly infusions for 3 weeks, and either 2 or 4 weekly infusions for 8 weeks. Serum concentrations of total anti‐E2 obtained during daily infusions of 120‐240 mg HCV‐AbXTL68 were 50‐200 μg/mL above concentrations in the placebo group. Median serum concentration of HCV RNA dropped below baseline in all groups immediately after transplantation. On day 2, median change from baseline in HCV RNA was −1.8 and −2.4 log in the 120‐mg and 240‐mg groups, respectively, compared with −1.5 log with placebo. The difference was lost after day 7 when the dosing frequency was reduced. The coincidence of increases in anti‐E2 with decreases in HCV RNA concentration indicate that the dose‐related changes in HCV RNA concentration were a result of HCV‐AbXTL68 administration in the 120‐ and 240‐mg groups. The overall incidence of nonfatal serious adverse events was higher with placebo (60%) vs. all active treatments combined (42%). In conclusion, HCV‐AbXTL68 may decrease serum concentrations of HCV RNA in patients after liver transplantation. Studies evaluating more frequent daily dosing at doses >120 mg are necessary to investigate sustained viral suppression in this population. Liver Transpl 12:1381–1389. 2006.

Human monoclonal antibodies specific to hepatitis B virus generated in a human/mouse radiation chimera: the Trimera system.

An approach to develop fully human monoclonal antibodies in a human/mouse radiation chimera, the Trimera system, is described. In this system, functional human lymphocytes are engrafted in normal strains of mice which are rendered immuno‐incompetent by lethal total body irradiation followed by radioprotection with severe combined immunodeficient (SCID) mouse bone marrow. Following transplantation, human lymphocytes colonize murine lymphatic organs and secrete human immunoglobulins. We have established this system as a tool to develop fully human monoclonal antibodies, and applied it for the generation of monoclonal antibodies specific for hepatitis B virus surface antigen. A strong memory response to hepatitis B surface antigen was elicited in Trimera engrafted with lymphocytes from human donors positive for antibodies to hepatitis B surface antigen. The human specific antibody fraction in the Trimera was 102–103‐fold higher as compared with that found in the donors. Spleens were harvested from Trimera mice showing high specific‐antibody titres and cells were fused to a human–mouse heteromyeloma fusion partner. Several stable hybridoma clones were isolated and characterized. These hybridomas produce high‐affinity, IgG, anti‐hepatitis B surface antigen antibodies demonstrating the potential of the Trimera system for generating fully human monoclonal antibodies. The biological function and the neutralizing activity of these antibodies are currently being tested.

The Trimera mouse: generating human monoclonal antibodies and an animal model for human diseases.

Monoclonal antibodies of human origin may have great therapeutic value in the treatment of cancer, autoimmune disorders and viral or bacterial infections. Several methods for generating human monoclonal antibodies exist; some are based on the transplantation of a functioning human immune system into severe combined immunodeficient (scid) mice or into Trimera mice, which are mice that have been lethally irradiated and radioprotected by transplantation of bone-marrow cells from scid mice. Trimera mice could be also used to develop animal models for human diseases by transplanting infected human tissue fragments and for creating models for cell therapy.

Novel mechanism of antibodies to hepatitis B virus in blocking viral particle release from cells

Antibodies are thought to exert antiviral activities by blocking viral entry into cells and/or accelerating viral clearance from circulation. In particular, antibodies to hepatitis B virus (HBV) surface antigen (HBsAg) confer protection, by binding circulating virus. Here, we used mathematical modeling to gain information about viral dynamics during and after single or multiple infusions of a combination of two human monoclonal anti‐HBs (HepeX‐B) antibodies in patients with chronic hepatitis B. The antibody HBV‐17 recognizes a conformational epitope, whereas antibody HBV‐19 recognizes a linear epitope on the HBsAg. The kinetic profiles of the decline of serum HBV DNA and HBsAg revealed partial blocking of virion release from infected cells as a new antiviral mechanism, in addition to acceleration of HBV clearance from the circulation. We then replicated this approach in vitro, using cells secreting HBsAg, and compared the prediction of the mathematical modeling obtained from the in vivo kinetics. In vitro, HepeX‐B treatment of HBsAg‐producing cells showed cellular uptake of antibodies, resulting in intracellular accumulation of viral particles. Blocking of HBsAg secretion also continued after HepeX‐B was removed from the cell culture supernatants. Conclusion: These results identify a novel antiviral mechanism of antibodies to HBsAg (anti‐HBs) involving prolonged blocking of the HBV and HBsAg subviral particles release from infected cells. This may have implications in designing new therapies for patients with chronic HBV infection and may also be relevant in other viral infections. (HEPATOLOGY 2010;)

Cell-based and animal models for hepatitis B and C viruses.

Reliable cell-based assays and animal models have been developed for evaluating agents against hepatitis B virus. Although much progress has been made, in vitro and in vivo assays for hepatitis C virus are still on the horizon. Advances towards establishing inexpensive and reliable experimental models have accelerated the development of therapeutic modalities for these life-threatening viral infections. The characterization of well-defined viral targets coupled with improved molecular diagnostic technologies have illuminated this field.

Treatment of Recent-Onset Type 1 Diabetic Patients With DiaPep277: Results of a Double-Blind, Placebo-Controlled, Randomized Phase 3 Trial

OBJECTIVE To evaluate safety and efficacy of DiaPep277 in preserving β-cell function in type 1 diabetic patients. RESEARCH DESIGN AND METHODS DIA-AID 1 is a multinational, phase 3, balanced-randomized, double-blind, placebo-controlled, parallel-group clinical study. Newly diagnosed patients (N = 457, aged 16–45 years) were randomized to subcutaneous injections of DiaPep277 or placebo quarterly for 2 years. The primary efficacy end point was the change from baseline in the area under the glucagon-stimulated C-peptide curve. Secondary end points were the change from baseline in mixed-meal stimulated C-peptide secretion and in fasting C-peptide and achieving target HbA1c ≤7% (≤53 mmol/mol). Partial remission (target HbA1c on insulin ≤0.5 units/kg/day) and hypoglycemic event rate were exploratory end points. RESULTS DiaPep277 was safe and well tolerated. Significant preservation of C-peptide secretion was observed in the DiaPep277-treated group compared with the placebo (relative treatment effects of 23.4%, P = 0.037, and 29.2%, P = 0.011, in the modified intent-to-treat [mITT] and per-protocol [PP] populations, respectively). The mixed-meal stimulation failed to distinguish between the groups. There was a trend toward efficacy in fasting C-peptide levels, though not statistically significant. Significantly more DiaPep277-treated than placebo-treated patients maintained target HbA1c (mITT 56% versus 44%, P = 0.03; PP 60% versus 45%, P = 0.0082) and entered partial remission (mITT 38% versus 29%, P = 0.08; PP 42% versus 30%, P = 0.035). DiaPep277 treatment reduced the relative hypoglycemic event risk (mITT by 20%; PP by 28%). CONCLUSIONS DiaPep277 safely contributes to preservation of β-cell function and to improved glycemic control in patients with type 1 diabetes.

Therapy with anti-flagellin A monoclonal antibody limits Pseudomonas aeruginosa invasiveness in a mouse burn wound sepsis model

BACKGROUND The aim of this study was to evaluate the effect of an anti-flagellin sub-type monoclonal antibody (anti-fla-a) on Pseudomonas aeruginosa infection in a mouse burn model and to assay bacterial dissemination and invasiveness. METHODS After immediate post-burn infection with P. aeruginosa, mortality and morbidity (daily weight changes) were monitored in mice treated with anti-fla-a as compared to untreated mice. Bacterial dissemination and invasiveness were monitored by bacterial counts at the burn site and spleen. Three different timing regimens for anti-fla-a treatment were studied: (a) prophylaxis (pre-infection), (b) therapeutic (post-infection), and (c) combined mode. RESULTS Combined regimen of anti-fla-a markedly improved survival of mice infected with P. aeruginosa from 6% to 96% (p<0.0001), similar to treatment with Imipenem. Furthermore, a significant improvement in survival was obtained when anti-fla-a was given prior to (75% survival) or post-infection (50% survival). It reduced bacterial load in the spleen (p=0.01), preventing bacterial sepsis. CONCLUSION Anti-fla-a is effective in reducing mortality and morbidity in murine P. aeruginosa-infected burn model.

Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5′ and/or 3′ end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all VH families/segments was observed showing that ONCL did not introduce cloning biases for or against any VH family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 × 1010 and by selecting five unique Fabs against GAPDH antigen.

Efficacy of antibodies against the N-terminal of Pseudomonas aeruginosa flagellin for treating infections in a murine burn wound model.

Background: In an era of increasing drug resistance, immunotherapy is a desirable treatment against Pseudomonas aeruginosa infections. The flagellum, which is an important pseudomonal virulence factor, was targeted for immunotherapy. The aim of the study was to evaluate the efficacy of polyclonal immunotherapy targeted against the N-terminal of flagellin (anti-N′-fla-b) for treating severe P. aeruginosa infection in a murine burn wound model. Methods: Groups of 12 mice were infected (subeschar) with P. aeruginosa strain PA01, and were treated either with systemic anti-N′-fla-b immunoglobulin G (IgG), nonspecific IgG, or imipenem. The control groups included mice with burn alone, mice with untreated infected burn, and mice without burn infected with P. aeruginosa. Three separate regimens were examined: prophylaxis (preinfection), therapeutic (postinfection), and combined. The efficacy of anti-N′-fla-b was evaluated by monitoring the mortality and morbidity (relative weight loss) during a period of 2 weeks. Results: Anti-N′-fla-b IgG immunotherapy significantly decreased the mortality rate of infected burned mice followed by severe P. aeruginosa infection. The mortality rate in the anti-N′-fla-b–treated groups ranged from 0 to 17 percent compared with 58 to 83 percent in nontreated groups infected with 2 to 5 × 106 colony-forming units of P. aeruginosa (p < 0.05). The mortality rate in the anti-N′-fla-b–treated groups was similar to that of groups treated with imipenem. The three tested regimens yielded similar results. Morbidity paralleled survival results. Histopathologic examination revealed an earlier reepithelialization of the infected wound in the anti-N′-fla-b–treated mice compared with untreated mice. Conclusion: Immunotherapy with anti-N′-fla-b IgG, given either as prophylaxis or therapeutically, effectively reduced mortality and morbidity and improved wound healing in a severely P. aeruginosa–infected murine burn model.

Evaluation of Long-Term Treatment Effect in a Type 1 Diabetes Intervention Trial: Differences After Stimulation With Glucagon or a Mixed Meal

OBJECTIVE Endogenous insulin secretion, measured by C-peptide area under the curve (AUC), can be tested using both the glucagon stimulation test (GST) and the mixed-meal tolerance test (MMTT). This study compares these two stimulation methods using long-term data from patients newly diagnosed with type 1 diabetes or with latent autoimmune diabetes. RESEARCH DESIGN AND METHODS A recently completed phase 3 intervention study with DiaPep277 demonstrated improved glycemic control and a significant treatment effect of glucagon-stimulated C-peptide secretion. Unexpectedly, MMTT failed to detect differences between the treated and control groups. Data from 343 patients in two balanced-randomized, double-blind, placebo-controlled, parallel-group trials of DiaPep277 were used to compare and correlate between GST- and MMTT-derived C-peptide AUC. Pearson’s correlations were calculated for absolute C-peptide AUC at baseline and 12 and 24 months and for long-term changes in AUC (∆AUC). RESULTS The absolute AUC values obtained at any single time point by the two tests were well correlated in both data sets (r = 0.74–0.9). However, the correlations between the ∆AUC were much weaker (r = 0.39–0.58). GST-stimulated C-peptide secretion was stable over the fasting glucose range permitted for the test (4–11.1 mmol/L), but MMTT-stimulated C-peptide secretion decreased over the same range, implying differences in sensitivity to glucose. CONCLUSIONS Measurement of long-term changes in stimulated C-peptide, reflecting endogenous insulin secretion, during the course of intervention trials may be affected by the method of stimulation, possibly reflecting different sensitivities to the physiological status of the tested subject.