CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma.

Clinical development of chimeric antigen receptor (CAR)-T-cell therapy has been enabled by advances in synthetic biology, genetic engineering, clinical-grade manufacturing, and complex logistics to distribute the drug product to treatment sites. A key ambition of the CARAMBA project is to provide clinical proof-of-concept for virus-free CAR gene transfer using advanced Sleeping Beauty (SB) transposon technology. SB transposition in CAR-T engineering is attractive due to the high rate of stable CAR gene transfer enabled by optimized hyperactive SB100X transposase and transposon combinations, encoded by mRNA and minicircle DNA, respectively, as preferred vector embodiments. This approach bears the potential to facilitate and expedite vector procurement, CAR-T manufacturing and distribution, and the promise to provide a safe, effective, and economically sustainable treatment. As an exemplary and novel target for SB-based CAR-T cells, the CARAMBA consortium has selected the SLAMF7 antigen in multiple myeloma. SLAMF7 CAR-T cells confer potent and consistent anti-myeloma activity in preclinical assays in vitro and in vivo. The CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) investigates the feasibility, safety, and anti-myeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial with virus-free CAR-T cells in Europe, and the first clinical trial that uses advanced SB technology worldwide.

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.

Statins stimulate the production of a soluble form of the receptor for advanced glycation end products.

The beneficial effects of statin therapy in the reduction of cardiovascular pathogenesis, atherosclerosis, and diabetic complications are well known. The receptor for advanced glycation end products (RAGE) plays an important role in the progression of these diseases. In contrast, soluble forms of RAGE act as decoys for RAGE ligands and may prevent the development of RAGE-mediated disorders. Soluble forms of RAGE are either produced by alternative splicing [endogenous secretory RAGE (esRAGE)] or by proteolytic shedding mediated by metalloproteinases [shed RAGE (sRAGE)]. Therefore we analyzed whether statins influence the production of soluble RAGE. Lovastatin treatment of either mouse alveolar epithelial cells endogenously expressing RAGE or HEK cells overexpressing RAGE caused induction of RAGE shedding, but did not influence secretion of esRAGE from HEK cells overexpressing esRAGE. Lovastatin-induced secretion of sRAGE was also evident after restoration of the isoprenylation pathway, demonstrating a correlation of sterol biosynthesis and activation of RAGE shedding. Lovastatin-stimulated induction of RAGE shedding was completely abolished by a metalloproteinase ADAM10 inhibitor. We also demonstrate that statins stimulate RAGE shedding at low physiologically relevant concentrations. Our results show that statins, due to their cholesterol-lowering effects, increase the soluble RAGE level by inducing RAGE shedding, and by doing this, might prevent the development of RAGE-mediated pathogenesis.

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.

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.