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.

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 interferong (IFN-g). 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.

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 genetransfer 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 IntroductionThe intrinsic coagulation cascade is a tightly regulated proteaseand cofactor-dependent amplification system that ensures the formation of stable clots after injury. 1 Within this system, deficiencies of the coagulation cofactor, factor VIII (FVIII), or the corresponding coagulation protease, factor IX (FIX), lead to the X-chromosomal inherited bleeding disorders hemophilia A and hemophilia B, respectively. Hemophilia A occurs approximately in 1 of 5000 newborn boys, whereas hemophilia B is less common. 2 Untreated, hemophilia presents with spontaneous bleeding preferentially into large joints and skeletal muscle, and internal and intracranial bleedings can also occur. Substitution of deficient coagulation factors by intravenous infusion of plasma-derived or recombinant coagulation factor concentrates is the therapy of choice. In the last decades, treatment has evolved from so-called on-demand treatment for acute injury/hemorrhage to a secondary preventative approach with regular prophylactic infusions. Prophylactic treatment, securing plasma levels above 1% of normal, already prevents the major long-term consequences of the disease: joint damage and muscular atrophy. 3,4 A major obstacle for protein substitution therapy is the occurrence of neutralizing Abs directed against the FVIII protein. This can either be a result of an immune response after exogenous protein exposure 5 or may appear in adult patients as a spontaneous auto-immune event. 6 In these cases, FVIII infusion is often ineffective and so-called bypassing agents are used. These agents consist of constitutively activated proteases such as activated factor VII (FVIIa), and promote clot formation directly without restoring the intrinsic amplification loop. Although these therapeutics are efficient at stopping acute bleeding, limitations include the relatively short half-lives of activated proteases in the circulation and potential vascular risks in long-term treatment. 7,8 In the present study, we report on the generation of FIX variants with FVIII-independent clotting activity that we...