Regina Heidenreich

Microtumor growth initiates angiogenic sprouting with simultaneous expression of VEGF, VEGF receptor-2, and angiopoietin-2

Tumors have been thought to initiate as avascular aggregates of malignant cells that only later induce vascularization. Recently, this classic concept of tumor angiogenesis has been challenged by the suggestion that tumor cells grow by co-opting preexisting host vessels and thus initiate as well-vascularized tumors without triggering angiogenesis. To discriminate between these two mechanisms, we have used intravital epifluorescence microscopy and multi-photon laser scanning confocal microscopy to visualize C6 microglioma vascularization and tumor cell behavior. To address the mechanisms underlying tumor initiation, we assessed the expression of VEGF, VEGF receptor-2 (VEGFR-2), and angiopoietin-2 (Ang-2), as well as endothelial cell proliferation. We show that multicellular aggregates (<< 1 mm(3)) initiate vascular growth by angiogenic sprouting via the simultaneous expression of VEGFR-2 and Ang-2 by host and tumor endothelium. Host blood vessels are not co-opted by tumor cells but rather are used as trails for tumor cell invasion of the host tissue. Our data further suggest that the established microvasculature of growing tumors is characterized by a continuous vascular remodeling, putatively mediated by the expression of VEGF and Ang-2. The results of this study suggest a new concept of vascular tumor initiation that may have important implications for the clinical application of antiangiogenic strategies.

Design, synthesis, and biological evaluation of novel 3-aryl-4-(1H-indole-3yl)-1,5-dihydro-2H-pyrrole-2-ones as vascular endothelial growth factor receptor (VEGF-R) inhibitors.

In this study we report on the design, synthesis, and biological evaluation of pyrrole-2-one 2 to be a highly potent VEGF-R2/3 inhibitor with IC 50 of 31/37 nM. The novel 3,4-diaryl-2 H-pyrrole-2-ones were designed on the basis of the modeled binding mode of the corresponding 1 H-pyrrole-2,5-dione (maleimide) VEGF-R2/3 inhibitor 1 indicating two H-bond ligand-protein interactions in the ATP pocket for the amide 2 but not for the isomer 3. Flexible synthetic routes to 3,4-diaryl-2 H-pyrrole-2-ones and structure-activity relationships for the compounds in a panel of 24 therapeutically relevant protein kinases (IC 50 values) are presented. Accordingly to the in vitro data, compounds 1 and 2 were found to possess highly potent antiangiogenic activities in the cellular HLMEC sprouting assay and also slightly induced apoptosis in HDMECs whereas 3 was determined to be significantly less active. Hence, the pyrrole-2-one moiety was dissected from the corresponding maleimide protein kinase inhibitor as a suitable key pharmacophore.

EpCAM, a human tumor-associated antigen promotes Th2 development and tumor immune evasion

Experimental tumor vaccination and adoptive T-cell therapies show that interferon-gamma (IFN-gamma)-producing CD4(+) T helper cells (Th1) can be highly effective in tumor prevention and therapy. Unexpectedly, first vaccine trials in humans revealed that tumor immune therapy may not only be protective, but, on the contrary, even promote tumor progression. Here, we analyzed T-cell immune responses to the epithelial cell adhesion molecule (EpCAM), one of the most common tumor-associated antigens (TAA) serving as immune target in colon cancer patients. Th-cell priming against EpCAM inevitably resulted in interleukin-4 (IL-4)-dominated Th2 responses, even under most stringent Th1-inducing conditions. These EpCAM-reactive Th2 cells rather promoted growth of EpCAM-expressing tumors. To analyze the role of IL-4 in tumor immune evasion, we generated EpCAM-reactive Th1 cells from IL-4.ko mice. These Th1 cells provided tumor-specific protection and established highly protective Th1 memory responses, even in naive BALB/c mice. Inhibition of tumor growth by Th1 cells resulted in intra-tumoral expression of cytokines of the IL-12 family and of IFN-gamma. Preventing activation-associated death of Th1 cells further increased intratumoral IFN-gamma expression and improved therapeutic efficacy. Thus, human TAA may promote tumor immune evasion by strongly favoring Th2 development.

Inhibition of solid tumor growth by gene transfer of VEGF receptor‐1 mutants

Vascular endothelial growth factor (VEGF) and the high‐affinity VEGF receptor Flk‐1/KDR (VEGFR‐2) are key regulators of tumor angiogenesis. Strategies to block VEGF/VEGFR‐2 signaling were successfully used to inhibit experimental tumor growth and indicated that VEGFR‐2 is the main signaling VEGF receptor in proliferating tumor endothelium. Here, we investigated the role of the VEGF receptor‐1 (VEGFR‐1/Flt‐1) in the vascularization of 2 different experimental tumors in vivo. VEGFR‐1 mutants were generated that lack the intracellular tyrosine kinase domain. Retrovirus‐mediated gene transfer of the VEGFR‐1 mutants led to a strong reduction of tumor growth and angiogenesis in xenografted C6 glioma and in syngeneic BFS‐1 fibrosarcoma. Histological analysis of the inhibited fibrosarcoma revealed reduced vascular density, decreased tumor cell proliferation as well as increased tumor cell apoptosis and the formation of necrosis. The retroviral gene transfer of the full length VEGFR‐1 also caused a significant reduction of tumor growth in both models. The inhibitory effects of the VEGFR‐1 mutants and the full length VEGFR‐1 in BFS‐1 fibrosarcoma were mediated through host tumor endothelial cells because the BFS‐1 fibrosarcoma cells were not infected by the retrovirus. The formation of heterodimers between VEGFR‐2 and full length or truncated VEGFR‐1 was observed in vitro and might contribute to the growth inhibitory effect by modulating distinct signal transduction pathways. The results of our study underline the central role of the VEGF/VEGFR‐1 signaling system in tumor angiogenesis and demonstrate that VEGFR‐1 can serve as a target for anti‐angiogenic gene therapy.

Simultaneous blockade of VEGFR-1 and VEGFR-2 activation is necessary to efficiently inhibit experimental melanoma growth and metastasis formation

Metastasis continues to be the major cause of morbidity and mortality in malignant melanoma. In our study, we explored whether inhibition of VEGFR‐1 or VEGFR‐2 signaling conveys distinct suppressive effects on B16 melanoma subcutaneous growth and metastasis formation. The inhibition of VEGFR‐1 or ‐2 alone had no significant influence on both melanoma growth and metastasis formation. In contrast, simultaneous blockade of VEGFR‐1 and ‐2 signaling strongly suppressed progression in both B16 tumor models. There was no expression of VEGFR‐1 or ‐2 detectable on the B16 cells used, excluding the melanoma cells as direct therapeutic targets. Analyzing the contribution of progenitor‐like cells during melanoma metastasis formation, we observed an enhanced proliferation and mobilization of VEGFR‐1+ myeloid and VEGFR‐2+ endothelial cells with progenitor potential by the induction of melanoma lung metastasis, which was not influenced by interference with VEGFR signaling. These results indicate that the antimetastatic effects exerted by combined inhibition of VEGFR‐1 and ‐2 signaling were mediated via targeting cell populations other than progenitors only. Sole inhibition of VEGFR‐1 signaling led to a strong reduction of the CD45‐positive inflammatory infiltrate in the tumor tissue. However, the formation of lung metastasis was not affected, indicating that inhibition of the inflammatory response was not sufficient to efficiently block B16 melanoma metastasis development. Taken together, our data suggest that in the utilized B16 tumor models the blockade of both the inflammatory and the VEGFR‐2‐dependent angiogenic response are necessary to effectively inhibit solid tumor growth and formation of lung metastasis by B16 melanoma cells.

A novel RNA-based adjuvant combines strong immunostimulatory capacities with a favorable safety profile

Protein‐ and peptide‐based tumor vaccines depend on strong adjuvants to induce potent immune responses. Here, we demonstrated that a recently developed novel adjuvant based on a non‐coding, long‐chain RNA molecule, termed RNAdjuvant®, profoundly increased immunogenicity of both antigen formats. RNAdjuvant® induced balanced, long‐lasting immune responses that resulted in a strong anti‐tumor activity. A direct comparison to Poly(I:C) showed superior efficacy of our adjuvant to enhance antigen‐specific multifunctional CD8+ T‐cell responses and mediate anti‐tumor responses induced by peptide derived from HPV‐16 E7 protein in the syngeneic TC‐1 tumor, a murine model of human HPV‐induced cervical cancer. Moreover, the adjuvant was able to induce functional memory responses that mediated complete tumor remission. Despite its remarkable immunostimulatory activity, our RNA‐based adjuvant exhibited an excellent pre‐clinical safety profile. It acted only locally at the injection site where it elicited a transient but strong up‐regulation of pro‐inflammatory and anti‐viral cytokines as well as cytoplasmic RNA sensors without systemic cytokine release. This was followed by the activation of immune cells in the draining lymph nodes. Our data indicate that our RNA‐based adjuvant is a safe and potent immunostimulator that may profoundly improve the efficacy of a variety of cancer vaccines.

Self-adjuvanted mRNA vaccines induce local innate immune responses that lead to a potent and boostable adaptive immunity.

mRNA represents a new platform for the development of therapeutic and prophylactic vaccines with high flexibility with respect to production and application. We have previously shown that our two component self-adjuvanted mRNA-based vaccines (termed RNActive® vaccines) induce balanced immune responses comprising both humoral and cellular effector as well as memory responses. Here, we evaluated the early events upon intradermal application to gain more detailed insights into the underlying mode of action of our mRNA-based vaccine. We showed that the vaccine is taken up in the skin by both non-leukocytic and leukocytic cells, the latter being mostly represented by antigen presenting cells (APCs). mRNA was then transported to the draining lymph nodes (dLNs) by migratory dendritic cells. Moreover, the encoded protein was expressed and efficiently presented by APCs within the dLNs as shown by T cell proliferation and immune cell activation, followed by the induction of the adaptive immunity. Importantly, the immunostimulation was limited to the injection site and lymphoid organs as no proinflammatory cytokines were detected in the sera of the immunized mice indicating a favorable safety profile of the mRNA-based vaccines. Notably, a substantial boostability of the immune responses was observed, indicating that mRNA can be used effectively in repetitive immunization schedules. The evaluation of the immunostimulation following prime and boost vaccination revealed no signs of exhaustion as demonstrated by comparable levels of cytokine production at the injection site and immune cell activation within dLNs. In summary, our data provide mechanistic insight into the mode of action and a rational for the use of mRNA-based vaccines as a promising immunization platform.

Tracking Adult Neovascularization during Ischemia and Inflammation Using Vegfr2-LacZ Reporter Mice

The vascular endothelial growth factor/vascular endothelial growth factor receptor 2 (VEGF/VEGFR-2) signal transduction system plays a key role during embryonic vascular development and adult neovascularization. In contrast to many endothelial genes, VEGFR-2 is expressed at low levels in most adult vessels but is strongly upregulated during neovascularization, leading to a pro-angiogenic response. Here, we analyzed the activity of regulatory sequences of the murine Vegfr2 gene during neovessel formation in vivo under ischemic and inflammatory conditions. Hindlimb ischemia was induced in transgenic mice, expressing the LacZ reporter gene under the control of Vegfr2 promoter/enhancer elements. Most vessels in the ischemic muscle tissue showed strong endothelium-specific reporter gene expression, whereas nearly no LacZ-expressing capillaries were observed in untreated control tissue. Cutaneous punch wounds were created to induce angiogenesis under inflammatory conditions, leading to robust LacZ expression in the majority of the blood vessels in the wound tissue. Since the cornea is physiologically avascular, the functionality of these promoter/enhancer elements exclusively in newly formed vessels was confirmed using the cornea micropocket assay. Taken together, our results show that these Vegfr2 regulatory elements are active during adult neovessel formation in general. Therefore, these sequences may prove to be valuable targets for novel endothelium-specific anti-angiogenic as well as pro-angiogenic treatment strategies. They may especially allow directing therapeutic gene expression to sites of adult neovascularization. Moreover, the Vegfr2/LacZ reporter mice represent a powerful model to generally analyze the transcriptional control mechanisms involved in the induction of Vegfr2 expression during adult neovascularization.

RNActive® Technology: Generation and Testing of Stable and Immunogenic mRNA Vaccines

Developing effective mRNA vaccines poses certain challenges concerning mRNA stability and ability to induce sufficient immune stimulation and requires a specific panel of techniques for production and testing. Here, we describe the production of stabilized mRNA with enhanced immunogenicity, generated using conventional nucleotides only, by introducing changes to the mRNA sequence and by complexation with the nucleotide-binding peptide protamine (RNActive® technology). Methods described here include the synthesis, purification, and protamine complexation of mRNA vaccines as well as a comprehensive panel of in vitro and in vivo methods for evaluation of vaccine quality and immunogenicity.

Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines

AbstractmRNA represents a promising new vaccine technology platform with high flexibility in regard to development and production. Here, we demonstrate that vaccines based on sequence optimized, chemically unmodified mRNA formulated in optimized lipid nanoparticles (LNPs) are highly immunogenic and well tolerated in non-human primates (NHPs). Single intramuscular vaccination of NHPs with LNP-formulated mRNAs encoding rabies or influenza antigens induced protective antibody titers, which could be boosted and remained stable during an observation period of up to 1 year. First mechanistic insights into the mode of action of the LNP-formulated mRNA vaccines demonstrated a strong activation of the innate immune response at the injection site and in the draining lymph nodes (dLNs). Activation of the innate immune system was reflected by a transient induction of pro-inflammatory cytokines and chemokines and activation of the majority of immune cells in the dLNs. Notably, our data demonstrate that mRNA vaccines can compete with licensed vaccines based on inactivated virus or are even superior in respect of functional antibody and T cell responses. Importantly, we show that the developed LNP-formulated mRNA vaccines can be used as a vaccination platform allowing multiple, sequential vaccinations against different pathogens. These results provide strong evidence that the mRNA technology is a valid approach for the development of effective prophylactic vaccines to prevent infectious diseases.Vaccine technology: Repurposing the genetic messengerVaccines based on mRNA provoke strong immune responses after a single dose. mRNA is commonly known as the ‘genetic messenger’ cousin of DNA and a crucial mediator of protein production. Now, research led by Mariola Fotin-Mleczek, of Germany’s CureVac AG, demonstrates that mRNA can be developed to produce virus fragments, called antigens, that can prime a vaccinee’s immune system against a pathogen. Testing their vaccine platform, the team created mRNA coding for rabies and influenza antigens, and used intramuscular injection to inoculate non-human primates. A single dose elicited strong immune responses, which the team then successfully maintained through booster vaccinations for an observation period of 1 year. The responses outperformed those of licensed vaccines against rabies and influenza type H3N2. This research shows that mRNA has promise as a versatile, cost-effective, rapidly scalable vaccine technology.