Jörg Schüttrumpf

Galectin-9 is a suppressor of T and B cells and predicts the immune modulatory potential of mesenchymal stromal cell preparations.

Therapeutic approaches using multipotent mesenchymal stromal cells (MSCs) are advancing in regenerative medicine, transplantation, and autoimmune diseases. The mechanisms behind MSC immune modulation are still poorly understood and the prediction of the immune modulatory potential of single MSC preparations remains a major challenge for possible clinical applications. Here, we highlight galectin-9 (Gal-9) as a novel, important immune modulator expressed by MSCs, which is strongly upregulated upon activation of the cells by interferon-γ (IFN-γ). Further, we demonstrate that Gal-9 is a major mediator of the anti-proliferative and functional effects of MSCs not only on T cells but also on B cells. Here, Gal-9 and activated MSCs contribute to the suppression of antigen triggered immunoglobulin release. Moreover, we determined that Gal-9 expression could serve as a marker to predict a higher or lower immune modulatory potential of single cell preparations and therefore to distinguish the therapeutic potency of MSCs derived from different donors. Also in vivo co-administration of MSCs or murine Gal-9 resulted in significantly reduced IgG titers in mice immunized with human coagulation factor VIII (FVIII). In conclusion, Gal-9 acts as an immune modulator interfering with multiple cell types including B cells and Gal-9 may serve as a predictive indicator for clinical MSC therapy.

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.

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.

Transgene Loss and Changes in the Promoter Methylation Status as Determinants for Expression Duration in Nonviral Gene Transfer for Factor IX

Advances in delivery techniques and in expression construct design have renewed interest in nonviral gene transfer. Here, we test plasmid or bacterial backbone free minicircle vectors for factor IX (FIX) expression by hydrodynamic liver-directed delivery. Both constructs are driven by a hepatic control region, the human α(1)-antitrypsin promoter, which results in long-term expression in FIX knockout mice. However, levels of expression were higher and expression loss over time was reduced when using minicircles. Even at the highest expression levels (>700% of normal) FIX was fully functional. Transgene loss was the main determinant for expression loss over time for both vector types. A significant effect of gene silencing was observed only for the plasmid, not for the minicircle vector. To determine the influence of promoter methylation, we performed bisulfite-mediated conversion and sequencing of vector DNA on days 14 and 100 after gene transfer. We determined a higher frequency of methyl-protected cytosines in CpGs and a lower degree of demethylation at bacterial Dcm methylation sequences near transcription factor-binding sites in the α(1)-antitrypsin promoter in plasmid compared with minicircle mice on day 100. Therefore, the methylation status might reflect differences in the levels and durability of expression. Judging from the high levels of functional FIX obtained, small fractions of liver or single liver segments should be sufficient to reach therapeutic efficacy in translating hydrodynamic delivery to humans. However, transgene loss remains to be addressed to further guarantee sustained expression over time.

Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.

Tregalizumab - A Monoclonal Antibody to Target Regulatory T Cells.

Regulatory T cells (Tregs) represent a subpopulation of CD4+ T cells, which are essential for the maintenance of immunological tolerance. The absence or dysfunction of Tregs can lead to autoimmunity and allergies. The restoration of functional Tregs and/or Treg cell numbers represents a novel and attractive approach for the treatment of autoimmune diseases, e.g., rheumatoid arthritis (RA). The CD4 cell surface receptor is a target for modulation of T cell function. Monoclonal antibodies (mAbs) against CD4 have previously been tested for the treatment of autoimmune diseases, including RA. Furthermore, in model systems, anti-CD4 antibodies are able to induce tolerance and mediate immunomodulatory effects through a variety of mechanisms. Despite the availability of innovative and effective therapies for RA, many patients still have persistently active disease or experience adverse events that can limit use. A growing body of evidence suggests that Treg modulation could offer a new therapeutic strategy in RA and other autoimmune disorders. Here, we describe tregalizumab (BT-061), which is a novel, non-depleting IgG1 mAb that binds to a unique epitope of CD4. Tregalizumab represents the first humanized anti-CD4 mAb that selectively induces Treg activation.

Oral gene therapy for hemophilia B using chitosan-formulated FIX mutants

Oral gene delivery of non‐viral vectors is an attractive strategy to achieve transgene expression. Although expected efficacy from non‐viral delivery systems is relatively low, repeated vector administration is possible and may help to obtain durable transgene expression in a therapeutic range.

Factor VIII-eGFP fusion proteins with preserved functional activity for the analysis of the early secretory pathway of factor VIII

Considering the difficulty in detecting factor (F)VIII in vivo, fluorescently labelled FVIII protein provides a tool to analyse the intracellular localisation, bio distribution, and pharmacokinetics of the protein in living organisms. Here, we report the use of FVIII full length and B-domain deleted proteins, fused to enhanced green fluorescent protein (eGFP) at the C-terminus of the coagulation protein via a nine amino acid spanning linker. Comparison of the FVIII-eGFP fusion proteins to their unlabelled counterparts showed no impairment with respect to recombinant expression levels, intracellular processing, specific coagulant activity and decay at physiological temperature. Confocal live cell imaging demonstrated ER-Golgi-transport of B-domain deleted FVIII-eGFP in vesicular tubular carriers. Using temperature blocks and release experiments, imaging of FVIII-eGFP fusion proteins enabled for the first time the visualisation of the early secretory pathway of B-domain deleted FVIII in living cells and in particular highlighted the apparent deficit of active transport carriers, an observation consistent with the low rates of FVIII secretion seen in recombinant expression systems.

High thioredoxin-1 levels in rheumatoid arthritis patients diminish binding and signalling of the monoclonal antibody Tregalizumab

The humanized non‐depleting anti‐CD4 monoclonal antibody Tregalizumab (BT‐061) is able to selectively activate the suppressive function of regulatory T cells and has been investigated up to phase IIb in clinical trials in patients suffering from rheumatoid arthritis (RA). A pharmacokinetic–pharmacodynamic model based on clinical data from RA and healthy volunteers, which used the cell surface CD4 downmodulation as marker of activity, confirmed a stronger effect in healthy volunteers compared with RA patients. We tried to understand this phenomenon and evaluated the influence of the small oxidoreductase thioredoxin‐1 (Trx1). To counteract oxidative stress that is strongly associated with RA pathophysiology, the organism employs Trx1. Therefore, increased expression and secretion of Trx1 is found in the synovial fluid and plasma of RA patients. Moreover, the binding site of Tregalizumab is in close proximity to a disulphide bond in domain 2 (D2) of CD4, which is a known target for a reduction by oxidoreductase Trx1. With the experiments reported herein, we demonstrated that specific reduction of the D2 disulphide bond by Trx1 led to diminished binding of Tregalizumab to recombinant human soluble CD4 and membrane‐bound CD4 on T cells. Moreover, we showed that this caused changes in the Tregalizumab‐induced CD4 signalling pathway via the lymphocyte‐specific protein tyrosine kinase p56Lck and CD4 downmodulation. In summary, we provide evidence that high Trx1 levels in RA patients compared with healthy subjects are a potential reason for diminished binding of Tregalizumab to CD4‐positive T cells and offer an explanation for the observed decreased CD4 downmodulation in RA patients in comparison to healthy subjects.

Next generation FIX muteins with FVIII‐independent activity for alternative treatment of hemophilia A

FVIII neutralizing antibodies are the main complication of substitution therapy in hemophilia A (HA); auto‐antibodies against FVIII causing acquired HA can also occur. Treatment of inhibitor patients remains challenging because prophylactic treatment with existing FVIII bypassing agents, all based on constitutively active coagulation factors, is difficult due to their short half‐life.