Human MoAb Research Articles

Identification immune response genes in psoriasis after treatment with secukinumab

BackgroundSecukinumab is a fully human IgG1κ MoAb that selectively binds to IL-17A with high affinity, and it has been proven effective for the treatment of psoriasis. However, the immune response pathways and mechanisms during the treatment are still masked. Therefore, the current study was designed to investigate the potential immune response genes via bioinformatics approaches.MethodsGene expression data of severe plaque-type psoriasis was retrieved from the GEO database. Quantification of immune infiltration by ssGSEA and identification of differentially infiltrated immune cells were conducted to validate the treatment effect of secukinumab. After data processing, differentially expressed genes were identified between the treatment and untreated group. TC-seq was employed to analyze the trend of gene expression and clustering analysis. IL-17 therapeutic immune response genes were selected by taking the intersection of the genes inside the key cluster set and the MAD3-PSO geneset. Based on these therapeutic response genes, protein–protein interaction networks were built for key hub gene selection. These hub genes would work as potential immune response genes, and be validated via an external dataset.ResultsEnrichment scores calculated by ssGSEA illustrated that the immune infiltration level of T cells had a strong difference before and after medication, which validated the treatment effect of Secukinumab. 1525 genes that have significantly different expression patterns before and after treatment were extracted for further analysis, and the enrichment result shows that these genes have the function related to epidermal development, differentiation, and keratinocytes differentiation. After overlapping candidate genes with MAD3-PSO gene set, 695 genes were defined as anti-IL7A treatment immune response genes, which were mainly enriched in receptor signaling and IL-17 signaling pathways. Hub gene were pinpointed from the PPI network constructed by anti-IL7A treatment immune response genes, their expression pattern fits TC-seq gene expression pattern.ConclusionOur study revealed the potential anti-IL7A treatment immune response genes, and the central hub genes, which may act critical roles in Secukinumab, induced immune response. This would open up a novel and effective avenue for the treatment of psoriasis.

Activity and Safety of Daratumumab Monotheray in Patients with Relapsed and Refractory Multiple Myeloma Requiring Dialysis: Preliminary Results of Spanish, Retrospective, Multicenter Trial

Introduction:Daratumumab (DARA) is a first-in-class, fully human IgG1 kappa MoAb that targets to a unique CD38 epitope in plasma cells and has been recently approved for patients with relapsed and refractory myeloma multiple (RRMM) that have received ≥3 prior lines of therapy including proteasome (IP) inhibitor and an immunomodulatory agent (IMiD). Based on pharmacokinetic analysis it is not necessary to adjust the dosage of DARA in patients with creatinine clearance 30-60 mL/min. However there are no published data available in patients with RRMM requiring dialysis.Aims:The objective of this retrospective, multicenter, open-label study is to report the safety and efficacy of DARA in patients with RRMM with end-stage renal failure requiring dialysis.Methods:The eligibility criteria to participate in this study included patients ≥18 years, documented progression of MM by IMWG criteria following the most recent therapy including a PI and an IMID or double-refractory to a PI and an IMID, ECOG performance status score 0-2, absence of chronic obstructive pulmonary disease or persistent asthma, and no prior exposure to anti-CD38 antibody therapy. Patients received DARA at the standard dose (16 mg/kg IV weekly for 8 weeks, then every 2 weeks for 16 weeks, and then every 4 weeks) until disease progression or unacceptable toxicity. Pre-medications with antihistamines, antipyretics and corticosteroids and post-infusion medications with corticosteroids were administered including the use of montelukast. Anti-viral prophylaxis was considered. Data on anti-MM activity and adverse events (AEs) including grade 3-4 AEs and infusion related reactions (IRRs) were collected.Results:The study, including eight Spanish centers is ongoing and we report here the preliminary results of the first eight patients included from January 2017 to July, 2017. There were 4 male and 4 female with a median age at the time of the beginning of DARA therapy of 63 years (range, 52 -71) and with a median time from diagnosis to initiation of DARA of 4.61 years (range, 1 - 6). MM was Bence Jones in 3 patients, IgA type in 2, IgD in 2, and IgG in 1 patient. Distribution of disease according to the International Stage System was: 5 patients were in stage II, 2 patients were in stage I, and the remaining patient was in stage III. The median number (range) of prior lines of therapy was 4 (2 -7); 37% of patients received >3 prior lines. All patients were double refractory to both a PI and IMiD, 2 patients were also refractory to carfilzomib, and 1 patient was triple refractory including pomalidomide. Four patients had received previous stem-cell transplant (3 autologous and 1 allogeneic).At the time of diagnosis, 2 out of the 8 patients had high-risk cytogenetics [1 patient with add(1q) and 1 with del(17p)]. Detailed patient characteristics with respect to baseline clinical features and laboratory parameters are provided in Table 1. In 6 patients, end-stage renal failure was myeloma-related (2 patients with acute renal injury at time of diagnosis), while the remaining 2 patients were already on dialysis at the time of MM diagnosis. The median time from the diagnosis of MM and the beginning of the dialysis was 5 years (range, 1-6). The median time on dialysis at study entry was 24 months (range, 8-97).The median (range) number of cycles of DARA administered was 4 (1-7). The overall response rate and the clinical benefit rate (stable disease or better) was 37.5% (1 VGPR and 2 PR) and 62.5%, respectively. No patient has become dialysis independent.With a median follow-up of 4 months, 2 patients have died because of disease progression (1 patient) and due to myeloma-related causes (1 patient) and one patient suffered disease progression. The remaining 5 patients are still alive with ongoing DARA treatment. Regarding safety, no patient discontinued treatment due to AEs and the most common non-hematological AEs included grade 1 or 2 IRRs observed only during the first cycle, (4 patients). Anemia (n=2, grade 2 and 3), thrombocytopenia (n=2, grade 1 and 2), and neutropenia (n=2, grade 1 and 4), were the hematological AEs most frequently observed.Conclusions:Although still preliminary, our data suggest that DARA monotherapy is an efficacious and safe therapeutic option for patients with RRMM and end-stage renal failure including dialysis. AE profile seems to be similar to that previously observed in patients with normal or moderately impaired renal function. [Display omitted] Disclosuresde la Rubia:Celgene: Other: Honoraria; Janssen: Other: Honoraria; Amgen: Other: Honoraria.

Naxitamab-based chemoimmunotherapy for resistant high-risk neuroblastoma: Preliminary results of HITS pilot/phase II study.

10025 Background: Chemoresistant and relapsed disease are major obstacles to curing high-risk neuroblastoma (HR-NB). Anti-GD2 monoclonal antibody (MoAb) is effective in preventing relapse after remission but responses in relapsed or progressive disease (PD) are rare. We investigated the combination of humanized anti-GD2 MoAb naxitamab, (previously termed Hu3F8), irinotecan, temozolomide and sargramostim (GM-CSF): a pilot HITS protocol against resistant HR-NB now expanded to a phase II study (NCT03189706). Methods: Salient eligibility criteria included evaluable or measurable chemoresistant disease. Prior anti-GD2 MoAb and/or irinotecan/temozolomide (I/T) therapy was permitted. Each cycle comprised of irinotecan 50 mg/m2/day intravenously (IV) plus temozolomide 150 mg/m2/day IV or orally (days 1-5); naxitamab 2.25 mg/kg/day IV over 30 minutes, days 2, 4, 8 and 10 (total 9 mg/kg or 270 mg/m2 per cycle), and GM-CSF 250 mg/m2/day subcutaneously, days 6-10. Toxicity was measured by CTCAE v4.0 and responses by modified International Neuroblastoma Response Criteria. Results: Forty-six (23 enrolled on protocol and 23 on compassionate-use basis) heavily prior-treated patients (median age at enrollment: 6.6 years; median number of prior relapses: 2) have received 175 (median 2; range 1-12) cycles to date. At enrollment, 7 patients had HR-NB refractory to induction chemotherapy while 39 had prior relapse. Toxicities included myelosuppression and diarrhea expected with I/T, and pain and hypertension expected with naxitamab. No other > grade 2 related toxicities occurred; treatment was outpatient. Early responses, assessed after 2 cycles, were documented in 18 (39%) patients and were complete (n = 9), partial (n = 8), and mixed (n = 1); 13 patients had stable disease. Responses were achieved in refractory (3/7;43%) and PD (15/39;38%) subgroups, in patients who had previously received I/T (12/34;35%) and/or anti-GD2 MoAb (14/36;39%), and in soft tissue (6/22; 27%) MIBG-avid skeletal sites (20/36;56%) and on bone marrow histology (9/12; 75%). Conclusions: High-dose naxitamab-based chemoimmunotherapy is safe and effective against chemoresistant HR-NB. This ongoing phase II study may define a broader role for naxitamab which was recently granted breakthrough designation by the FDA. Clinical trial information: NCT03189706.

Human IgM monoclonal antibodies block HIV-transmission to immune cells in cervico-vaginal tissues and across polarized epithelial cells in vitro

The importance of natural IgM antibodies in protection against infections is still emerging and these antibodies have a potential role in the maintenance of homeostasis through clearance of apoptotic bodies, complement-dependent mechanisms, inflammation and exclusion of misfolded proteins. Natural IgM act as a first line of defence against unknown hazardous factors and are present in most vertebrates. We investigated the functional capacity of anti-HIV-1 IgM monoclonal antibodies, from a combinatorial Fab library derived from healthy individuals, and evaluated their protective role in inhibiting HIV-1 in vitro when passing across the human mucosal epithelial barrier. Primary HIV-1 isolates were efficiently transmitted over the tight polarized epithelial cells when added to their apical surface. Efficient inhibition of HIV-1 transmission was achieved when anti-HIV-1 IgM monoclonal antibodies were added to the basolateral side of the cells. Two of these human IgM MoAbs had the ability to neutralize HIV and reduced infection of dendritic cells in primary cervico-vaginal tissue biopsies in vitro. This indicates a potential role of natural IgM antibodies in the reduction of HIV-1 transmission in mucosal tissues and improve our understanding of how natural IgM antibodies against a neutralizing epitope could interfere with viral transmission.

The multi-faceted potential of CD38 antibody targeting in multiple myeloma.

CD38, an adenine dinucleotide phosphate (ADP) ribose cyclase and a cyclic ADP ribose hydrolase, is widely expressed on the surface of multiple myeloma (MM) cells. It is known to play a pivotal role in the downstream pathways that mediate MM cell growth, signal transduction, and adhesion. The clinical use of CD38 monoclonal antibodies (MoAbs), such as daratumumab, either as monotherapy or in combination with other anti-MM agents, has produced impressive results in patients who have failed standard MM therapy. CD38 MoAbs exhibit several cytotoxic mechanisms on MM cells. In addition to the classical effector mechanisms associated with antibody therapy, CD38 MoAbs induce MM apoptosis and clonal T-cell expansion. Here, we summarize the results of some pivotal clinical studies using a human CD38 MoAb, daratumumab, in patients with MM, discuss the anti-MM effector mechanisms induced by CD38 MoAbs, and review the potential tumor antigens that may be suitable targets for immunotherapy of MM. Finally, we present a paradigm of immunotherapy for MM patients using CD38 MoAbs followed by GM-CSF and an immune checkpoint inhibitor in patients who have undergone high dose chemotherapy and autologous stem cell transplant. CD38 MoAbs have emerged as a novel and ultimately very promising immunotherapeutic agent for MM because of its ability to induce MM cytotoxicity through both arms of the adaptive immune responses.

Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis C virus glycoprotein E1.

Although both envelope glycoproteins of the hepatitis C virus, E1 and E2/NS1, show a high degree of sequence variation, the E1 protein includes a well conserved domain, which may be functionally important. We have analysed the human B cell response to a peptide fragment from amino acid residues 314-330 (EP3) covering the central conserved sequence of this domain. Anti-hepatitis C virus-positive blood donors were screened for anti-EP3 antibodies with an ELISA based on immobilized peptide. Thirty out of 92 (32%) RIBA-confirmed donors displayed a significant antibody response to EP3. From three of these blood donors we established four anti-EP3-producing heterohybridoma cell lines: Ul/F30 and Ul/F31 produced IgM-kappa, whereas Ul/F32 and Ul/F33 secreted the isotypes IgG1-lambda and IgG1-kappa, respectively. Epitope analysis with overlapping nonapeptides suggests the existence of different antigenic determinants within the EP3 fragment. Although both IgG antibodies Ul/F32 and Ul/F33 have dissociation constants to the peptide of approximately 10(-9) M, binding to recombinant E1 protein expressed in COS-7 cells was different. Only Ul/F33 detected envelope protein of approximately 24-35 kD in Western blot. This human MoAb will be useful for further investigations on the hepatitis C virus glycoprotein E1.

Complement Activation and Cytotoxicity In CLL Cells Treated with Alemtuzumab and Ofatumumab: Evidence for Multiple Mechanisms of Resistance to Complement Dependent Cytotoxicity

AbstractAbstract 3575Unconjugated monoclonal antibodies (MoAb) have proven value in the therapy of CLL, and alemtuzumab (ALEM) has efficacy in patients with purine analog refractory/p53 defective disease. Although the mechanisms of action of ALEM in vivo are not fully defined, complement dependant cytotoxicity (CDC) appears to play an important role. ALEM CDC resistance in a subpopulation of CLL cells in vivo could thus contribute to treatment failure. We hypothesized that the CD20 targeted complement activating human MoAb ofatumumab (OFA) would increase complement activation (as manifested by C3b deposition) as well as CDC in CLL cells treated with ALEM in vitro. Methods:We have completed experiments on 4 of 15 specimens collected from patients with progressive untreated CLL. CLL cells were isolated from blood by density gradient centrifugation and negative selection with magnetic beads. CD52 and CD20 expression were measured with fluorescent beads (Quantum MESF, Bangs Laboratories, IN). CLL cells were treated at a concentration of 2 × 106/ml with 10 mcg/ml of ALEM (Genzyme) and/or OFA (G.S.K.) in AIM V medium (Invitrogen CA) and 10% standard normal human serum (NHS) (Sigma, MO) as a source of complement for 1 hour at 37C. Absolute viable cell counts were assayed with counting beads (TruCOUNT, BD, CA) and propidium iodide (PI) staining (Sigma) using a FACSCalibur (BD) and CellQuest Pro software (BD). CDC was determined by comparing counts to CLL cells treated with NHS alone. Membrane levels of CD59, bound MoAb, and deposited C3b on the CLL cells membranes were measured by flow cytometry using using the anti-CD59 antibody FITC-CD59 (BD), mouse anti-human Fc antibody FITC-HB43, and the anti-C3b antibody FITC-7C12. To study ALEM CDC resistant cells, CLL cells were first treated with ALEM and NHS as detailed above. Surviving viable cells were isolated using density gradient centrifugation and then retreated. C5 deficient serum (Sigma) was used in experiments to determine MoAb binding and C3b deposition in the entire CLL cell population. Results:Binding of either ALEM or OFA to the CLL cells in NHS resulted in activation of complement and deposition of large amounts of membrane C3b on the cells. However, ALEM induced higher levels of C3b deposition (delta MFI 220) and caused more CDC (73% vs. 18%) compared to OFA. This correlated well with the higher levels of expression of CD52 vs. CD20 expression in CLL cells (143 ×103, range 131 ×103 – 159 ×103 vs. mean 20 ×103 molecules/cell, range 10 ×103 – 28 ×103). Studies with the anti-Fc antibody showed that only a small subpopulation of CLL cells had weak MoAb binding. Addition of OFA to ALEM increased complement-mediated C3b deposition. In three patients with high initial ALEM CDC, addition of OFA did not appreciably increase CDC (81% to 87%). However, in one patient with cells that were less sensitive to ALEM CDC, addition of OFA did appreciably increase CDC (51% to 86%). There was no relationship between levels of CD59 expression and CDC. All four CLL specimens studied contained a subpopulation of cells that were resistant to CDC (mean 13%, range 9–18%) despite robust C3b deposition. Conclusions:These data suggests that there are multiple mechanisms of resistance to MoAb-mediated CDC including: 1. Low MoAb binding because of low target antigen expression; 2. Insufficient C3b deposition after MoAb ligation and; 3. Intrinsic resistance in some cells in cells in which high levels of C3b deposition were clearly demonstrable. OFA can increase MoAb binding, C3b deposition, and CDC in CLL cells treated with ALEM. However, addition of OFA does not overcome the intrinsic resistance of a small subpopulation of CLL cells to CDC. Future studies will be needed to examine these resistance mechanisms and to develop interventions to disrupt them. In addition, membrane C3b could be a target for complement dependent cellular cytotoxity in these CDC resistant cells. Disclosures:Taylor:G.S.K.: Research Funding; Genmab: Research Funding. Zent:Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

Ofatumumab: a new CD20 monoclonal antibody therapy for B-cell chronic lymphocytic leukemia.

Though most patients with chronic lymphocytic leukemia (CLL) respond to first-line therapy, all patients eventually relapse, after which therapeutic options are limited. Fludarabine-refractory patients have a particularly poor prognosis. The addition of the CD20 monoclonal antibody (MoAb) rituximab to chemotherapy in CLL has improved outcomes, particularly in early lines of therapy; however, the efficacy of rituximab monotherapy in CLL is limited, potentially in part because of reduced cell lysis via complement-dependent cytotoxicity (CDC) in this setting. Rituximab CDC is dependent on CD20 expression; CLL cells underexpress CD20. Ofatumumab is a human MoAb that targets an epitope encompassing the membrane-proximal small-loop on the CD20 molecule, which differs from the binding location of rituximab. In vitro studies with ofatumumab have demonstrated that it is significantly more effective than rituximab at corresponding dose levels at lysing CLL cells and B-cell lines, especially those with low CD20 copy numbers. In patients with CLL refractory to both fludarabine and alemtuzumab or refractory to fludarabine with bulky lymphadenopathy and, therefore, less suitable for treatment with the CD52 MoAb alemtuzumab, results from the planned interim analysis showed an encouraging response rate with ofatumumab (Independent Endpoint Review Committee evaluated) and survival parameters, which seemed to be higher than those reported from a historical assessment of other salvage therapies in a corresponding group of patients. Ofatumumab was also well tolerated; the most common adverse events were transient grade 1 or 2 infusion reactions and infections. Ongoing trials will help confirm the role of ofatumumab in CLL, in addition to the effect of this agent in combination with chemotherapies and other MoAbs.

Efficacy of the monoclonal antibody EGFR inhibitors for the treatment of metastatic colorectal cancer.

Two anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs) have been approved in Canada for the treatment of metastatic colorectal cancer (mCRC) - cetuximab, a mouse-human chimeric MoAb, and panitumumab, a fully human MoAb. This paper reviews the efficacy of the anti-EGFR monoclonal antibodies cetuximab and panitumumab - both as monotherapy and in combination with cytotoxic chemotherapy - in the treatment of mCRC. Both cetuximab and panitumumab have demonstrated clinical efficacy in monotherapy in patients with mCRC, an advantage that has recently been found to be limited largely to those with wild-type KRAS tumors. Advantages of using these agents in monotherapy include reduced cost and toxicity. While the addition of cetuximab to irinotecan has shown superior progression-free survival and response compared with cetuximab monotherapy, there is currently no evidence for a benefit of panitumumab in combination with irinotecan.

Treatment of MDS with Excess Blasts by Bevacizumab Is Well Tolerated and Is Associated with a Decrease of VEGF Plasma Level.

Abstract 2770Poster Board II-746 Background:AML and MDS are associated with increased in BM vascularity and increased levels of various angiogenic factors including VEGF, which is implicated in the proliferation and survival of leukemic cells and whose autocrine production may contributes to leukemia self-renewal and resistance to apoptotic and maturation stimuli (Kerbauy DB, 2007). Higher VEGF plasma levels have been observed in MDS with excess blasts (Brunner B, 2002). The humanized MoAb against VEGF, Bevacizumab (BEV) has proved an effective treatment targeting VEGF. Methods:The GFM conducted a multicentric phase II trial testing the efficacy and tolerance of BEV in MDS with excess of marrow blasts and the impact of BEV on angiogenesis (ClinicalTrials.gov Identifier: NCT00565656). BEV was administered at 5 mg/kg IV every 2 weeks for 12 weeks. Patients with no response could receive, in the absence of major toxicities, 4 additional cycles with dose escalation of BEV to 10 mg/kg every 2 weeks. Results:20 patients (pts) were included of whom 17 were currently evaluable for efficacy: 14M/3F, median age 70 years (62–84), RAEB-1 (8), RAEB-2 (9), IPSS INT-1 (6), INT-2 (9), and high (2).No patient stopped treatment before week 12 because of side effects and no treatment related SAE was reported. Preliminary results on haematological adverse events have shown mild toxicity. Four pts with grade 0–2 neutropenia and 3 pts with grade 0–2 anemia have progressed in a grade 3–4 during treatment.One of 17 evaluable pts was responder (RBC transfusion independence in a pt requiring 2 RBC concentrates/month), during 2 months.Bone marrow microvessel density, VEGF plasma level, VEGF mRNA expression, HIF-1a expression were determined before the treatment to characterise angiogenesis in the pts included and after treatment to evaluate the impact of Bevacizumab on this angiogenesis. Median plasma VEGF was 56.6 pg/ml (range: 13.2–151.7 pg/ml) at inclusion. After 12 weeks of treatment VEGF plasma level was significantly decreased to 30.4 pg/ml (range: 13.9–53.1 pg/ml). This decrease was observed in all pts and has been confirmed for bone marrow VEGF mRNA in the first analysed patients (n=4) by RTQPCR. Further results of this study will be presented. Conclusion:In our study, BEV was well tolerated but had limited efficacy. On the other hand, in colorectal cancer, BEV alone has limited efficacy and mainly plays a therapeutic role by enhancing the efficacy of chemotherapy. The in vivo decrease of plasma VEGF with BEV in this study, and its limited toxicity, suggest that it could be associated in higher risk with agents having different mechanisms of action, especially hypomethylating agents, to produce synergistic effects while maintaining tolerable safety profiles.Brunner B et al. Blood levels of angiogenin and vascular endothelial growth factor are elevated in myelodysplastic syndromes and in acute myeloid leukemia. J Hematother Stem Cell Res. 2002 Feb;11(1):119–25Dias S et al. Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration. J Clin Invest. 2000 Aug;106(4):511–21.Kerbauy DB, Deeg HJ. Apoptosis and antiapoptotic mechanisms in the progression of myelodysplastic syndrome. Exp Hematol. 2007 Nov;35(11):1739–46. Disclosures:No relevant conflicts of interest to declare.

Successful Administration of Panitumumab Alone After Severe Infusion Reaction to Cetuximab in a Patient with Metastatic Colorectal Cancer

The administration of monoclonal antibodies (MoAbs) can result in infusion reactions of various grades; severe reactions are seen in approximately 3% of patients after infusions of cetuximab (fatalities occur in approximately 0.1% of patients). We report a case of a patient who had a severe reaction to cetuximab (a murine MoAb) who then received panitumumab (a human MoAb) and tolerated it without any infusion reaction.

Safety and Efficacy of the Terminal Complement Inhibitor Eculizumab in Japanese Patients with Paroxysmal Nocturnal Hemoglobinuria: AEGIS Phase II Clinical Study Results.

In patients with paroxysmal nocturnal hemoglobinuria (PNH), lack of the GPI-anchored terminal complement inhibitor CD59 on hematopoietic stem cells and subsequently matured blood cells results in chronic intravascular hemolysis and thrombosis. The patients also show kidney disease and pulmonary hypertension in addition to disabling fatigue, abdominal pain and impaired quality of life. Eculizumab is a humanized MoAb against a terminal complement molecule C5, and has been evaluated in 2 phase III studies in North America, Western Europe and Australia. Eculizumab significantly reduced hemolysis, anemia, transfusion requirements, and thrombotic events, and improved fatigue, renal impairment and quality of life. We conducted an open-label single-arm phase II study (AEGIS) to evaluate the safety and efficacy of eculizumab in Japanese patients with PNH relative to the two phase III eculizumab studies previously reported. The AEGIS study criteria included patients with significant thrombocytopenia (platelet counts ≥ 30x109/L) and/or minimal transfusion requirements (1 or more transfusion episodes in the preceding 2 years). Eculizumab was dosed as follows: 600mg weekly for 4 weeks; 900mg one week later; and then 900mg every other week for a total of 12 weeks of therapy. Patients received meningococcal vaccine 2 weeks prior to treatment. Eculizumab was administered to 29 Japanese patients at 9 institutions. The median patient age was 47 years (range 26–70 years), median platelet count was 150x109/L (range 28–291x109/L), 45% had a history of aplastic anemia or MDS, and 48% were taking corticosteroids. Eculizumab serum levels were sufficient to completely block complement. Twenty seven out of 29 patients completed the study. Intravascular hemolysis, the primary efficacy endpoint of the trial, was rapidly and significantly reduced with eculizumab treatment. Lactate dehydrogenase (LDH) decreased 86% from a median of 1,814 U/L at baseline to a median of 244 U/L at 12 weeks of treatment (P<0.001; normal range 103–223 U/L). Control of hemolysis resulted in improvement in anemia; hemoglobin levels increased from baseline (p=0.002 respectively). Transfusion requirements decreased 71% from a mean (SE) of 5.2 (±1.04) PRBC units/patient during the 12-week pre-treatment period to 1.5 (±0.67) units/patient during 12 weeks of eculizumab treatment (P<0.001 for the prespecified median change). Transfusion independence was achieved in 67% (14/21) of patients who were transfusion-dependent prior to treatment (P<0.001). Fatigue levels, as measured by the FACIT-Fatigue instrument, significantly improved within one week of eculizumab treatment, with a median increase of 5.0 points at 12 weeks (P<0.001). A change of 3 or more points is considered clinically meaningful. A post hoc analysis was performed to evaluate the effect of eculizumab on chronic kidney disease (CKD), measured as an improvement or worsening in CKD stage during treatment according to the KDOQI CKD published guidelines. Eculizumab improved CKD in 41% (12/29) of patients, while 55% (16/29) maintained stable kidney function, and only 3.4% (1/29) showed worsening (P<0.001). There were five reported thrombotic events in patients prior to eculizumab and no reported events in treated patients to-date. The drug was safe and well tolerated in all patients. The most frequent adverse events (AEs) were headache (52%), nasopharyngitis (41%), and nausea (21%). Most AEs were mild to moderate in severity and not considered related to eculizumab. No serious AEs were reported as probably related to drug. In summary, this trial demonstrates that eculizumab is safe and well tolerated in Japanese patients with PNH and provides beneficial effects in PNH similar to those observed in previous phase III trials.Table 1: Efficacy of EculizumabBaseline12 weeks TreatmentChange From BaselineP-ValueaLDH, U, L Median (mean ± SE)1,814 (1,845 ±115)244 (399 ± 99)P < 0.001Transfusions (PRBC Units/Patient)bmedian (mean ± SE)2.0 (5.2 ±1.04)0.0 (1.5 ± 0.67)P < 0.001Hemoglobin (g/dL) Median (mean ± SE)7.5 (7.3±0.3)9.0 (8.9 ± 0.4)P =0.002FACIT-fatigue scorec Median (mean ± SE)41.0 (38.5 ±1.9)43.0 (42.6± 1.5)+5.0 (4.1 ±2.3)P < 0.001aOverall mixed model analysisbBaseline represents the number of units transfused during the 12 week period prior to treatment. One PRBC unit is prepared from 200 ml of donor peripheral blood.cA change of ≥ 3 is considered clinically meaningful

Role of Molecular Mimickry of Hepatitis C Virus (HCV) Protein with Platelet GPIIIa in Hepatitis C-Related Immunologic Thrombocytopenia

HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). Since HCV-ITP, like HIV-ITP, is associated with circulating immune complexes, we asked whether the complexes could contain platelet fragments induced by oxidative platelet fragmentation and whether HCV-ITP could be induced by molecular mimickry with an HCV peptide. The incidence of Hepatitis-C related ITP varies from 10–40% increasing with severity of liver disease. HIV-ITP is more frequent in drug abusers compared to non-drug abusers (37% vs 16%); and more severe in HIV-drug abusers than non-drug abusers (platelets <10,000/ul in 52% vs 9%). A striking feature of HIV-infection in drug abusers is the frequent co-infection with HCV. HCV infection in non-drug abusers (~30%) increase to 90% in drug abusers. We asked whether coinfection with HCV facilitates ITP, and if so, which would be the mechanism. We screened a 7 mer peptide phage library with anti-GPIIIa49-66 Ab as bait for peptides showing homology sequences with HCV protein. Three clones had 70% homology with HCV protein, as well as a non-conserved core envelope 1 peptide, PHC09 (SAIHIRNASG). Three additional non-conserved envelope 1 peptides with 70% homology for PHC09 were found in the NIH data base (PHC09-H4, PHC09-H5 and PHC09-H6). The envelope 1 protein is a known neutralizing HCV epitope reactive with human MoAb vs 12 different HCV Envelope 1 isolates, containing the candidate linear epitope _ _ RN_SG_Y_. Sera from dually infected thrombocytopenic patients with HCV and HIV-ITP reacted strongly with PHC09 and correlated inversely with platelet count r2=0.7, p<0.01, n=15. Ab raised against this peptide in GPIIIa −/− mice induced severe thrombocytopenia in wild-type mice, n=4. Affinity-purified IgG against PHC09 induced oxidative platelet fragmentation in vitro, n=5. Drug abusers dually infected with HCV and HIV had a greater incidence and severity of thrombocytopenia (<30,000/ul) in 8/15 dually infected patients compared to 2/15 HCV-ITP (p<0.025) or 3/15 HIV-ITP patients (p=0.06). This was associated with a greater incidence and titer of anti-GPIIIa49-66 Ab in 12/15 HCV-HIV-ITP compared to 2/15 HCV-ITP (P=0.001) or 6/15 HIV-ITP patients (p<0.05). They also had a greater incidence of anti PHC09 Ab in 10/15 HCV-HIV-ITP patients compared to 3/15 HCV-ITP (p=0.004) or 1/15 HIV-ITP patients (p=0.001). Similar findings were obtained with PHC09-H5. NZB/W F1 mice injected with recombinant core envelope 1 developed Ab titers vs PHC09 and significantly lowered their platelet count by 60%, compared to controls, P<0.001. Anti-PHC09 Ab correlated inversely with platelet count, r2=0.63, n=15, P<0.05, n=8. Thus, 1) HCV-ITP complexes contain anti-PHC09 Ab capable of inducing oxidative platelet fragmentation; 2) HCV-ITP can be induced by molecular mimickry with HCV core envelope 1 protein, PHC09; 3) Dually infected ITP drug abusers with HIV and HCV have greater titers of anti-GPIIIa49-66/PHC09 Ab than HCV-ITP or HIV-ITP patients and more severe thrombocytopenia.

Autoantibodies to cerebroside sulphate (sulphatide) in leprosy.

Sera from 40 leprosy patients were screened for autoantibodies to cerebroside sulphate (sulphatide). Anti-sulphatide IgM in groups of patients with lepromatous (LL) and borderline (BL + BB + BT), but not with tuberculoid (TT) disease, were significantly elevated above the levels found in endemic control subjects. Eight-six percent (18 out of 21; mean 1.59 OD units) of LL, 33% (four out of 12; mean 1.08 OD units) of borderline and 13% (one out of eight; mean 0.69 OD units) of tuberculoid patients had anti-sulphatide IgM in their sera above a cut-off value of 2 s.d. above the mean value (0.66 OD units) for control sera. Elevated anti-sulphatide IgG was detected in only one patient's serum, an individual with LL disease. The level of anti-sulphatide IgM was strongly correlated to expression of the TH3 idiotype, an idiotype previously defined by a human MoAb that bound Mycobacterium leprae phenolic glycolipid, Klebsiella capsular polysaccharide, polynucleotides and human tissues. The purified, TH3 MoAb was found in this study to bind sulphatide, but not cholesterol-3-sulphate or cerebroside. It is suggested that anti-sulphatide IgM is elevated in leprosy, in relation to the bacterial load. Anti-sulphatide IgM fell at the onset of erythema nodosum leprosum (ENL) reaction, consistent with the deposition of serum antibodies, and thus may play a part in pathology during periods of inflammation, particularly in multibacillary patients.

Plasma half-lives and bioavailability of human monoclonal Rh D antibodies BRAD-3 and BRAD-5 following intramuscular injection into Rh D-negative volunteers.

Two human MoAbs, BRAD-3 (an IgG3 anti-D) and BRAD-5 (an IgG1 anti-D), were produced from Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines grown in hollow fibre bioreactors. Six Rh D-negative male volunteers were injected intramuscularly with anti-D; two received BRAD-3 (approx. 1500 micrograms, 2600 IU), two were given BRAD-5 (300 micrograms, 2000 IU), and two had polyclonal anti-D immunoglobulin (500 IU, approx. 100 micrograms anti-D). Levels of anti-D in plasma samples taken up to 42 days later were measured by a sensitive AutoAnalyser method. The half life of BRAD-5 (mean 22.2 days) was greater, and that of BRAD-3 (mean 10.2 days) less than that of polyclonal anti-D (mean 15.6 days). The bioavailability (plasma uptake) of the MoAbs (mean 33.9%) was less than that of the polyclonal anti-D (mean 60.3%). BRAD-3 and BRAD-5 may be suitable for use in antenatal and post-natal prophylaxis against Rh D haemolytic disease of the newborn.

Human MoAbs produced from normal, HIV-1-negative donors and specific for glycoprotein gp120 of the HIV-1 envelope.

Human MoAbs of IgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts.

Common idiotypes expressed on human, monoclonal, abnormal immunoglobulins and cryoglobulins with polyreactive autoantibody activities.

Several human monoclonal immunoglobulins with the same autoantibody activity have been shown to have cross-reactive idiotypes (CRI). In this study, using polyclonal anti-idiotypic antibodies, we found that 28% of human monoclonal immunoglobulins with polyreactive autoantibody activity from myeloma, Waldenström's macroglobulinaemia and cryoglobulinaemia patients shared common idiotype(s). Furthermore, the latter were expressed on human and murine natural MoAbs (respectively in 12% and 22% of the clones tested) and on human IgG preparations used for therapeutic intravenous injections (IVIg) and which contain natural antibodies. These findings suggest that monoclonal immunoglobulins could arise from the proliferation of a clone that normally produces a natural antibody. The existence of common idiotype(s) between monoclonal immunoglobulins and IVIg could be relevant to the improvement noted after treatment with IVIg in patients suffering from peripheral neuropathies associated with monoclonal gammopathy.

Induction of experimental systemic lupus erythematosus (SLE) in mice with severe combined immunodeficiency (SCID).

A model in which experimental SLE is induced in normal mice by the injection of a human anti-DNA MoAb expressing a common idiotype 16/6 Id has been established in our laboratory. In the present study we have attempted the induction of experimental SLE in mice with SCID by the transfer of lymphocytes obtained from mice with experimental SLE. Disease could not be induced by direct immunization of SCID mice with the 16/6 Id nor by transfusion of normal splenocytes and immunization with the 16/6 Id thereafter. In contrast, disease was induced in SCID mice which were transplanted with splenic lymphocytes obtained from SLE afflicted BALB/c mice. The disease was expressed by the presence of high titres of antibodies and glomerular immune complex deposits were present in the kidney sections of these mice. Mice that received spleen cells from donors with experimental SLE together with the 16/6 Id developed higher titres of autoantibodies and had, in addition to the immune complex deposits, glomerular histological pathology. The model of experimental SLE induction in SCID mice should help in the elucidation of the role of different cell types in the pathogenesis of SLE.

Methotrexate treatment in murine experimental systemic lupus erythematosus (SLE); clinical benefits associated with cytokine manipulation.

The objective of this study was to determine the effects of Methotrexate (MTX) on the development and the course of experimental murine SLE, as well as on the cytokine profile involved in the disease. SLE was induced in naive BALB/c female mice by injection of the human anti-DNA MoAb bearing a common idiotype (16/6 Id). Six weeks following immunization, when high levels of autoantibodies were demonstrated, the mice were treated with MTX (2 mg/kg once a week) for a period of 10 months. MTX treatment had no effect on 16/6 Id-induced autoantibody production. However, MTX treatment had beneficial effects on the clinical manifestations of the experimental disease (i.e. leucocyte counts, levels of protein in the urine and immune complex deposits in the kidneys). Thus, only 20% of 16/6 Id-immunized BALB/c mice that were treated with MTX had immune complex deposits in their kidneys compared with 100% of SLE-afflicted BALB/c mice that were not treated. We have observed a significant elevation in IL-1, tumour necrosis factor (TNF) and IL-10 secretion in BALB/c mice afflicted with experimental SLE. IL-2, IL-4, IL-6 and interferon-gamma (INF-gamma) levels were decreased in these mice compared with the levels detected in healthy controls. Treatment with MTX reversed the levels of all the above cytokines to normal levels observed in control mice. These studies demonstrate therapeutic effects of MTX on murine experimental SLE. The normal cytokine profile observed following treatment with MTX is suggested to play a role in the amelioration of the clinical manifestations of experimental SLE.

Efficacy of HIV-specific and 'antibody-independent' mechanisms for complement activation by HIV-infected cells.

Previous studies in this laboratory have shown that efficient activation of complement (C) on HIV isolates and HIV-infected cells requires the binding of specific anti-HIV antibodies, while other investigators have observed 'antibody-independent' C activation. In an attempt to clarify these disparate findings, we investigated the effect of several variables on C activation by HIV-infected cells using flow cytometric analysis of C3 deposition. Antibody-mediated C activation using pooled sera from infected persons or human MoAbs directed against the V3 region of gp120 was always substantially higher than activation without antibody. Normal human serum (NHS) from a subset of HIV antibody-negative donors did, however, induce low levels of C3 deposition. Differences in C3 activation between the various NHS did not correlate with total haemolytic C levels or mannose-binding protein (MBP) levels. IgM isolated from NHS that induced high levels of C activation was at least partly responsible for the 'antibody-independent' C activation. Although there appeared to be a correlation between NHS that induced C activation and the presence of anti-blood type B IgM, absorption of anti-B did not abrogate the C3 deposition. Additionally, MoAb to the B antigen did not induce C3 deposition. These studies show that IgM in sera from HIV-uninfected donors can induce C3 deposition on HIV-infected cells, but that specific antibody-dependent C activation is substantially more efficient. Therefore, 'antibody-independent' C activation on HIV-infected cells may, in some cases, be more accurately described as HIV-cross-reactive antibody-dependent C activation.