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The clinical application of immune effector cells genetically modified to express chimeric antigen receptors (CARs) has shown impressive results including complete remissions of certain malignant hematological diseases. However, their application can also cause severe side effects such as cytokine release syndrome (CRS) or tumor lysis syndrome (TLS). One limitation of currently applied CAR T cells is their lack of regulation. Especially, an emergency shutdown of CAR T cells in case of life-threatening side effects is missing. Moreover, targeting of tumor-associated antigens (TAAs) that are not only expressed on tumor cells but also on vital tissues requires the possibility of a switch allowing to repeatedly turn the activity of CAR T cells on and off. Here we summarize the development of a modular CAR variant termed universal CAR (UniCAR) system that promises to overcome these limitations of conventional CARs.

Ziel/Aim Although CAR T-cell therapy has demonstrated tremendous clinical efficacy particularly in hematological malignancies, the success in solid tumors is still limited. The combination of the immunotherapeutic (Uni)CAR T-cell therapy with target modules (TM) in solid tumors and radiotherapy could be an additional treatment option. Therefore, we developed a human prostate stem cell antigen (PSCA)-specific, IgG4-based TM radiolabeled with copper-64 (Cu-64) for imaging and with actinium-225 (Ac-225) for treatment. Methodik/Methods A novel human PSCA-specific IgG4-based TM was conjugated with DOTAGA and radiolabeled with (Cu-64) and (Ac-255). The imaging and therapy were studied in NMRI Foxn1 nu/nu mice with xenotransplanted PSCA-expressing PC-3 tumors. Metabolic changes of the tumors were visualized with 18F-PSMA (PET), the physical size of the tumors and the distance between the vessels in the tumors (US) were measured. Ergebnisse/Results The radiochemical yield for the Cu-64/Ac-255 TM was 96 % and 52 % respectively, the RCP of the products were g.t. 97 %. The xenotransplanted mice were intravenously injected with the Cu-64 labeled TM given as single dose. After fast distribution, the blood activity concentration decreased very slowly. At 31 h p.i. the activity was maximal in the tumors thereby reaching the optimal tumor to background ratios and only the liver was also visible with much lower activity. The Ac-225 treatment resulted in significant lower tumor size as compared with the control group after 40 days. Doubling time of tumor volume was increased from 15.0 days (control) to 38.6 days of the treatment group. The 18F-PSMA ligand uptake was decreased in the tumor periphery. In comparison to the tumor size the vessel density decreased to a lower extent. Schlussfolgerungen/Conclusions We here present a Cu-64/Ac-225 labeled TM that allows the combination of (Uni)CAR T-cell immunotherapy with imaging with the Cu-64 labeled TM version and radiotherapy with the Ac-225 labeled TM version combined as radioimmunotheranostics.

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

Due to its overexpression on the surface of prostate cancer (PCa) cells, the prostate stem cell antigen (PSCA) is a potential target for PCa diagnosis and therapy. Here we describe the development and functional characterization of a novel IgG4-based anti-PSCA antibody (Ab) derivative (anti-PSCA IgG4-TM) that is conjugated with the chelator DOTAGA. The anti-PSCA IgG4-TM represents a multimodal immunotheranostic compound that can be used (i) as a target module (TM) for UniCAR T cell-based immunotherapy, (ii) for diagnostic positron emission tomography (PET) imaging, and (iii) targeted alpha therapy. Cross-linkage of UniCAR T cells and PSCA-positive tumor cells via the anti-PSCA IgG4-TM results in efficient tumor cell lysis both in vitro and in vivo. After radiolabeling with 64Cu2+, the anti-PSCA IgG4-TM was successfully applied for high contrast PET imaging. In a PCa mouse model, it showed specific accumulation in PSCA-expressing tumors, while no uptake in other organs was observed. Additionally, the DOTAGA-conjugated anti-PSCA IgG4-TM was radiolabeled with 225Ac3+ and applied for targeted alpha therapy. A single injection of the 225Ac-labeled anti-PSCA IgG4-TM was able to significantly control tumor growth in experimental mice. Overall, the novel anti-PSCA IgG4-TM represents an attractive first member of a novel group of radio-/immunotheranostics that allows diagnostic imaging, endoradiotherapy, and CAR T cell immunotherapy.

The exonuclease three prime repair exonuclease 1 (TREX1) safeguards the cell against DNA accumulation in the cytosol and thereby prevents innate immune activation and autoimmunity. TREX1 mutations lead to chronic DNA damage and cell-intrinsic type I interferon (IFN) response. Associated disease phenotypes include Aicardi-Goutieres syndrome, familial chilblain lupus and systemic lupus erythematosus. Given the role of ultraviolet (UV) light in lupus pathogenesis, we assessed sensitivity to UV light in lupus patients with TREX1 mutation by phototesting which revealed an enhanced photosensitivity. TREX1-deficient fibroblasts and keratinocytes generated increased levels of reactive oxygen species in response to UV irradiation as well as increased levels of 8-oxo-guanine lesions after oxidative stress. Likewise, the primary UV-induced DNA lesions cyclobutane pyrimidine dimers (CPD) were induced more strongly in TREX1-deficient cells. Further analysis revealed that single-stranded DNA regions, frequently formed during DNA replication and repair, promote CPD formation. Together, this resulted in a strong UV-induced DNA damage response that was associated with a cyclic GMP-AMP synthase (cGAS)-dependent type I IFN activation. In conclusion, these findings link chronic DNA damage to photosensitivity and type I IFN production in TREX1 deficiency and explain the induction of disease flares upon UV exposure in lupus patients with TREX1 mutation.

Antigen-specific redirection of immune effector cells with chimeric antigen receptors (CARs) demonstrated high therapeutic potential for targeting cancers of different origins. Beside CAR-T cells, natural killer (NK) cells represent promising alternative effectors that can be combined with CAR technology. Unlike T cells, primary NK cells and the NK cell line NK-92 can be applied as allogeneic off-the-shelf products with a reduced risk of toxicities. We previously established a modular universal CAR (UniCAR) platform which consists of UniCAR-expressing immune cells that cannot recognize target antigens directly but are redirected by a tumour-specific target module (TM). The TM contains an antigen-binding moiety fused to a peptide epitope which is recognized by the UniCAR molecule, thereby allowing an on/off switch of CAR activity, and facilitating flexible targeting of various tumour antigens depending on the presence and specificity of the TM. Here, we provide proof of concept that it is feasible to generate a universal off-the-shelf cellular therapeutic based on UniCAR NK-92 cells targeted to tumours expressing the disialoganglioside GD2 by GD2-specific TMs that are either based on an antibody-derived single-chain fragment variable (scFv) or an IgG4 backbone. Redirected UniCAR NK-92 cells induced specific killing of GD2-expressing cells in vitro and in vivo, associated with enhanced production of interferon-γ. Analysis of radiolabelled proteins demonstrated that the IgG4-based format increased the in vivo half-life of the TM markedly in comparison to the scFv-based molecule. In summary, UniCAR NK-92 cells represent a universal off-the-shelf platform that is highly effective and flexible, allowing the use of different TM formats for specific tumour targeting.

Bifunctional derivatives of bis(phosphinate)-bearing cyclam (BPC) chelators bearing a carboxylate, amine, isothiocyanate, azide, or cyclooctyne in the BP side chain were synthesized. Conjugations required no protection of phosphinate or ring secondary amine groups. The ring amines were not reactive (proton protected) at pH < ∼8. For isothiocyanate coupling, oligopeptide N-terminal α-amines were more suitable than alkyl amines, e.g., Lys ω-amine (pKa ∼7.5–8.5 and ∼10–11, respectively) due to lower basicity. The Cu-64 labeling was efficient at room temperature (specific activity ∼100 GBq/μmol; 25 °C, pH 6.2, ∼100 ligand equiv, 10 min). A representative Cu-64-BPC was tested in vivo showing fast clearance and no nonspecific radioactivity deposition. The monoclonal anti-PSCA antibody 7F5 conjugates with thiocyanate BPC derivative or NODAGA were radiolabeled and studied in PC3-PSCA tumor bearing mice by PET. The radiolabeled BPC conjugate was accumulated in the prostate tumor with a low off-target uptake, unlik...

Digital, Chimeric antigen receptor (CAR) T cells have shown impressive therapeutic potential. Due to the lack of direct control mechanisms, therapy-related adverse reactions including cytokine release- and tumor lysis syndrome can even become life-threatening. In case of target antigen expression on non-malignant cells, CAR T cells can also attack healthy tissues. To overcome such side effects, we have established a modular CAR platform termed UniCAR: UniCAR T cells per se are inert as they recognize a peptide epitope (UniCAR epitope) that is not accessible on the surface of living cells. Bifunctional adapter molecules termed target modules (TM) can cross-link UniCAR T cells with target cells. In the absence of TMs, UniCAR T cells automatically turn off. Until now, all UniCAR TMs were constructed by fusion of the UniCAR epitope to an antibody domain. To open up the wide field of low-molecular-weight compounds for retargeting of UniCAR T cells to tumor cells, and to follow in parallel the progress of UniCAR T cell therapy by PET imaging we challenged the idea to convert a PET tracer into a UniCAR-TM. For proof of concept, we selected the clinically used PET tracer PSMA-11, which binds to the prostate-specific membrane antigen overexpressed in prostate carcinoma. Here we show that fusion of the UniCAR epitope to PSMA-11 results in a low-molecular-weight theranostic compound that can be used for both retargeting of UniCAR T cells to tumor cells, and for non-invasive PET imaging and thus represents a member of a novel class of theranostics., 1 ed.

There are several reasons for drying of polyacrylamide gels after gel electrophoresis; for example, it is necessary if autoradiography has to be performed using radioactive labeled proteins. Another reason may be to simply store the gel in the laboratory book. Aside laborious commercial solutions, the simple and cheap drying protocol presented here may be sufficient especially for storage of the dried gel in the lab book.

Protein-based preparations for biomedical research, and radiolabeled pharmaceuticals for nuclear medical diagnostics and therapy require highest chemical, radiochemical, and in particular protein purity. For detection of contaminations most commonly size exclusion high-performance liquid chromatography and polyacrylamide gel analysis in combination with silver staining is used. Here we describe a by far more sensitive radiolabeling method for the detection of traces of contaminating proteins.

The common acute lymphoblastic leukemia antigen CD10 is a marker for several hematological malignancies, including acute lymphoblastic leukemia as well as T and B cell lymphomas, Burkitt lymphomas, and some solid tumors like renal cell carcinomas, pancreatic tumors and melanomas. Because of its tumor related expression pattern, CD10 is an attractive target for adoptively transferred T cells that are genetically modified to express chimeric antigen receptors (CARs). Recently, conventional CAR T cell therapy targeting CD19-positive hematological malignancies was clinically approved because of its impressive effectiveness in patients. However, CAR T cells can also cause severe side effects like on-target, off-tumor reactions, tumor lysis syndrome and cytokine release syndrome. Most critically, activity of conventional CAR T cells cannot be controlled, once they are applied in patients. As CD10 is also widely expressed on normal tissues, CAR T cell reactivity has to be controllable in order to stop CAR T cell therapy in case of on-target, off-tumor toxicities occur. Especially for this purpose, we have recently established a switchable, modular and universal CAR platform technology, named UniCAR system, which can be repeatedly turned on and off. In contrast to conventional CARs, that directly recognize a tumor-associated antigen (TAA) on the tumor cell surface via their extracellular single-chain variable fragment (scFv), the UniCAR system is structured in a modular manner of two components. The first component are T cells genetically engineered to express UniCARs and the second component are target modules (TMs). Most importantly, UniCARs cannot directly bind to a TAA because their extracellular scFv is directed against the peptide epitope E5B9 which is not present on the surface of living cells. Consequently, UniCAR armed T cells are per se inert. They can be redirected towards tumor cells only via a TM. TMs consist of a scFv targeting a TAA and the epitope E5B9 recognized by UniCARs allowing a cross-linkage of UniCAR T cells with tumor cells which results in T cell activation. As TMs have a very short half-life, UniCAR T cell activity can be controlled by dosing of the TM. Once the TM is administered, UniCAR T cells can be switched on, but once the TM injection is stopped and the TM is eliminated, UniCAR T cells are switched off immediately. Here, we show proof of concept for functionality of the UniCAR system targeting CD10-positive malignancies. Therefor, a novel anti-CD10 TM was constructed which is able to redirect UniCAR T cells to eliminate CD10-expressing tumor cells. In summary, we have established a universal, switchable, modular UniCAR platform technology that can be used to target CD10-positive malignancies. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

The clinical efficacy of CAR T cell therapies has been widely recognized, particularly in the treatment of hematologic malignancies. Nevertheless, CAR T cells also have the capability to elicit undesired effects such as on-target/off-tumor recognition and cytokine release syndrome. To increase clinical safety of CAR T cell therapy, a novel modular universal CAR platform termed UniCAR was developed by our group. In the UniCAR system, antigen-binding specificity and signaling features are two distinct moieties, in which the antigen specificity is provided by targeting modules (TMs) to redirect UniCAR T cells in an individualized time- and target-dependent manner. In this way, UniCAR T-cells acquire killing potential only in the presence of a tumour-specific TM. Given the reduced size of such molecules, they are rapidly eliminated and therefore, need to be continuously infused. Thus, possible side effects and activation of UniCAR T cells can be easily monitored and controlled by TM dosing. During the onset of therapy, tumor burden and the risk for severe side effects are high and regulation of CAR T cell activity is particularly important at this stage. For this reason, TMs with extended half-life may play an important role by improving eradication of residual tumor cells in late phases of treatment and further expedite clinical application. In this line of thought, a set of novel short-lived and longer-lasting TMs directed against several tumor-associated antigens was developed. Short-lived TMs are composed of a tumor-specific binding moiety fused to the La peptide epitope (E5B9) which is recognized by UniCAR T cells. In order to generate extended half-life TMs, these two components are fused via an Fc domain derived from the human IgG4 molecule. In vitro and in vivo assays have shown that both short-lived and longer-lasting TMs efficiently redirect UniCAR T cells to cancer cells in a highly target-specific manner, thereby promoting the secretion of pro-inflammatory cytokines and tumor cell lysis. Further assays using PET-imaging, demonstrated that all TM formats specifically enriched at the tumor site presenting either short or prolonged serum half-lives. From a clinical point of view, after the initial reduction of tumor burden promoted by the small TMs, IgG4-based TMs could be subsequently administrated allowing a more convenient and personalized treatment of the patients avoiding the continuous infusion of the short-lived TMs. Furthermore, the specific accumulation of such IgG4-based TMs at the tumor site sets these molecules as attractive candidates for in vivo imaging and endoradiotherapy. Taken together, combination of switchable UniCAR T cells and TMs with different sizes, specificities and half-lives represent a flexible and individualized approach at different stages of cancer treatment. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

Hematological malignancies are successfully treated with chimeric antigen receptor (CAR) armed T cells. Despite the clinical success, CAR T cell therapy struggles still with some problems including the selection of tumor escape variants and on-target, off-tumor side reactions as well as massive cytokine release and uncontrollability of CAR T cell activity in the patients. In order to enable controllability of CAR T cells and to avoid unspecific side effects, we established a novel switchable, split and adaptable CAR platform technology, termed RevCAR system. The novel RevCARs lack the single chain variable fragment (scFv) commonly used as extracellular domain in conventional CARs. Instead of the scFv, RevCARs contain only a small peptide epitope as extracellular portion. This design reduces the CAR size, avoids unspecific antigen binding and prevents antigen independent tonic signaling caused by scFv dimerization. As RevCAR T cells do not recognize anything, they are per se inert. Only in the presence of a corresponding bispecific antibody based target module (RevTM) they can be specifically redirected to tumor cells. Therefor RevTMs consist of two scFvs. One recognizes the RevCAR peptide epitope and the other one simultaneously binds to a tumor associated antigen (TAA). By dosing of the RevTM, which has a very short half-life, the reactivity of RevCAR T cells can be switched on and off reversibly. Another advantage is that the RevCAR system can be flexibly adapted to any tumor antigen simply by exchanging the RevTM. Furthermore, the small RevCAR size is favorable for inserting more than one RevCAR in the same T cell thus facilitating the mode of gated targeting which is a highly attractive approach to minimize the risk for on-target, off-tumor toxicities against healthy tissues and to increase tumor specificity of conventional CAR T cells. For 'AND' gate targeting via the RevCAR system, two different RevCARs were constructed and expressed simultaneously in the same T cell. The two RevCARs differed with respect to the extracellular peptide epitope and the intracellular signaling domain. Moreover, the respective transmembrane domain was selected to isolate the respective RevCAR signal. The first RevCAR is designed to transmit the activation signal, the second RevCAR to deliver a costimulatory signal. For efficient RevCAR T cell activation, both RevCARs must be engaged via their respective RevTM which on the one hand binds to one of the two RevCAR epitopes and on the other hand to one of two TAAs expressed on the same target cell. Here, we present two RevCAR/RevTM systems for retargeting of AML cells as well as solid tumor cells including via gated targeting. In summary, we show proof of concept for a novel switchable RevCAR system that can be used for retargeting of AML cells as well as solid tumors. The novel modular RevCAR platform is characterized by small size, lacks unwanted tonic signaling effects, allows the control of RevCAR T cell activity, enables gated targeting strategies, and can be adapted to any tumor antigen and tumor type. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

Long-term survival of adoptively transferred chimeric Ag receptor (CAR) T cells is often limited. Transplantation of hematopoietic stem cells (HSCs) transduced to express CARs could help to overcome this problem as CAR-armed HSCs can continuously deliver CAR+ multicell lineages (e.g., T cells, NK cells). In dependence on the CAR construct, a variable extent of tonic signaling in CAR T cells was reported; thus, effects of CAR-mediated tonic signaling on the hematopoiesis of CAR-armed HSCs is unclear. To assess the effects of tonic signaling, two CAR constructs were established and analyzed 1) a signaling CAR inducing a solid Ag-independent tonic signaling termed CAR-28/ζ and 2) a nonstimulating control CAR construct lacking intracellular signaling domains termed CAR-Stop. Bone marrow cells from immunocompetent mice were isolated, purified for HSC-containing Lin−cKit+ cells or the Lin−cKit+ Sca-1+ subpopulation (Lin−Sca-1+cKit+), and transduced with both CAR constructs. Subsequently, modified bone marrow cells were transferred into irradiated mice, in which they successfully engrafted and differentiated into hematopoietic progenitors. HSCs expressing the CAR-Stop sustained normal hematopoiesis. In contrast, expression of the CAR-28/ζ led to elimination of mature CAR+ T and B cells, suggesting that the CAR-mediated tonic signaling mimics autorecognition via the newly recombined immune receptors in the developing lymphocytes.

CAR-modified T cells show impressive results in clinical trials. However, cytokine release syndrome and “on-target, off-tumor” reactions represent most concerning side effects. To improve the safety of CAR-T cell therapy, we established a switchable CAR platform termed UniCAR system consisting of two components: UniCAR-modified T cells and tumor-specific target modules (TM). For treatment of EGFR+ epithelial tumors, we recently described a monovalent nanobody-based α-EGFR TM, either expressed in bacteria or eukaryotic cells. In spite of the identical primary sequence the eukaryotic TM showed a reduced killing capability and affinity. Here we describe a novel bivalent α-EGFR-EGFR TM. As expected, the avidity of the bivalent TM is higher than that of its monovalent counterpart. Binding of neither the monovalent α-EGFR TM nor the bivalent α-EGFR-EGFR TM to EGFR effected the EGF-mediated signaling. While the monovalent α-EGFR TM could only mediate the killing of tumor cells expressing high levels of EGFR, the bivalent α-EGFR-EGFR TM could redirect UniCAR T cells to tumor cells expressing low levels of EGFR. According to PET experiments in vivo, the increased avidity of the bivalent α-EGFR-EGFR TM improves the enrichment at the tumor site and its use for PET imaging.

There are several reasons for drying of polyacrylamide gels after gel electrophoresis. E.g. if autoradiography should be performed from radioactive-labeled proteins. Another reason may be to simply store the gel in the laboratory book. Aside laborious commercial solutions, especially for storage of the dried gel in the lab book the here presented simple and cheap drying protocol may be sufficient.

Ubiquitylation (ubi) by the E3-ligases Mindbomb1 (Mib1) and Neuralized (Neur) is required for activation of the DSL ligands Delta (Dl) and Serrate (Ser) to activate Notch signalling. These ligases transfer ubiquitin to lysines of the ligands' intracellular domains (ICDs), which sends them into an Epsin-dependent endocytic pathway. Here, we have tested the requirement of ubi of Dl for signalling. We found that Dl requires ubi for its full function, but can also signal in two ubi-independent modes, one dependent and one independent of Neur. We identified two neural lateral specification processes where Dl signals in an ubi-independent manner. Neur, which is needed for these processes, was shown to be able to activate Dl in an ubi-independent manner. Our analysis suggests that one important role of DSL protein ubi by Mib1 is their release from cis-inhibitory interactions with Notch, enabling them to trans-activate Notch on adjacent cells., eLife digest Cells use chemical signals to communicate, setting off chains of reactions known as signalling pathways. One key signalling pathway, thought to be required for the development of all animals, is called Notch. In fruit flies, signal proteins known as Delta and Serrate activate the Notch pathway by binding to receptors on the outside of the cell. To do so, the signal proteins first need to be activated themselves. Two enzymes known as Mindbomb1 and Neuralized activate Delta and Serrate. Both enzymes add a small unit called ubiquitin to specific locations on the signal proteins, but the effect that ubiquitin has on Notch signalling is not yet fully understood. Berndt, Seib, Kim et al. have now examined fruit flies that had a variety of genetic mutations. These included some flies that could produce mutant versions of the Serrate and Delta proteins that lacked the locations to which ubiquitin normally attaches. The results of the experiments reveal that Delta requires ubiquitin, Mindbomb1 and Neuralized to work at full capacity. However, Delta could still perform some of its roles without ubiquitin. Neuralized and Delta can partner up to send some signals independently of ubiquitin, and Delta can even send some signals on its own. Serrate, on the other hand, does not work at all without ubiquitin. The results presented by Berndt et al. help us to understand the role that ubiquitin plays in activating Notch signalling. Further work that builds on these findings could help to shed light on how uncontrolled Notch activation can contribute to a variety of diseases, including cancer, cardiovascular diseases and multiple sclerosis.

Huriez syndrome is a rare dominant genodermatosis characterized by congenital palmoplantar keratosis, scleroatrophic changes of the hands and feet, and an increased risk for cutaneous squamous cell carcinoma (CSCC) in approximately 15% of affected individuals (Figure 1a, Supplementary Table S1 online). CSCC in Huriez syndrome is characterized by early onset, localization on scleroatrophic skin, and aggressive metastasis formation (Delaporte et al., 1995; Hamm et al., 1996) suggesting that the local disease-specific skin changes promote malignant transformation.

As the expression of a tumor associated antigen (TAA) is commonly not restricted to tumor cells, adoptively transferred T cells modified to express a conventional chimeric antigen receptor (CAR) might not only destroy the tumor cells but also attack target-positive healthy tissues. Furthermore, CAR T cells in patients with large tumor bulks will unpredictably proliferate and put the patients at high risk of adverse side effects including cytokine storms and tumor lysis syndrome. To overcome these problems, we previously established a modular CAR technology termed UniCAR: UniCAR T cells can repeatedly be turned on and off via dosing of a target module (TM). TMs are bispecific molecules which cross-link UniCAR T cells with target cells. After elimination of the respective TM, UniCAR T cells automatically turn off. Here we describe novel TMs against the disialoganglioside GD2 which is overexpressed in neuroectodermal but also many other tumors. In the presence of GD2-specific TMs, we see a highly efficient target-specific and -dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and tumor cell lysis both in vitro and experimental mice. According to PET-imaging, anti-GD2 TM enrich at the tumor site and are rapidly eliminated thus fulfilling all prerequisites of a UniCAR TM.

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity., La lista completa de autores que integran el documento puede consultarse en el archivo, Instituto de Física de Líquidos y Sistemas Biológicos

Importance Familial chilblain lupus is a rare, autosomal dominant form of lupus erythematosus characterized by cold-induced inflammatory lesions at acral locations presenting in early childhood. Familial chilblain lupus is usually caused by a mutation inTREX1(3′ repair exonuclease 1). Observations We report on a family with dominant chilblain lupus segregating a novelTREX1mutation (c.585C>G; H195Q) within the highly conserved exonuclease (Exo) III domain. Affected family members experienced cold-induced chilblain lesions of varying degrees, ranging from bluish-red infiltrations to mutilating necrotic ulcerations. In addition, all patients showed signs of systemic disease, such as arthritis, lymphopenia, or antinuclear antibodies. An increased expression of myxovirus resistance protein A in the skin and induction of interferon-stimulated genes in peripheral blood cells demonstrated activation of type I interferon. Conclusions and Relevance This case further implicates type I interferon–dependent innate immune activation in the pathogenesis ofTREX1-associated familial chilblain lupus. Unlike previously reportedTREX1mutations, which affect the Exo I or Exo II domains, the mutation presented here alters the Exo III domain, suggesting a particular role of mutations within the catalytic Exo domains in the pathogenesis of familial chilblain lupus. The high prevalence of extracutaneous manifestations, along with activation of type I interferon, underlines the systemic nature of familial chilblain lupus.

Owing to their clinical success, there is growing interest in novel bispecific antibodies (bsAbs) for retargeting of T cells to tumor cells including for the treatment of acute myeloid leukemia (AML). One potential target for retargeting of T cells to AML blasts is the surface molecule CD33. Here we describe a novel modular targeting platform that consists of a universal effector module (EM) and individual target modules (TMs). Both modules can form an immune complex via a peptide epitope. The resulting targeting complex can functionally replace a conventional bsAb. By fusion of a costimulatory domain (for example, the extracellular CD137 ligand domain) to the TM, the targeting complex can even provide a costimulatory signal to the redirected T cells at their side of interaction with the tumor cell. Furthermore, we observed that an efficient killing of tumor cells expressing low levels of the tumor target CD33 becomes critical at low effector-to-target cell ratios but can be improved by costimulation via CD137 using our novel targeting system.

Mycosis fungoides (MF) is the most frequent form of cutaneous T-cell lymphoma (CTCL), which can deteriorate from patch stage to dermal-based tumors and systemic involvement in years. The interaction of chemokines in the skin with CTCL cells might have implications for the pathogenesis of the disease. In this study, we show by PCR analysis and immunofluorescence staining that the chemokine CCL18 is present in skin biopsy specimens of patients with MF and its precursor form parapsoriasis en plaque but not in healthy tissue. In addition, the serum levels of CCL18 were increased threefold in MF patients compared with those in healthy controls. In skin, CCL18 was specifically expressed by CD163(+) CD209(+) macrophages at the invasive margin of the tumor and not expressed by mature CD208(+) dendritic cells in the center of the tumor. The chemokine CCL17 was, by contrast, ubiquitously expressed. Furthermore, CCL18 promoted the chemotaxis but not the proliferation of CTCL cells. CCL18 inhibited proliferation of tumor cells and abolished the CXCL12-induced growth of a CTCL cell line. These data link the increased expression of CCL18 with CTCL and suggest an immunomodulatory effect of the chemokine in the pathogenesis of CTCL.

We have recently described a novel modular targeting platform for T cell recruitment that not only efficiently replaces but also is superior to conventional T cell-engaging bispecific antibodies as it allows for the flexible targeting of several antigens and the delivery of co-stimulatory ligands to malignant lesions, thereby enhancing the antitumor potential of redirected T cells.

Regulatory T cells (Tregs) play an inevitable role in immune homeostasis by maintaining self-tolerance as well as regulating the magnitude of immune responses against foreign antigens. Over the last few years, the enormous potential of adoptive Treg transfer for treatment of auto- and alloimmunity including Graft-versus-Host disease (GvHD) has been validated in a vast number of in vitro and in vivo studies. For their clinical application, all modes of action should be well understood. Regarding their cytotoxic potential, only few and conflicting data exist. On the one hand, it is assumed that Tregs are capable of inducing apoptosis of effector T cells (Teff) utilizing granzyme/perforin or FasL expression. Others claim that Tregs are not capable of suppressing Teff via programmed cell death pathways but rather induce apoptosis by cytokine deprivation. However, it is of importance to clarify whether Tregs possess a cytotoxic potential particularly when activating the cells antigen-specifically using bispecific antibodies (bsAb). In recent years, bsAb have emerged as promising tools for an antigen-specific immunotherapy of malignant diseases. Their tremendous potential for tumor therapy has been verified in a plethora of in vitro and in vivo studies as well as in first clinical trials. So far, our group was able to demonstrate that not only Teff but also Tregs can be redirected by CD3-engaging bsAb (Koristka et al., J Immunol. 2012; J Autoimmun. 2013). According to a recent presentation (Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research, 2012, abstract nr 4841), bsAb-redirected Tregs can act as killer cells and efficiently mediate cancer cell lysis. In order to shed light onto this controversial issue, we decided to analyze this question in more detail. According to our investigations tumor cell elimination of bsAb-engaged Tregs is largely dependent on the purity of isolated Treg fractions. Tregs isolated on the basis of CD25 expression exhibited a remarkable killing capacity which is most probably due to contaminating CD25+FOXP3- Teff, as highly pure (> 99 %), FACS-isolated CD4+CD25+CD127low Tregs did not display any considerable cytotoxic effect upon cross-linkage to tumor cells via bsAb. The same applies for CD45RA-sorted, expanded Tregs. In comparison to autologous, expanded Teff, tumor cell lysis was negligible. Moreover, the lack of cytotoxicity was independent of the chosen target antigen, as redirecting Tregs with two different bsAb did not result in tumor cell eradication. Besides, upon polyclonal stimulation with conventional aCD3/CD28-coated beads Tregs were not capable of eliminating target cells. Furthermore, as opposed to autologous Teff, Tregs showed only a marginal upregulation of the degranulation marker CD107a when being activated either antigen-specifically via bsAb or polyclonally via beads. Taken together, our findings clearly demonstrate that Tregs bear no considerable cytotoxic potential and hence do not contribute to cancer cell lysis, as recently claimed. On the other hand, the results show that Tregs can be activated by bsAb without the risk of cytotoxic effects against the recognized target cells. This provides the basis for the application of bsAb for a site-specific recruitment of Tregs aiming at attenuating Teff-mediated proinflammatory immune responses and tissue destruction in order to treat auto- and alloimmune diseases including GvHD. Disclosures: No relevant conflicts of interest to declare.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease in which environmental exposures like virus infection and UV-irradiation trigger activation of the innate and adaptive immune system in genetically predisposed individuals. Heterozygous mutations of the 3' repair exonuclease 1 (TREX1) are associated with SLE. Biallelic mutations in TREX1 and the three subunits of ribonuclease H2 (RNASEH2A-C) cause Aicardi-Goutieres syndrome, an inflammatory encephalopathy with clinical overlap with SLE. We therefore investigated the role of RNase H2 in SLE pathogenesis. RNase H2 is responsible for the removal of misincorporated ribonucleotides from DNA and is indispensable for genome integrity. We demonstrated a genetic association for rare RNase H2 sequence variants with SLE. RNase H2-deficient fibroblasts of AGS and SLE patients accumulated ribonucleotides in genomic DNA resulting in chronic low-level DNA damage, constitutive p53 phosphorylation and senescence. Patient fibroblasts proliferated slower than fibroblasts from healthy individuals and showed impairment of cell cycle progression. In addition, patient fibroblasts exhibited constitutive up-regulation of interferon-stimulated genes and an enhanced type I interferon response to the nucleic acid poly(I:C) and UV-irradiation. UV-irradiation induced enhanced cyclobutane pyrimidine dimer formation in ribonucleotide-containing DNA. This suggests that innate immune activation may be caused by immune recognition of DNA metabolites of DNA damage repair and may also explain photosensitivity in SLE patients with RNase H2 mutation. In summary, our findings implicate RNase H2 in the pathogenesis of SLE, and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.