Pharmacodynamic and Antitumor Activity of BI 836880, a Dual Vascular Endothelial Growth Factor and Angiopoietin 2 Inhibitor, Alone and Combined with Programmed Cell Death Protein-1 Inhibition.

Vascular endothelial growth factor (VEGF) and angiopoietin (ANG)-2 have complementary roles in angiogenesis and promote an immunosuppressive tumor microenvironment. It is anticipated that the combination of VEGF and ANG2 blockade could provide superior activity to the blockade of either pathway alone and that the addition of VEGF/ANG2 inhibition to an anti-programmed cell death protein-1 (PD-1) antibody could change the tumor microenvironment to support T-cell-mediated tumor cytotoxicity. Here, we describe the pharmacologic and antitumor activity of BI 836880, a humanized bispecific nanobody comprising two single-variable domains blocking VEGF and ANG2, and an additional module for half-life extension in vivo. BI 836880 demonstrated high affinity and selectivity for human VEGF-A and ANG2, resulting in inhibition of the downstream signaling of VEGF/ANG2 and a decrease in endothelial cell proliferation and survival. In vivo, BI 836880 exhibited significant antitumor activity in all patient-derived xenograft models tested, showing significantly greater tumor growth inhibition (TGI) than bevacizumab (VEGF inhibition) and AMG386 (ANG1/2 inhibition) in a range of models. In a Lewis lung carcinoma syngeneic tumor model, the combination of PD-1 inhibition with VEGF inhibition showed superior efficacy versus the blockade of either pathway alone. TGI was further increased with the addition of ANG2 inhibition to VEGF/PD-1 blockade. VEGF/ANG2 inhibition had a strong antiangiogenic effect. Our data suggest that the blockade of VEGF and ANG2 with BI 836880 may offer improved antitumor activity versus the blockade of either pathway alone and that combining VEGF/ANG2 inhibition with PD-1 blockade can further enhance antitumor effects. SIGNIFICANCE STATEMENT: Vascular endothelial growth factor (VEGF) and angiopoietin (ANG)-2 play key roles in angiogenesis and have an immunosuppressive effect in the tumor microenvironment. This study shows that BI 836880, a bispecific nanobody targeting VEGF and ANG2, demonstrates substantial antitumor activity in preclinical models. Combining VEGF/ANG2 inhibition with the blockade of the PD-1 pathway can further improve antitumor activity.

Café-au-lait spots in neurofibromatosis type 1 and in healthy control individuals: hyperpigmentation of a different kind?

Solitary café-au-lait spots are quite common in the general population but multiple café-au-lait macules (CALM) are often indicative of an underlying genetic disorder. The frequency of having more than five CALM is rare in normal individuals and is therefore considered as a cut-off for the diagnosis of neurofibromatosis type 1 (NF1). The etiopathogenesis of these macules is still very obscure. In this study we compared epidermal melanocyte and dermal mast cell numbers between four groups: control normal and control CALM skin, and NF1 normal and NF1 CALM skin and elaborated a possible role for stem cell factor (SCF) in CALM formation. The groups were analyzed by immunohistochemistry for numerical analysis of the melanocyte and mast cell population and by ELISA, western blot analysis and real-time quantitative PCR for further determination of the role of SCF. We found a significant increase in melanocyte density in NF1 CALM skin compared with the isolated CALM in control individuals. However, both groups displayed a similar increase in mast cell density. In addition, we found increased levels of soluble SCF in NF1 CALM and in NF1 normal fibroblast supernatant. We conclude that SCF is an important cytokine in NF1 skin, but that additional (growth) factors and/or genetic mechanisms are needed to induce NF1-specific CALM hyperpigmentation.

Neurofibromatosis type 1 protein and amyloid precursor protein interact in normal human melanocytes and colocalize with melanosomes.

The neurofibromatosis type 1 (NF1) gene product, neurofibromin, is known to interact with Ras, thereby negatively regulating its growth-promoting function. Although this is a well-established interaction, the discovery of other neurofibromin interacting partners could reveal new functional properties of this large protein. Using yeast two-hybrid analysis against a brain cDNA library, we identified a novel interaction between the amyloid precursor protein and the GTPase activating protein-related domain of neurofibromin. This interaction was further analyzed in human melanocytes and confirmed by immunoprecipitation and colocalization studies. In addition, we observed a colocalization of amyloid precursor protein and neurofibromin with melanosomes. Amyloid precursor protein has been proposed to function as a vesicle cargo receptor for the motor protein kinesin-1 in neurons. This colocalization of amyloid precursor protein and neurofibromin with melanosomes was lost in melanocytes obtained from normal skin of a NF1 patient. We suggest that a complex between amyloid precursor protein, neurofibromin, and melanosomes might be important in melanosome transport, which could shed a new light on the etiopathogenesis of pigment-cell-related manifestations in NF1.

Gene expression profiling of cultured human NF1 heterozygous (NF1+/-) melanocytes reveals downregulation of a transcriptional cis-regulatory network mediating activation of the melanocyte-specific dopachrome tautomerase (DCT) gene.

One of the major primary features of the neurocutaneous genetic disorder Neurofibromatosis type 1 are the hyperpigmentary café-au-lait macules where disregulation of melanocyte biology is supposed to play a key etiopathogenic role. To gain better insight into the possible role of the tumor suppressor gene NF1, a transcriptomic microarray analysis was performed on human NF1 heterozygous (NF1+/-) melanocytes of a Neurofibromatosis type 1 patient and NF1 wild type (NF1+/+) melanocytes of a healthy control patient, both cultured from normally pigmented skin and hyperpigmented lesional café-au-lait skin. From the magnitude of gene effects, we found that gene expression was affected most strongly by genotype and less so by lesional type. A total of 137 genes had a significant twofold or more up- (72) or downregulated (65) expression in NF1+/- melanocytes compared with NF1+/+ melanocytes. Melanocytes cultured from hyperpigmented café-au-lait skin showed 37 upregulated genes whereas only 14 were downregulated compared with normal skin melanocytes. In addition, significant genotype xlesional type interactions were observed for 465 genes. Differentially expressed genes were mainly involved in regulating cell proliferation and cell adhesion. A high number of transcription factor genes, among which a specific subset important in melanocyte lineage development, were downregulated in the cis-regulatory network governing the activation of the melanocyte-specific dopachrome tautomerase (DCT) gene. Although the results presented have been obtained with a restricted number of patients (one NF1 patient and one control) and using cDNA microarrays that may limit their interpretation, the data nevertheless addresses for the first time the effect of a heterozygous NF1 gene on the expression of the human melanocyte transcriptome and has generated several interesting candidate genes helpful in elucidating the etiopathology of café-au-lait macules in NF1 patients.

Pigment cell-related manifestations in neurofibromatosis type 1: an overview.

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder, affecting approximately 1 in 3500 individuals. The most commonly seen tumors in NF1 patients are the (sub)cutaneous neurofibromas. However, individuals with NF1 typically present in childhood with well-defined pigmentary defects, including cafe-au-lait macules (CALMs), intertriginous freckling and iris Lisch nodules. NF1 is considered a neurocristopathy, primarily affecting tissues derived from the neural crest. Since the pigment producing melanocyte originates in the neural crest, the presence of (hyper)pigmentary lesions in the NF1 phenotype because of changes in melanocyte cell growth and differentiation is to be expected. We want to discuss the pigmentary cutaneous manifestations of NF1 represented by CALMs and intertriginous freckles and the pigmentary non-cutaneous manifestations represented by iris Lisch nodules. Several hypotheses have been suggested in explaining the poorly understood etiopathogenesis of CALMs. Whether other pigmentary manifestations might share similar etiopathogenic mechanisms remains obscure. Additional attention will be drawn to a readily seen phenomenon in NF1: hyperpigmentation overlying (plexiform) neurofibromas, which could suggest common etiopathogenetic-environmental cues or mechanisms underlying CALMs and neurofibromas. Finally, we want to address the relationship between malignant melanoma and NF1.