Local immunotherapy with the RNA-based immune stimulator CV8102 induces substantial anti-tumor responses and enhances checkpoint inhibitor activity.

Immunotherapy has revolutionized cancer treatment in recent years. Although currently approved checkpoint inhibitors (CPIs) yield remarkable anti-tumoral responses in several cancer types, a substantial proportion of patients do not benefit from such therapies. Local activation of innate immune signaling pathways is a promising approach to overcome the immunosuppressive tumor microenvironment, induce anti-tumor immunity, and improve the efficacy of CPI therapies. Here, we assessed the mode of action and efficacy of the RNA-based innate immune stimulator CV8102 for local immunotherapy in preclinical models. Intratumoral (i.t.) administration of CV8102 activated innate immune responses in the tumor microenvironment and draining lymph nodes, resulting in a dose-dependent anti-tumoral response. Combining i.t. CV8102 with systemic anti-programmed death protein 1 (PD-1) treatment further enhanced anti-tumoral responses, inducing tumor infiltration and activation of CD8+ T cells. The resulting memory response prevented tumor growth in rechallenged animals and impaired the growth of non-injected distal tumors. Therefore, i.t. CV8102 delivery is a promising approach for local cancer immunotherapy, especially in combination with CPIs. Clinical testing of CV8102 is ongoing (NCT03291002).

mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells.

mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables efficient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-of-experiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarifies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNA-LNP-based antitumor immunotherapy.

Differential Requirement of Vav Proteins for Btk-dependent and -Independent Signaling During B Cell Development.

Btk and Vav proteins are all components of the signalosome that builds upon B cell receptor (BCR) activation. However, the role of Vav proteins within the signalosome is quite complex and not yet fully understood. Until now, studies of these have focused predominantly on a deficiency of Vav proteins alone or in combination with other Vav protein family members. Since a physical association of Btk with Vav was shown previously, we asked whether these molecules lie in the same or independent signaling pathways. By analyzing Vav1 and Vav3 single knock-out mice and generating double-knock-out animals deficient for either Vav1 or Vav3 and Btk, we observed, in line with previous publications, no severe B cell developmental defects when either Vav1 or Vav3 alone are not expressed. However, a simultaneous deficiency of Btk together with either Vav1 or Vav3 leads to a severe reduction of splenic B cells, which exhibit an immature phenotype. B cell developmental defects of Btk/Vav1-double deficient mice in the periphery were more severe than those observed in Btk-single-deficient animals. Additionally, morphological changes in splenic microarchitecture were observed in double- but also in single-knock-out mutants. These observations were accompanied by reduced BCR-induced Ca2+ mobilization, proliferation, germinal center formation and immunoglobulin secretion. Although deletion of Btk alone impaired Ca2+ mobilization upon BCR activation, the defect was even more severe when Vav1 or Vav3 were also mutated, indicating that Btk and the Vav proteins act in separate pathways that converge on Ca2+ signaling. In vitro ASC differentiation suggests that both B and T cells contribute to the observed phenotype of a Btk/Vav-double deficiency. Our results show that Vav proteins and Btk are both components of the BCR-activated signalosome but control separate signaling pathways important for B cell development.

BOB.1/OBF.1 is required during B-cell ontogeny for B-cell differentiation and germinal center function.

BOB.1/OBF.1 is a lymphocyte-specific transcriptional co-activator of octamer-dependent transcription. It regulates the expression of genes important for lymphocyte physiology together with the Oct-1 and Oct-2 transcription factors. So far, BOB.1/OBF.1 has been studied in conventional knockout mice, whereby a function of BOB.1/OBF.1 in B but also in T cells was described. The main characteristic of BOB.1/OBF.1-deficient mice is the complete absence of germinal centers. However, it is entirely unsolved at which stage of B-cell development BOB.1/OBF.1 expression is essential for germinal center formation. Still, it is not known whether defects observed late in B-cell development of BOB.1/OBF.1-deficient mice are merely a consequence of defective early B-cell development. To answer the question, whether BOB.1/OBF.1 expression is required before or during the process of germinal center formation, we established a mouse system, which allows the conditional deletion of BOB.1/OBF.1 at different stages of B-cell development. Our data reveal a requirement for BOB.1/OBF.1 during both early antigen-independent and late antigen-dependent B-cell development, and further a requirement for efficient germinal center reaction during complete B-cell ontogeny. By specifically deleting BOB.1/OBF.1 in germinal center B cells, we provide evidence that the failure to form germinal centers is a germinal center B-cell intrinsic defect and not exclusively a consequence of defective early B-cell maturation.

Impaired Peyer's patch development in BOB.1/OBF.1-deficient mice.

BOB.1/OBF.1 expression regulates the transcription of direct and indirect target genes. We propose that BOB.1/OBF.1 affects CXCL13-CXCR5 signaling of LTinducer and LTorganizer cells during embryonic Peyer's patch organogenesis as well as of B cells and follicular dendritic cells during lymphocyte homing at postnatal stages of secondary lymphoid organ development.

mRNA: A Novel Avenue to Antibody Therapy?

First attempts to use exogenous mRNA for protein expression in vivo were made more than 25 years ago. However, widespread appreciation of in vitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such in vivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient in vivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.

mRNA mediates passive vaccination against infectious agents, toxins, and tumors.

The delivery of genetic information has emerged as a valid therapeutic approach. Various reports have demonstrated that mRNA, besides its remarkable potential as vaccine, can also promote expression without inducing an adverse immune response against the encoded protein. In the current study, we set out to explore whether our technology based on chemically unmodified mRNA is suitable for passive immunization. To this end, various antibodies using different designs were expressed and characterized in vitro and in vivo in the fields of viral infections, toxin exposure, and cancer immunotherapies. Single injections of mRNA-lipid nanoparticle (LNP) were sufficient to establish rapid, strong, and long-lasting serum antibody titers in vivo, thereby enabling both prophylactic and therapeutic protection against lethal rabies infection or botulinum intoxication. Moreover, therapeutic mRNA-mediated antibody expression allowed mice to survive an otherwise lethal tumor challenge. In conclusion, the present study demonstrates the utility of formulated mRNA as a potent novel technology for passive immunization.

mRNA Cancer Vaccines.

mRNA cancer vaccines are a relatively new class of vaccines, which combine the potential of mRNA to encode for almost any protein with an excellent safety profile and a flexible production process. The most straightforward use of mRNA vaccines in oncologic settings is the immunization of patients with mRNA vaccines encoding tumor-associated antigens (TAAs). This is exemplified by the RNActive® technology, which induces balanced humoral and cellular immune responses in animal models and is currently evaluated in several clinical trials for oncologic indications. A second application of mRNA vaccines is the production of personalized vaccines. This is possible because mRNA vaccines are produced by a generic process, which can be used to quickly produce mRNA vaccines targeting patient-specific neoantigens that are identified by analyzing the tumor exome. Apart from being used directly to vaccinate patients, mRNAs can also be used in cellular therapies to transfect patient-derived cells in vitro and infuse the manipulated cells back into the patient. One such application is the transfection of patient-derived dendritic cells (DCs) with mRNAs encoding TAAs, which leads to the presentation of TAA-derived peptides on the DCs and an activation of antigen-specific T cells in vivo. A second application is the transfection of patient-derived T cells with mRNAs encoding chimeric antigen receptors, which allows the T cells to directly recognize a specific antigen expressed on the tumor. In this chapter, we will review preclinical and clinical data for the different approaches.

Cardiac-Specific Activation of IKK2 Leads to Defects in Heart Development and Embryonic Lethality.

The transcription factor NF-κB has been associated with a range of pathological conditions of the heart, mainly based on its function as a master regulator of inflammation and pro-survival factor. Here, we addressed the question what effects activation of NF-κB can have during murine heart development. We expressed a constitutively active (CA) mutant of IKK2, the kinase activating canonical NF-κB signaling, specifically in cardiomyocytes under the control of the α-myosin heavy chain promoter. Expression of IKK2-CA resulted in embryonic lethality around E13. Embryos showed defects in compact zone formation and the contractile apparatus, and overall were characterized by widespread inflammation with infiltration of myeloid cells. Gene expression analysis suggested an interferon type I signature, with increased expression of interferon regulatory factors. While apoptosis of cardiomyocytes was only increased at later stages, their proliferation was decreased early on, providing an explanation for the disturbed compact zone formation. Mechanistically, this could be explained by activation of the JAK/STAT axis and increased expression of the cell cycle inhibitor p21. A rescue experiment with an IκBα superrepressor demonstrated that the phenotype was dependent on NF-κB. We conclude that activation of NF-κB is detrimental during normal heart development due to excessive activation of pro-inflammatory pathways.

Long-term IKK2/NF-κB signaling in pancreatic ß-cells induces immune-mediated diabetes.

Type 1 diabetes is a multifactorial inflammatory disease in genetically susceptible individuals characterized by progressive autoimmune destruction of pancreatic ß-cells initiated by yet unknown factors. Although animal models of type 1 diabetes have substantially increased our understanding of disease pathogenesis, heterogeneity seen in human patients cannot be reflected by a single model and calls for additional models covering different aspects of human pathophysiology. Inhibitor of κB kinase (IKK)/nuclear factor-κB (NF-κB) signaling is a master regulator of inflammation; however, its role in diabetes pathogenesis is controversially discussed by studies using different inhibition approaches. To investigate the potential diabetogenic effects of NF-κB in ß-cells, we generated a gain-of-function model allowing conditional IKK2/NF-κB activation in ß-cells. A transgenic mouse model that expresses a constitutively active mutant of human IKK2 dependent on Pdx-1 promoter activity (IKK2-CA(Pdx-1)) spontaneously develops full-blown immune-mediated diabetes with insulitis, hyperglycemia, and hypoinsulinemia. Disease development involves a gene expression program mimicking virus-induced diabetes and allergic inflammatory responses as well as increased major histocompatibility complex class I/II expression by ß-cells that could collectively promote diabetes development. Potential novel diabetes candidate genes were also identified. Interestingly, animals successfully recovered from diabetes upon transgene inactivation. Our data give the first direct evidence that ß-cell-specific IKK2/NF-κB activation is a potential trigger of immune-mediated diabetes. Moreover, IKK2-CA(Pdx-1) mice provide a novel tool for studying critical checkpoints in diabetes pathogenesis and mechanisms governing ß-cell degeneration/regeneration.

MyD88 is involved in myeloid as well as lymphoid hematopoiesis independent of the presence of a pathogen.

MyD88 was originally described as a primary response gene up-regulated during myeloid differentiation after IL-6 induction. Later, MyD88 was shown to be a key molecule necessary for IL1, IL18 and Toll-like receptor signaling. Since these receptors recognize abundantly produced cytokines during infection or molecular patterns of pathogens, MyD88 itself was suggested to be an important regulator of the first line of defense against invading pathogens, including the differentiation and maturation of myeloid cells. Here we describe that MyD88 is important for early and late hematopoietic events that occur independently of antigen under steady-state conditions. In MyD88-deficient mice the earliest alteration in hematopoiesis was found at the level of long-term hematopoietic stem cells. Moreover, we found that MyD88 influences not only the development of the myeloid lineage but also the differentiation of B cells. The B cell defect observed in Btk-deficient mice is further enhanced when both molecules, Btk and MyD88, are not expressed. Therefore, MyD88 affects myeloid as well as lymphoid hematopoiesis. Since Btk and MyD88 deficiencies influence differentially myeloid and lymphoid development, both molecules seem to act in different signaling pathways important for appropriate developmental events during myelo- and lymphopoiesis.

C2-streptavidin mediates the delivery of biotin-conjugated tumor suppressor protein p53 into tumor cells.

We have previously generated a recombinant C2-streptavidin fusion protein for the delivery of biotin-labeled molecules of low molecular weight into the cytosol of mammalian cells. A nontoxic moiety of Clostridium botulinum C2 toxin mediates the cellular uptake, whereas the streptavidin unit serves as a binding platform for biotin-labeled cargo molecules. In the present study, we used the C2-streptavidin transporter to introduce biotin-conjugated p53 protein into various mammalian cell lines. The p53 tumor suppressor protein is inactivated in many human cancers by multiple mechanisms and therefore the restoration of its activity in tumor cells is of great therapeutic interest. Recombinant p53 was expressed in insect cells and biotin-labeled. Biotin-p53 retained its specific high-affinity DNA-binding as revealed by gel-shift analysis. Successful conjugation of biotin-p53 to the C2-streptavidin transporter was monitored by an overlay blot technique and confirmed by real-time surface plasmon resonance, providing a KD-value in the low nM range. C2-streptavidin significantly enhanced the uptake of biotin-p53 into African Green Monkey (Vero) epithelial cells as shown by flow cytometry. Using cell fractionation, the cytosolic translocation of biotin-p53 was detected in Vero cells as well as in HeLa cervix carcinoma cells. In line with this finding, confocal microscopy displayed cytoplasmic staining of biotin-p53 in HeLa and HL60 leukemia cells. Internalized biotin-p53 partially colocalized with early endosomes, as confirmed by confocal microscopy. In conclusion, our results demonstrate the successful conjugation of biotin-p53 to C2-streptavidin and its subsequent receptor-mediated endocytosis into different human tumor cell lines.

Octamer-dependent transcription in T cells is mediated by NFAT and NF-κB.

The transcriptional co-activator BOB.1/OBF.1 was originally identified in B cells and is constitutively expressed throughout B cell development. BOB.1/OBF.1 associates with the transcription factors Oct1 and Oct2, thereby enhancing octamer-dependent transcription. In contrast, in T cells, BOB.1/OBF.1 expression is inducible by treatment of cells with PMA/Ionomycin or by antigen receptor engagement, indicating a marked difference in the regulation of BOB.1/OBF.1 expression in B versus T cells. The molecular mechanisms underlying the differential expression of BOB.1/OBF.1 in T and B cells remain largely unknown. Therefore, the present study focuses on mechanisms controlling the transcriptional regulation of BOB.1/OBF.1 and Oct2 in T cells. We show that both calcineurin- and NF-κB-inhibitors efficiently attenuate the expression of BOB.1/OBF.1 and Oct2 in T cells. In silico analyses of the BOB.1/OBF.1 promoter revealed the presence of previously unappreciated combined NFAT/NF-κB sites. An array of genetic and biochemical analyses illustrates the involvement of the Ca(2+)/calmodulin-dependent phosphatase calcineurin as well as NFAT and NF-κB transcription factors in the transcriptional regulation of octamer-dependent transcription in T cells. Conclusively, impaired expression of BOB.1/OBF.1 and Oct2 and therefore a hampered octamer-dependent transcription may participate in T cell-mediated immunodeficiency caused by the deletion of NFAT or NF-κB transcription factors.

Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation.

UNLABELLED: Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-κB (NF-κB) system is activated in response to several of these stresses, we hypothesized that NF-κB activation in hepatocytes may contribute to fibrosis development. To activate the NF-κB signaling pathway in a time- and cell-type-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-κB-induced liver fibrosis in a liver-injury-independent manner. CONCLUSION: Our results revealed that hepatic activation of IKK/NF-κB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases.

The role of transcription factors in the guidance of granulopoiesis.

In recent years, the prospective isolation of hematopoietic stem and progenitor cells has identified the hierarchical structure of hematopoietic development and lineage-commitment. Moreover, these isolated cell populations allowed the elucitation of the molecular mechansims associated with lineage choice and revealed the indispensable functions of transcription factors as lineage determinants. This review summarizes current concepts regarding adult murine granulopoiesis and illustrates the importance of the transcription factors C/EBPα, PU.1 and GATA-2 for the development of neutrophil, eosinophil and basophil granulocytes.

Neutrophil development and function critically depend on Bruton tyrosine kinase in a mouse model of X-linked agammaglobulinemia.

Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice, the development of granulocytes is favored at the expense of monocytes. However, Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras, we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils, Btk plays a role in GM-CSF- and Toll-like receptor-induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα, C/EBPß, and PU.1, depends on Btk. In addition, expression of several granule proteins, including myeloperoxidase, neutrophilic granule protein, gelatinase and neutrophil elastase, is Btk-dependent. In the Arthus reaction, an acute inflammatory response, neutrophil migration into tissues, edema formation, and hemorrhage are significantly reduced in Btk-deficient animals. Together, our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo.

MYC stimulates EZH2 expression by repression of its negative regulator miR-26a.

The MYC oncogene, which is commonly mutated/amplified in tumors, represents an important regulator of cell growth because of its ability to induce both proliferation and apoptosis. Recent evidence links MYC to altered miRNA expression, thereby suggesting that MYC-regulated miRNAs might contribute to tumorigenesis. To further analyze the impact of MYC-regulated miRNAs, we investigated a murine lymphoma model harboring the MYC transgene in a Tet-off system to control its expression. Microarray-based miRNA expression profiling revealed both known and novel MYC targets. Among the miRNAs repressed by MYC, we identified the potential tumor suppressor miR-26a, which possessed the ability to attenuate proliferation in MYC-dependent cells. Interestingly, miR-26a was also found to be deregulated in primary human Burkitt lymphoma samples, thereby probably being of clinical relevance. Although today only few miRNA targets have been identified in human disease, we could show that ectopic expression of miR-26a influenced cell cycle progression by targeting the bona fide oncogene EZH2, a Polycomb protein and global regulator of gene expression yet unknown to be regulated by miRNAs. Thus, in addition to directly targeting protein-coding genes, MYC modulates genes important to oncogenesis via deregulation of miRNAs, thereby vitally contributing to MYC-induced lymphomagenesis.