Christoph Königs

FVIII-specific human chimeric antigen receptor T-regulatory cells suppress T- and B-cell responses to FVIII.

Replacement therapy with factor VIII (FVIII) is used in patients with hemophilia A for treatment of bleeding episodes or for prophylaxis. A common and serious problem with this therapy is the patients immune response to FVIII, because of a lack of tolerance, leading to the formation of inhibitory antibodies. Development of tolerogenic therapies, other than standard immune tolerance induction (ITI), is an unmet goal. We previously generated engineered antigen-specific regulatory T cells (Tregs), created by transduction of a recombinant T-cell receptor (TCR) isolated from a hemophilia A subjects T-cell clone. The resulting engineered T cells suppressed both T- and B-cell effector responses to FVIII. In this study, we have engineered an FVIII-specific chimeric antigen receptor (ANS8 CAR) using a FVIII-specific scFv derived from a synthetic phage display library. Transduced ANS8 CAR T cells specific for the A2 domain proliferated in response to FVIII and ANS8 CAR Tregs were able to suppress the proliferation of FVIII-specific T-effector cells with specificity for a different FVIII domain in vitro. These data suggest that engineered cells are able to promote bystander suppression. Importantly, ANS8 CAR-transduced Tregs also were able to suppress the recall antibody response of murine splenocytes from FVIII knockout mice to FVIII in vitro and in vivo. In conclusion, CAR-transduced Tregs are a promising approach for future tolerogenic treatment of hemophilia A patients with inhibitors.

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: implications for the tertiary structure of the receptor and for an N-terminal binding site for HIV-1 gp120.

The chemokine receptor CCR5 contains seven transmembrane‐spanning domains. It binds chemokines and acts as co‐receptor for macrophage (m)‐tropic (or R5) strains of HIV‐1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)‐constrained phage‐displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme‐linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C‐HASIYDFGS‐C (3A9 / 1), and 5C7 most frequently identified C‐PHWLRDLRV‐C (5C7 / 1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9 / 1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7 / 1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4‐dependent reactivity with gp120 of a primary, m‐tropic HIV‐1 isolate. Thus reactivity of antibodies raised to CCR5 against phage‐displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Humoral immune responsiveness to a defined epitope on factor VIII before and after B cell ablation with rituximab

In hemophilia A, up to 30% of patients develop neutralizing antibodies (inhibitors) to factor VIII (FVIII). Treatment of an inhibitor patient with anti-CD20 (rituximab) provided an opportunity to study the humoral immune response to the well defined and constantly administered antigen, FVIII, before therapy and after B cell repopulation. Levels of CD20(+) B cells, inhibitor titers as well as antibody titers to selected antigens and FVIII-specific IgG subclasses were monitored. Inhibitors were absent for 420 days after B cell depletion, whereas antibody titers to other monitored antigens remained constant. No changes of FVIII specific IgG subclass distribution were observed. In order to characterize specific epitopes phage displayed random peptide libraries were screened with plasma before treatment and after recurrence of inhibitors. A peptide corresponding to a dominant amino acid motif selected by phage display bound FVIII-specific IgG1 before and after anti-CD20 treatment and partially restored FVIII activity in vitro. This peptide mimics a conformational epitope in the A2 domain of factor VIII, which is still recognized after inhibitor relapse despite complete B cell depletion and long-term absence of inhibitors. Mapping of specific epitopes on a defined antigen gives further information on mechanisms underlying B cell repopulation after rituximab therapy.

Age-matched dendritic cell subpopulations reference values in childhood.

Dendritic cells (DCs) are the most potent antigen‐presenting cells and are the key link between the innate and adaptive immune response. Only a few reports with study populations of up to 50 individuals have been published with age‐based reference values for DC subpopulations in healthy children. Therefore, we aimed to establish reference ranges in a larger study population of 100 healthy children, which allowed age‐matched subgroups. Most previous studies were performed using a dual‐platform approach. In this study, a single‐platform approach in a lyse no‐wash procedure was used. DC subpopulations were defined as follows: CD45+CD85k+HLA‐DR+CD14−CD16−CD33+ cells as myeloid DCs (mDCs) and CD45+CD85k+HLA‐DR+CD14−CD16−CD123+ cells as plasmacytoid DCs (pDCs). Reference ranges were established using a semi‐parametric regression of age‐matched absolute and relative DC counts. We found a significant decline with increasing age in the medians of mDCs (P = 0.0003) and pDCs per μl peripheral blood (PB) (P = 0.004) and in the 50%, 90% and 95% reference ranges. We also identified significantly lower absolute cell counts of mDCs per μl PB in girls than in boys for all age groups (P = 0.0015). Due to the larger paediatric study population and single‐platform approach, this study may give a more precise overview of the normal age‐matched development of DC subpopulations and may provide a basis for analyzing abnormal DC counts in different illnesses or therapies such as post stem cell transplantation.

Peptide Ligands Selected with CD4-Induced Epitopes on Native Dualtropic HIV-1 Envelope Proteins Mimic Extracellular Coreceptor Domains and Bind to HIV-1 gp120 Independently of Coreceptor Usage

ABSTRACT During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance.

Epitope mapping via selection of anti-FVIII antibody-specific phage-presented peptide ligands that mimic the antibody binding sites

The most serious complication in todays treatment of congenital haemophilia A is the development of neutralising antibodies (inhibitors) against factor VIII (FVIII). Although FVIII inhibitors can be eliminated by immune tolerance induction (ITI) based on repeated administration of high doses of FVIII, 20-30% of patients fail to become tolerant. Persistence of FVIII inhibitors is associated with increased morbidity and mortality. Data from recent studies provide evidence for a potential association between ITI outcome and epitope specificity of FVIII inhibitors. Nevertheless the determination of epitopes and their clinical relevance has not yet been established. In this study a general strategy for the identification of anti-FVIII antibody epitopes in haemophilia A patient plasma was to be demonstrated. Phage-displayed peptide libraries were screened against anti-FVIII antibodies to isolate specific peptides. Peptide specificity was confirmed by FVIII-sensitive ELISA binding. Peptide residues essential for antibody binding were identified by mutational analysis and epitopes were predicted via FVIII homology search. The proposed mapping strategy was validated for the monoclonal murine antibody (mAb) 2-76. Binding studies with FVIII variants confirmed the location of the predicted epitope at the level of individual amino acids. In addition, anti-FVIII antibody-specific peptide ligands were selected for 10 haemophilia A patients with FVIII inhibitors. Detailed epitope mapping for three of them showed binding sites on the A2, A3 and C2 domains. Precise epitope mapping of anti-FVIII antibodies using antibody-specific peptide ligands can be a useful approach to identify antigenic sites on FVIII.

New predictive approaches for ITI treatment.

Immune tolerance induction (ITI) therapy in patients with haemophilia A and inhibitors constitutes a huge burden for affected patients and families and poses a large economic burden for a chronic disease. Concerted research efforts are attempting to optimize the therapeutic approach to the prevention and eradication of inhibitors. The Italian ITI Registry has provided data on 110 patients who completed ITI therapy as at July 2013. Analysis of independent predictors of success showed that, together with previously recognized factors – namely inhibitor titre prior to ITI, historical peak titre and peak titre on ITI – the type of causative FVIII gene mutation also contributes to the identification of patients with good prognosis and may be useful to optimize candidate selection and treatment regimens. Numerous studies have demonstrated that inhibitor reactivity against different FVIII products varies and is lower against concentrates containing von Willebrand factor (VWF). An Italian study compared inhibitor titres against a panel of FVIII concentrates in vitro and correlated titres with the capacity to inhibit maximum thrombin generation as measured by the thrombin generation assay (TGA). Observations led to the design of the PredictTGA study which aims to correlate TGA results with epitope specificity, inhibitor reactivity against different FVIII concentrates and clinical data in inhibitor patients receiving FVIII in the context of ITI or as prophylactic/on demand treatment. At the immunological level, it is known that T cells drive inhibitor development and that B cells secrete FVIII‐specific antibodies. As understanding increases about the immunological response in ITI, it is becoming apparent that modulation of T‐cell‐ and B‐cell‐mediated responses offers a range of potential new and specific approaches to prevent and eliminate inhibitors as well as individualize ITI therapy.

Pharmacokinetics and short-term safety and tolerability of etravirine in treatment-experienced HIV-1-infected children and adolescents

Objectives:To evaluate the pharmacokinetics, weight-based dose selection and short-term safety and tolerability of etravirine in HIV-1-infected children and adolescents. Design:Phase I, nonrandomized, open-label study in two stages. Methods:Children and adolescents aged at least 6 years to 17 years or less on a stable lopinavir/ritonavir-based antiretroviral regimen with HIV-1 RNA plasma viral load less than 50 copies/ml were enrolled. In both stages, etravirine (4 mg/kg twice daily in stage I, 5.2 mg/kg twice daily in stage II), added to the existing antiretroviral regimen, was administered for 7 days followed by a morning dose and 12-h pharmacokinetic assessment on day 8. Pharmacokinetic parameters were determined using noncompartmental analysis. Data were compared with those previously established in HIV-1-infected adults on a similar etravirine (200 mg twice daily) combination antiretroviral regimen. Results:Twenty-one patients were recruited to each stage; 19 and 20 had evaluable pharmacokinetics in stages I and II, respectively. Mean (SD) maximum plasma concentrations in stages I and II were 495 (453) and 757 ng/ml (680), respectively; area under the plasma concentration–time curve over 12 h was 4050 (3602) and 6141 ng h/ml (5586), respectively. Statistical/qualitative comparisons showed comparable exposures with adults in stage II; however, the upper 90% confidence interval fell outside the predefined range. Plasma viral load remained undetectable on day 8 in all patients, and etravirine was well tolerated at both doses. Conclusion:Etravirine 5.2 mg/kg was well tolerated in this study and this dose was selected for further investigation in clinical trials.

Development of three different NK cell subpopulations during immune reconstitution after pediatric allogeneic hematopoietic stem cell transplantation: prognostic markers in GvHD and viral infections

Natural killer (NK) cells play an important role following allogeneic hematopoietic stem cell transplantation (HSCT) exerting graft-versus-leukemia/tumor effect and mediating pathogen-specific immunity. Although NK cells are the first donor-derived lymphocytes reconstituting post-HSCT, their distribution of CD56++CD16− (CD56bright), CD56++CD16+ (CD56intermediate=int), and CD56+CD16++ (CD56dim) NK cells is explicitly divergent from healthy adults, but to some extent comparable to the NK cell development in early childhood. The proportion of CD56bright/CD56int/CD56dim changed from 15/8/78% in early childhood to 6/4/90% in adults, respectively. Within this study, we first compared the NK cell reconstitution post-HSCT to reference values of NK cell subpopulations of healthy children. Afterward, we investigated the reconstitution of NK cell subpopulations post-HSCT in correlation to acute graft versus host disease (aGvHD) and chronic graft versus host disease (cGvHD) as well as to viral infections. Interestingly, after a HSCT follow-up phase of 12 months, the distribution of NK cell subpopulations largely matched the 50th percentile of the reference range for healthy individuals. Patients suffering from aGvHD and cGvHD showed a delayed reconstitution of NK cells. Remarkably, within the first 2 months post-HSCT, patients suffering from aGvHD had significantly lower levels of CD56bright NK cells compared to patients without viral infection or without graft versus host disease (GvHD). Therefore, the amount of CD56bright NK cells might serve as an early prognostic factor for GvHD development. Furthermore, a prolonged and elevated peak in CD56int NK cells seemed to be characteristic for the chronification of GvHD. In context of viral infection, a slightly lower CD56 and CD16 receptor expression followed by a considerable reduction in the absolute CD56dim NK cell numbers combined with reoccurrence of CD56int NK cells was observed. Our results suggest that a precise analysis of the reconstitution of NK cell subpopulations post-HSCT might indicate the occurrence of undesired events post-HSCT such as severe aGvHD.