The immunomodulating activity of trimodulin (polyvalent IgM, IgA, IgG solution): a post hoc analysis of the phase II CIGMA trial.

BACKGROUND: The phase II CIGMA trial performed in 160 patients with severe community-acquired pneumonia (sCAP) found treatment with trimodulin (human polyvalent immunoglobulin [Ig]: ~ 23% IgM, ~ 21% IgA, ~ 56% IgG) was associated with a lower mortality in those patients with elevated baseline serum levels of C-reactive protein (CRP) and/or subnormal IgM. METHODS: In this post hoc analysis, the pharmacodynamic effects of trimodulin treatment (182.6 mg/kg/day for 5 days) were investigated on Ig replenishment, cellular markers of inflammation (absolute neutrophil [ANC] and lymphocyte [ALC] count, neutrophil-to-lymphocyte ratio [NLR]), and soluble markers of inflammation (procalcitonin [PCT] and CRP). The impact of these pharmacodynamic effects on mortality was also evaluated. RESULTS: Compared with healthy subjects, baseline serum levels of IgM, IgG, and ALC were significantly lower, and ANC, NLR, PCT and CRP significantly higher in sCAP patients (p < 0.0001). Low Ig concentrations increased with trimodulin. Normalization of ANC (analysis of variance [ANOVA] p = 0.016) and PCT (ANOVA p = 0.027) was more rapid with trimodulin compared with placebo. These and other effects were more evident in patients with low baseline IgM levels. Normalization of PCT and CRP levels was both steadier and faster with trimodulin treatment. In patients with low baseline ALC, trimodulin was associated with a lower 28-day all-cause mortality rate (14.5% vs 32.1% in placebo, p = 0.043) and more ventilator-free days ([VFD]; median VFD: 3.5 vs 11 in placebo, p = 0.043). These numerical differences were greater if baseline IgM was also low (low ALC, low IgM: 8.1% mortality vs 34.1% placebo, p = 0.006; 3 VFD vs 15 VFD, p = 0.009, respectively). Results were consistent in patients with high baseline CRP (low ALC, high CRP: 10.9% mortality vs 34.1% placebo, p = 0.011). CONCLUSIONS: This post hoc pharmacodynamic analysis of a blinded phase II trial suggests that trimodulin compensates for, and more rapidly modifies, the dysregulated inflammatory response seen in sCAP patients. Trimodulin was associated with significantly lower mortality and more VFD in subgroups with high CRP and low ALC. This effect was particularly marked in patients who also had low baseline IgM values. These findings require confirmation in prospective trials.

Serial Monitoring and Hyperimmunoglobulin versus Standard of Care to Prevent Congenital Cytomegalovirus Infection: A Phase III Randomized Trial.

INTRODUCTION: Nonrandomized studies support the potential of cytomegalovirus hyperimmunoglobulin (CMV-HyperIg) in preventing maternofetal CMV transmission, but prospective interventional studies show equivocal results. We pre-sent a prospective phase-III international randomized open-label trial on the potential effect of CMV-HyperIg following serial monitoring of CMV serostatus. METHODS: CMV-seronegative pregnant women (gestational age [GA] <14 weeks) were 1:1 randomized to monthly CMV-serostatus monitoring and CMV-HyperIg upon seroconversion (treatment), or routine prenatal care with CMV-serostatus testing at end of pregnancy (control). Ethical considerations required that control subjects with confirmed seroconversion be offered Cytotect®. The primary endpoint was the proportion of fetuses/newborns with congenital CMV infection. Secondary endpoints included neonatal CMV disease and safety during the 24-month follow-up. RESULTS: The treatment arm counted 4,800 randomized subjects: 52 seroconverted (median GA 24 [11-35] weeks), of which 45 completed follow-up. The control arm counted 4,735 randomized subjects: 42 seroconverted, of which 34 completed follow-up (evaluable data for 28 newborns) and 8 subjects chose off-label Cytotect®. Congenital CMV rates were 13/28 newborns (46.4% [CI 27.51; 66.13]) vs. 16/45 newborns (35.6% [CI 21.87; 51.22]) in control and treated arms, respectively (p = 0.46). Newborn CMV disease was mostly mild and spontaneously resolving. There were no major safety concerns. The target sample was not reached within an acceptable time frame. CONCLUSIONS: Serial monitoring of CMV serostatus with CMV-HyperIg treatment was associated with a mild nonsignificant reduction in the vertical CMV transmission rate. Studies on the optimal preventive strategy are hampered by epidemiological and ethical challenges and should focus on GA-dependent transmission rates and accurate dating of infection.

Long-term analysis of the benefit of prophylaxis for adult patients with severe or moderate haemophilia A.

INTRODUCTION: Prophylaxis with factor VIII (FVIII) concentrates in children with haemophilia A (HA) is current standard of care. The benefit of prophylactic treatment for adult HA patients is not commonly accepted. AIM: To investigate the benefit of prophylaxis over on-demand treatment in adult and elderly patients with severe or non-severe HA in a real-life setting. METHODS: Data from 163 patients comprising 1202 patient-years were evaluated for 7.5 (±5.3) years. The effects on the annual bleeding rate (ABR, including spontaneous and traumatic bleeds) of treatment with a plasma-derived FVIII concentrate, the patient's age and disease severity were investigated. The effect of changing the treatment from on demand to continuous prophylaxis on the patients' ABRs was further analysed. RESULTS: Prophylaxis had the greatest effect on the ABRs of patients of any age with severe or non-severe HA. The difference in ABR of all patients treated on demand (median 31.4; interquartile range (IQR) 27.6; N = 83) compared with those treated prophylactically (median 1.3; IQR 3.6; N = 122) was statistically significant (P < .05), even for patients with non-severe HA (median 8.4; IQR 15.5; N = 11) vs median 1.5; IQR 4.2 (N = 17), P < .05). Patients, aged up to 88 years, switching from on demand to continuous prophylaxis showed the lowest median ABR (1.1; N = 51) after their regimen change. CONCLUSION: Any (even low-frequency) prophylaxis results in lower ABR than on-demand treatment. Patients switching to prophylaxis benefitted the most, irrespective of age or HA severity. Prophylactic treatment-even tertiary-is the regimen of choice for patients of any age, including elderly patients, with severe or non-severe HA.

Activity of Indatuximab Ravtansine against Triple-Negative Breast Cancer in Preclinical Tumor Models.

PURPOSE: Triple-negative breast cancer (TNBC) is related with a poor prognosis as patients do hardly benefit from approved therapies. CD138 (Syndecan-1) is upregulated on human breast cancers. Indatuximab ravtansine (BT062) is an antibody-drug-conjugate that specifically targets CD138-expressing cells and has previously shown clinical activity in multiple myeloma. Here we show indatuximab ravtansine as a potential mono- and combination therapy for TNBC. METHODS: The effects of indatuximab ravtansine were assessed in vitro in SK-BR-3 and T47D breast cancer cell lines. The in vivo effects of indatuximab ravtansine alone and in combination with docetaxel or paclitaxel were assessed in MAXF401, MAXF1384 and MAXF1322 xenograft TNBC models. RESULTS: CD138+ SK-BR-3 and T47D cells were highly sensitive to indatuximab ravtansine. The high CD138-expressing MAXF401 xenograft model demonstrated strong inhibition of tumor growth with 4 mg/kg indatuximab ravtansine. High doses of indatuximab ravtansine (8 mg/kg), docetaxel and the combination of both led to complete remission. In the low CD138-expressing MAXF1384 xenograft model, only combination of indatuximab ravtansine and docetaxel demonstrated a significant efficacy. In the MAXF1322 xenograft model, indatuximab ravtansine alone and in combination with paclitaxel elicited complete remission. CONCLUSIONS: These data demonstrate potential use of indatuximab ravtansine in combination with docetaxel or paclitaxel for CD138-positive TNBC.

Broad neutralization of hepatitis C virus-resistant variants by Civacir hepatitis C immunoglobulin.

Hepatitis C virus (HCV)-induced end-stage liver disease is the major indication for liver transplantation (LT). However, reinfection of the liver graft is still common, especially in patients with detectable viral load at the time of LT. Limited data are available on direct-acting antivirals in the transplant setting for prevention of graft infection. The human hepatitis C immunoglobulin (HCIG) Civacir is an investigational drug that is currently being developed in an ongoing phase 3 clinical trial assessing its safety and efficacy at preventing HCV recurrence after liver transplantation (LT) in the United States. Using well-characterized patient-derived HCV variants selected during LT, we studied the molecular mechanism of action of Civacir. Inhibition of HCV infection was studied using infectious HCV models including HCV pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) containing patient-derived viral envelope glycoproteins from 22 HCV variants isolated from patients before and after LT. The human hepatitis C immune globulin Civacir is an investigational drug that is currently being developed in an ongoing phase 3 clinical trial assessing safety and efficacy to prevent HCV recurrence after LT in the United States. Using well-characterized patient-derived HCV variants selected during LT, we studied the molecular mechanism of action of Civacir. Inhibition of HCV infection was studied using infectious HCV models including HCV pseudoparticles and cell culture-derived HCV containing patient-derived viral envelope glycoproteins from 22 HCV variants isolated from patients before and after liver transplantation. Additionally, we studied neutralization of different HCV genotypes and of direct-acting antiviral-resistant viruses. Our results indicate that Civacir potently, broadly, and dose-dependently neutralizes all tested patient variants in HCV pseudoparticles and cell culture-derived HCV assays including variants displaying resistance to host neutralizing antibodies and antiviral monoclonal antibodies. The half-maximal inhibitory concentrations were independent of the phenotype of the viral variant, indicating that virus neutralization by Civacir is not affected by viral selection. Furthermore, Civacir is equally active against tested direct-acting antiviral-resistant HCV isolates in cell culture. CONCLUSION: Collectively, these results demonstrate broad neutralizing activity of Civacir against resistant viruses, likely due to synergy between anti-HCV antibodies derived from different plasma donors, and support its further clinical development for prevention of liver graft infection. (Hepatology 2016;64:1495-1506).

A specific CD4 epitope bound by tregalizumab mediates activation of regulatory T cells by a unique signaling pathway.

CD4(+)CD25(+) regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.

Specific gene delivery to liver sinusoidal and artery endothelial cells.

Different types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors.

Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice.

The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral gene-transfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In addition, we confirmed the therapeutic efficacy of FIX variants in mice with inhibitory Abs against FVIII. Further, mice tolerant to wild-type human FIX did not develop immune responses against the protein variants. Our results therefore indicate the feasibility of using variants of FIX to bypass FVIII as a novel treatment approach in hemophilia with and without neutralizing FVIII Abs.

No detection of the retrovirus xenotropic murine leukemia virus-related virus in individuals with hemophilia.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) is a retrovirus that has recently been related to prostate cancers and chronic fatigue syndrome. Since other human-pathogenic retroviruses, such as HIV, human T-lymphotropic virus type I (HTLV-I) and -II, are known blood-transmitted pathogens, XMRV might present another hazard associated with products derived from in vitro cultures of human or animal origin, or blood component-based therapeutics. Here, we investigated whether XMRV was transmitted to individuals with hemophilia and frequent exposure to plasma-derived or recombinant clotting factors. METHODS: We used highly sensitive real-time PCR to test plasma samples from 127 consecutive individuals with hemophilia who consulted our hemophilia center either for treatment or for a standard check-up. RESULTS: From the 127 hemophiliacs, 80 had prior contact to persons with either hepatitis B (n = 30), hepatitis C (n = 74) and/or HIV (n = 21), and 30 were currently being treated with plasma-derived and 97 with recombinant factor concentrates. None of the individuals tested positive for XMRV. CONCLUSIONS: Independent of the ongoing discussion on whether the positive XMRV testing in initial reports was a result of reagent, sample, or tissue contamination, and whether XMRV is a real threat or a testing artifact, our data suggest that XMRV might not play an important role for hemophiliacs.

ß-CATENIN stabilizes HIF2 through lncRNA and inhibits intravenous immunoglobulin immunotherapy.

Introduction: Tumor-initiating cells (TICs) are rare, stem-like, and highly malignant. Although intravenous hepatitis B and C immunoglobulins have been used for HBV and HCV neutralization in patients, their tumor-inhibitory effects have not yet been examined. Hepatitis B immunoglobulin (HBIG) therapy is employed to reduce hepatocellular carcinoma (HCC) recurrence in patients after living donor liver transplantations (LDLT). Hypothesis: We hypothesized that patient-derived intravenous immunoglobulin (IVIG) binding to HCC associated TICs will reduce self-renewal and cell viability driven by ß-CATENIN-downstream pathways. ß-CATENIN activity protected TICs from IVIG effects. Methods: The effects of HBIG and HCIG binding to TICs were evaluated for cell viability and self-renewal. Results: Inhibition of ß-CATENIN pathway(s) augmented TIC susceptibility to HBIG- and HCIG-immunotherapy. HBV X protein (HBx) upregulates both ß-CATENIN and NANOG expression. The co-expression of constitutively active ß-CATENIN with NANOG promotes self-renewal ability and tumor-initiating ability of hepatoblasts. HBIG bound to HBV+ cells led to growth inhibition in a TIC subset that expressed hepatitis B surface antigen. The HBx protein transformed cells through ß-CATENIN-inducible lncRNAs EGLN3-AS1 and lnc-ß-CatM. Co-expression of constitutively active ß-CATENIN with NANOG promoted self-renewal ability of TICs through EGLN3 induction. ß-CATENIN-induced lncRNAs stabilized HIF2 to maintain self-renewal of TICs. Targeting of EGLN3-AS1 resulted in destabilization of EZH2-dependent ß-CATENIN activity and synergized cell-killing of TICs by HBIG or HCIG immunotherapy. Discussion: Taken together, WNT and stemness pathways induced HIF2 of TICs via cooperating lncRNAs resulting in resistance to cancer immunotherapy. Therefore, therapeutic use of IVIG may suppress tumor recurrence through inhibition of TICs.

Sufficient blood, safe blood: can we have both?

The decision in September 2011 in the UK to accept blood donations from non-practicing men who have sex with men (MSM) has received significant public attention. Will this rule change substantially boost the number of blood donations or will it make our blood less safe? Clearly, most European countries have a blood procurement problem. Fewer young people are donating, while the population is aging and more invasive therapies are requiring more blood. Yet if that was the reason for allowing non-practicing MSM to donate, clearly re-admission of some other, much larger populations that are currently deferred from donation should likewise be considered. As far as risks for blood safety are concerned, evidence has been provided that the current quality of infectious disease marker testing significantly mitigates against, although does not completely eradicate, risks associated with admission of donors with a high risk of carrying certain blood-transmissible agents. However, it could be argued that more effective recruitment of the non-donor pool, which is substantially larger than the group of currently ineligible donors, would be a better strategy. Recruitment of this group will benefit the availability of blood without jeopardizing the current excellent safety profile of blood.

[Blood--a special resource]. / Rationaler Einsatz von Blutprodukten - Blut - eine besondere Ressource.

Haemotherapy is an integral part of modern high-tech medicine. Without supportive care including red blood cell (RBC), platelet concentrate (PC) and fresh frozen plasma (FFP) transfusion, invasive therapies such as high-dose chemotherapy regimens for haematological and solid malignancies, haematopoietic stem cell (HSC) and solid organ transplantation as well as major surgery and modern trauma management would not be possible. In this article we describe the current state of haemotherapy, the risk of adverse effects and risk minimization measures, specifically focussing on haemolytic transfusion reactions (HTR), transfusion-related lung injury (TRALI) and transfusion-transmitted infections (TTI). Aided by the introduction of NAT technology for blood component screening, the residual risk of transfusion transmitted infections was reduced to 1:10.8 million for HCV, to 1:4.3 million for HIV-1, and to 1:360,000 for HBV for blood products of the German Red Cross Blood Service.

Polyvalent Immunoglobulin Preparations Inhibit Pneumolysin-Induced Platelet Destruction.

Platelets play an important role in the development and progression of respiratory distress. Functional platelets are known to seal inflammatory endothelial gaps and loss of platelet function has been shown to result in loss of integrity of pulmonary vessels. This leads to fluid accumulation in the pulmonary interstitium, eventually resulting in respiratory distress. Streptococcus pneumoniae is one of the major pathogens causing community-acquired pneumonia. Previously, we have shown that its major toxin pneumolysin forms pores in platelet membranes and renders them nonfunctional. In vitro, this process was inhibited by polyvalent intravenous immunoglobulins (IVIGs). In this study, we compared the efficacy of a standard IVIG preparation (IVIG, 98% immunoglobulin G [IgG]; Privigen, CSL Behring, United States) and an IgM/IgA-enriched immunoglobulin preparation (21% IgA, 23% IgM, 56% IgG; trimodulin, Biotest AG, Germany) to inhibit pneumolysin-induced platelet destruction. Platelet destruction and functionality were assessed by flow cytometry, intracellular calcium release, aggregometry, platelet viability, transwell, and flow chamber assays. Overall, both immunoglobulin preparations efficiently inhibited pneumolysin-induced platelet destruction. The capacity to antagonize pneumolysin mainly depended on the final IgG content. As both polyvalent immunoglobulin preparations efficiently prevent pneumolysin-induced platelet destruction and maintain platelet function in vitro, they represent promising candidates for clinical studies on supportive treatment of pneumococcal pneumonia to reduce progression of respiratory distress.

Immunomodulation: Immunoglobulin Preparations Suppress Hyperinflammation in a COVID-19 Model via FcγRIIA and FcαRI.

The rapid spread of SARS-CoV-2 has induced a global pandemic. Severe forms of COVID-19 are characterized by dysregulated immune response and "cytokine storm". The role of IgG and IgM antibodies in COVID-19 pathology is reasonably well studied, whereas IgA is neglected. To improve clinical outcome of patients, immune modulatory drugs appear to be beneficial. Such drugs include intravenous immunoglobulin preparations, which were successfully tested in severe COVID-19 patients. Here we established a versatile in vitro model to study inflammatory as well as anti-inflammatory processes by therapeutic human immunoglobulins. We dissect the inflammatory activation on neutrophil-like HL60 cells, using an immune complex consisting of latex beads coated with spike protein of SARS-CoV-2 and opsonized with specific immunoglobulins from convalescent plasma. Our data clarifies the role of Fc-receptor-dependent phagocytosis via IgA-FcαRI and IgG-FcγR for COVID-19 disease followed by cytokine release. We show that COVID-19 associated inflammation could be reduced by addition of human immunoglobulin preparations (IVIG and trimodulin), while trimodulin elicits stronger immune modulation by more powerful ITAMi signaling. Besides IgG, the IgA component of trimodulin in particular, is of functional relevance for immune modulation in this assay setup, highlighting the need to study IgA mediated immune response.

Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach.

BACKGROUND: Up to 30% of hemophilia A patients develop inhibitory antibodies against the infused factor VIII (FVIII). The development of a deimmunized FVIII is an unmet high medical need. Although improved recombinant FVIII (rFVIII) products evolved within the last years, the immunogenicity has not been solved. A deimmunized FVIII could reduce the probability of inhibitor development, providing safer therapy. OBJECTIVE: To develop a deimmunized FVIII molecule by modifying major histocompatibility complex (MHC) class II presentation, leading to a functional but less immunogenic molecule. METHODS: We performed (1) in silico prediction of potentially immunogenic T cell epitopes and their modification by amino acid substitutions in the FVIII sequence, (2) evaluation of functional and structural similarity of the modified rFVIII to unmodified FVIII and registered products, and (3) confirmation of the reduced immunogenicity by in vitro testing. RESULTS: A partially deimmunized fully functional FVIII molecule incorporating 19 amino acid substitutions was generated. The substitutions led to a reduction of the immunogenicity score, indicating a reduced immunogenicity based on in silico calculations. This was confirmed in an in vitro dendritic cell (DC)--T cell assay. Using this assay, cells from healthy donors proved the significantly reduced immunogenicity of the modified FVIII variant by revealing less proliferation of T helper cells to this variant than to the unmodified FVIII. CONCLUSION: In silico predictions resulted in a partially deimmunized FVIII. This FVIII is fully functional and was demonstrated to be less immunogenic in in vitro testing. This approach may result in a reduction of the inhibitor risk for patients with hemophilia A.

The Functional Role of IgA in the IgM/IgA-Enriched Immunoglobulin Preparation Trimodulin.

In comparison to human immunoglobulin (Ig) G, antibodies of IgA class are not well investigated. In line with this, the functional role of the IgA component in IgM/IgA-enriched immunoglobulin preparations is also largely unknown. In recent years, powerful anti-pathogenic and immunomodulatory properties of human serum IgA especially on neutrophil function were unraveled. Therefore, the aim of our work is to investigate functional aspects of the trimodulin IgA component, a new plasma-derived polyvalent immunoglobulin preparation containing ~56% IgG, ~23% IgM and ~21% IgA. The functional role of IgA was investigated by analyzing the interaction of IgA with FcαRI, comparing trimodulin with standard intravenous IgG (IVIG) preparation and investigating Fc receptor (FcR)-dependent functions by excluding IgM-mediated effects. Trimodulin demonstrated potent immunomodulatory, as well as anti-pathogenic effects in our neutrophil model (neutrophil-like HL-60 cells). The IgA component of trimodulin was shown to induce a strong FcαRI-dependent inhibitory immunoreceptor tyrosine-based activation motif (ITAMi) signaling, counteract lipopolysaccharide-induced inflammation and mediate phagocytosis of Staphylococcus aureus. The fine-tuned balance between immunomodulatory and anti-pathogenic effects of trimodulin were shown to be dose-dependent. Summarized, our data demonstrate the functional role of IgA in trimodulin, highlighting the importance of this immunoglobulin class in immunoglobulin therapy.

The Dual Role of a Polyvalent IgM/IgA-Enriched Immunoglobulin Preparation in Activating and Inhibiting the Complement System.

Activation of the complement system is important for efficient clearance of a wide variety of pathogens via opsonophagocytosis, or by direct lysis via complement-dependent cytotoxicity (CDC). However, in severe infections dysregulation of the complement system contributes to hyperinflammation. The influence of the novel IgM/IgA-enriched immunoglobulin preparation trimodulin on the complement pathway was investigated in in vitro opsonophagocytosis, binding and CDC assays. Immunoglobulin levels before and after trimodulin treatment were placed in relation to complement assessments in humans. In vitro, trimodulin activates complement and induces opsonophagocytosis, but also interacts with opsonins C3b, C4b and anaphylatoxin C5a in a concentration-dependent manner. This was not observed for standard intravenous IgG preparation (IVIg). Accordingly, trimodulin, but not IVIg, inhibited the downstream CDC pathway and target cell lysis. If applied at a similar concentration range in healthy subjects, trimodulin treatment resulted in C3 and C4 consumption in a concentration-dependent manner, which was extended in patients with severe community-acquired pneumonia. Complement consumption is found to be dependent on underlying immunoglobulin levels, particularly IgM, pinpointing their regulative function in humans. IgM/IgA provide a balancing effect on the complement system. Trimodulin may enhance phagocytosis and opsonophagocytosis in patients with severe infections and prevent excessive pathogen lysis and release of harmful anaphylatoxins.

Pneumolysin induces platelet destruction, not platelet activation, which can be prevented by immunoglobulin preparations in vitro.

Community-acquired pneumonia by primary or superinfections with Streptococcus pneumoniae can lead to acute respiratory distress requiring mechanical ventilation. The pore-forming toxin pneumolysin alters the alveolar-capillary barrier and causes extravasation of protein-rich fluid into the interstitial pulmonary tissue, which impairs gas exchange. Platelets usually prevent endothelial leakage in inflamed pulmonary tissue by sealing inflammation-induced endothelial gaps. We not only confirm that S pneumoniae induces CD62P expression in platelets, but we also show that, in the presence of pneumolysin, CD62P expression is not associated with platelet activation. Pneumolysin induces pores in the platelet membrane, which allow anti-CD62P antibodies to stain the intracellular CD62P without platelet activation. Pneumolysin treatment also results in calcium efflux, increase in light transmission by platelet lysis (not aggregation), loss of platelet thrombus formation in the flow chamber, and loss of pore-sealing capacity of platelets in the Boyden chamber. Specific anti-pneumolysin monoclonal and polyclonal antibodies inhibit these effects of pneumolysin on platelets as do polyvalent human immunoglobulins. In a post hoc analysis of the prospective randomized phase 2 CIGMA trial, we show that administration of a polyvalent immunoglobulin preparation was associated with a nominally higher platelet count and nominally improved survival in patients with severe S pneumoniae-related community-acquired pneumonia. Although, due to the low number of patients, no definitive conclusion can be made, our findings provide a rationale for investigation of pharmacologic immunoglobulin preparations to target pneumolysin by polyvalent immunoglobulin preparations in severe community-acquired pneumococcal pneumonia, to counteract the risk of these patients becoming ventilation dependent. This trial was registered at www.clinicaltrials.gov as #NCT01420744.

Long-Term Safety and Efficacy Data of a Plasma-Derived Factor VIII Concentrate with von Willebrand Factor for Treatment of Patients with Hemophilia A Covering 18 Years.

We describe the results of the (to our knowledge) longest long-term noninterventional study so far performed to obtain real-life data on the treatment of hemophilia A patients with a single plasma-derived FVIII concentrate containing von Willebrand factor (pdFVIII; Haemoctin/Faktor VIII SDH Intersero). A total of 198 patients (146 in Germany and 52 in Hungary), of whom 160 had severe and 38 nonsevere hemophilia A, representing all age groups (0-88 years; mean ∼25 years at inclusion) were analyzed during prophylactic or on-demand treatment over 18 years (overall 1,418 patient-years; mean >7 years). pdFVIII was very effective and well tolerated. The mean annual bleeding rate, including spontaneous and traumatic bleeds, was considerably lower for patients treated prophylactically (mean 5.4; median 3.1) than for patients treated on demand (mean 26.1; median 21.9). Inhibitors were found in 13% (3/23) and high-titer inhibitors in 4% (1/23) of previously untreated patients with severe hemophilia A. Four previously treated patients with severe hemophilia A developed inhibitors, thereof three high-titer inhibitors (3.3 and 2.5 high-titer inhibitors in 1,000 patient-years). No unexpected adverse effect on the health of the patients, no pdFVIII-related thrombosis, thromboembolic event, or hypersensitivity reaction, and no suspected viral transmission related to pdFVIII were documented.

Functional relevance of in vivo half antibody exchange of an IgG4 therapeutic antibody-drug conjugate.

An increasing number of monoclonal antibodies and derivatives such as antibody-drug conjugates (ADC) are of the IgG1 and IgG4 isotype with distinct structural and functional properties. In cases where antibody-mediated cytotoxicity is not desired, IgG4 is often used, as its Fc region is relatively poor at inducing antibody-dependent cell-mediated or complement-dependent cytotoxicity. IgG4 ADCs with highly cytotoxic drugs against proliferating target cells but which lack or have diminished antibody effector functions against quiescent cells may have a favorable safety profile compared to IgG1. Another unique property of the IgG4 subclass is the capability to exchange half antibodies in vivo creating randomly bispecific antibodies. To investigate the functional properties of process-derived antibody species, and determine the influence of shuffling on the therapeutic efficacy, several model antibodies on the basis of the anti-CD138 antibody-drug conjugate BT062 (Indatuximab ravtansine) were generated: (I) A wild type nBT062, (II) a stable nBT062 comprising mutations to prevent half-antibody exchange, (III) a half nBT062 lacking covalent binding between two heavy chains and (IV) a stabilized, bispecific nBT062-natalizumab antibody with a second, monovalent specificity against CD49d. All nBT062 model variants were capable of CD138-specific binding and antigen-mediated internalization into cells. Furthermore, all nBT062 models inhibited tumor growth in vitro after conjugation with the maytansinoid DM4. The in vivo effects of the different molecular variants were assessed in the MAXF1322 xenograft model. The bispecific nBT062-natalizumab-DM4 demonstrated the least efficacy and was only moderately active even without the co-administration of a human IgG preparation. Wild type, stable and half nBT062-DM4 models demonstrated great anti-tumor activities. The efficacy of wild type and half nBT062-DM4 was reduced in the presence of IgG, while stable nBT062-DM4 was only marginally influenced. These pre-clinical data demonstrate the advantage of introducing half-antibody exchange-preventing mutations into therapeutic IgG4-based antibody drug-conjugates.