Combination of immune-checkpoint inhibitors and targeted therapies for melanoma therapy: The more, the better?

The approval of immune-checkpoint inhibitors (CPI) and mitogen activated protein kinase inhibitors (MAPKi) in recent years significantly improved the treatment management and survival of patients with advanced malignant melanoma. CPI aim to counter-act receptor-mediated inhibitory effects of tumor cells and immunomodulatory cell types on effector T cells, whereas MAPKi are intended to inhibit tumor cell survival. In agreement with these complementary modes of action preclinical data indicated that the combined application of CPI and MAPKi or their optimal sequencing might provide additional clinical benefit. In this review the rationale and preclinical evidence that support the combined application of MAPKi and CPI either in concurrent or consecutive regimens are presented. Further, we will discuss the results from clinical trials investigating the sequential or combined application of MAPKi and CPI for advanced melanoma patients and their implications for clinical practice. Finally, we outline mechanisms of MAPKi and CPI cross-resistance which limit the efficacy of currently available treatments, as well as combination regimens.

Immunomodulatory Properties of Immune Checkpoint Inhibitors-More than Boosting T-Cell Responses?

The approval of immune checkpoint inhibitors (ICI) that serve to enhance effector T-cell anti-tumor responses has strongly improved success rates in the treatment of metastatic melanoma and other tumor types. The currently approved ICI constitute monoclonal antibodies blocking cytotoxic T-lymphocyte-associated protein (CTLA)-4 and anti-programmed cell death (PD)-1. By this, the T-cell-inhibitory CTLA-4/CD80/86 and PD-1/PD-1L/2L signaling axes are inhibited. This leads to sustained effector T-cell activity and circumvents the immune evasion of tumor cells, which frequently upregulate PD-L1 expression and modulate immune checkpoint molecule expression on leukocytes. As a result, profound clinical responses are observed in 40-60% of metastatic melanoma patients. Despite the pivotal role of T effector cells for triggering anti-tumor immunity, mounting evidence indicates that ICI efficacy may also be attributable to other cell types than T effector cells. In particular, emerging research has shown that ICI also impacts innate immune cells, such as myeloid cells, natural killer cells and innate lymphoid cells, which may amplify tumoricidal functions beyond triggering T effector cells, and thus improves clinical efficacy. Effects of ICI on non-T cells may additionally explain, in part, the character and extent of adverse effects associated with treatment. Deeper knowledge of these effects is required to further develop ICI treatment in terms of responsiveness of patients to treatment, to overcome resistance to ICI and to alleviate adverse effects. In this review we give an overview into the currently known immunomodulatory effects of ICI treatment in immune cell types other than the T cell compartment.

Immunomodulatory Properties of BRAF and MEK Inhibitors Used for Melanoma Therapy-Paradoxical ERK Activation and Beyond.

The advent of mitogen-activated protein kinase (MAPK) inhibitors that directly inhibit tumor growth and of immune checkpoint inhibitors (ICI) that boost effector T cell responses have strongly improved the treatment of metastatic melanoma. In about half of all melanoma patients, tumor growth is driven by gain-of-function mutations of BRAF (v-rat fibrosarcoma (Raf) murine sarcoma viral oncogene homolog B), which results in constitutive ERK activation. Patients with a BRAF mutation are regularly treated with a combination of BRAF and MEK (MAPK/ERK kinase) inhibitors. Next to the antiproliferative effects of BRAF/MEKi, accumulating preclinical evidence suggests that BRAF/MEKi exert immunomodulatory functions such as paradoxical ERK activation as well as additional effects in non-tumor cells. In this review, we present the current knowledge on the immunomodulatory functions of BRAF/MEKi as well as the non-intended effects of ICI and discuss the potential synergistic effects of ICI and MAPK inhibitors in melanoma treatment.

Density of Conjugated Antibody Determines the Extent of Fc Receptor Dependent Capture of Nanoparticles by Liver Sinusoidal Endothelial Cells.

Despite considerable progress in the design of multifunctionalized nanoparticles (NPs) that selectively target specific cell types, their systemic application often results in unwanted liver accumulation. The exact mechanisms for this general observation are still unclear. Here we asked whether the number of cell-targeting antibodies per NP determines the extent of NP liver accumulation and also addressed the mechanisms by which antibody-coated NPs are retained in the liver. We used polysarcosine-based peptobrushes (PBs), which in an unmodified form remain in the circulation for >24 h due to the absence of a protein corona formation and low unspecific cell binding, and conjugated them with specific average numbers (2, 6, and 12) of antibodies specific for the dendritic cell (DC) surface receptor, DEC205. We assessed the time-dependent biodistribution of PB-antibody conjugates by in vivo imaging and flow cytometry. We observed that PB-antibody conjugates were trapped in the liver and that the extent of liver accumulation strongly increased with the number of attached antibodies. PB-antibody conjugates were selectively captured in the liver via Fc receptors (FcR) on liver sinusoidal endothelial cells, since systemic administration of FcR-blocking agents or the use of F(ab')2 fragments prevented liver accumulation. Cumulatively, our study demonstrates that liver endothelial cells play a yet scarcely acknowledged role in liver entrapment of antibody-coated NPs and that low antibody numbers on NPs and the use of F(ab')2 antibody fragments are both sufficient for cell type-specific targeting of secondary lymphoid organs and necessary to minimize unwanted liver accumulation.

Complement-Opsonized Nano-Carriers Are Bound by Dendritic Cells (DC) via Complement Receptor (CR)3, and by B Cell Subpopulations via CR-1/2, and Affect the Activation of DC and B-1 Cells.

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.

The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases.

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a "CPDCP-trunk" and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the "legumain-binding loop" of CY1 inhibit crayfish astacin following the "raised-elephant-trunk mechanism" recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and ß only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

Structure of mammalian plasma fetuin-B and its mechanism of selective metallopeptidase inhibition.

Mammalian fetuin-A and fetuin-B are abundant serum proteins with pleiotropic functions. Fetuin-B is a highly selective and potent inhibitor of metallo-peptidases (MPs) of the astacin family, which includes ovastacin in mammals. By inhibiting ovastacin, fetuin-B is essential for female fertility. The crystal structure of fetuin-B was determined unbound and in complex with archetypal astacin, and it was found that the inhibitor has tandem cystatin-type modules (CY1 and CY2). They are connected by an exposed linker with a rigid, disulfide-linked 'CPDCP-trunk', and are followed by a C-terminal region (CTR) with little regular secondary structure. The CPDCP-trunk and a hairpin of CY2 form a bipartite wedge, which slots into the active-site cleft of the MP. These elements occupy the nonprimed and primed sides of the cleft, respectively, but spare the specificity pocket so that the inhibitor is not cleaved. The aspartate in the trunk blocks the catalytic zinc of astacin, while the CY2 hairpin binds through a QWVXGP motif. The CY1 module assists in structural integrity and the CTR is not involved in inhibition, as verified by in vitro studies using a cohort of mutants and variants. Overall, the inhibition conforms to a novel 'raised-elephant-trunk' mechanism for MPs, which is reminiscent of single-domain cystatins that target cysteine peptidases. Over 200 sequences from vertebrates have been annotated as fetuin-B, underpinning its ubiquity and physiological relevance; accordingly, sequences with conserved CPDCP- and QWVXGP-derived motifs have been found from mammals to cartilaginous fishes. Thus, the raised-elephant-trunk mechanism is likely to be generally valid for the inhibition of astacins by orthologs of fetuin-B.

Mammalian plasma fetuin-B is a selective inhibitor of ovastacin and meprin metalloproteinases.

Vertebrate fetuins are multi-domain plasma-proteins of the cystatin-superfamily. Human fetuin-A is also known as AHSG, α2-Heremans-Schmid-glycoprotein. Gene-knockout in mice identified fetuin-A as essential for calcified-matrix-metabolism and bone-mineralization. Fetuin-B deficient mice, on the other hand, are female infertile due to zona pellucida 'hardening' caused by the metalloproteinase ovastacin in unfertilized oocytes. In wildtype mice fetuin-B inhibits the activity of ovastacin thus maintaining oocytes fertilizable. Here we asked, if fetuins affect further proteases as might be expected from their evolutionary relation to single-domain-cystatins, known as proteinase-inhibitors. We show that fetuin-A is not an inhibitor of any tested protease. In stark contrast, the closely related fetuin-B selectively inhibits astacin-metalloproteinases such as meprins and ovastacin, but not astacins of the tolloid-subfamily, nor any other proteinase. The analysis of fetuin-B expressed in various mammalian cell types, insect cells, and truncated fish-fetuin expressed in bacteria, showed that the cystatin-like domains alone are necessary and sufficient for inhibition. This report highlights fetuin-B as a specific antagonist of ovastacin and meprin-metalloproteinases. Control of ovastacin was shown to be indispensable for female fertility. Meprin inhibition, on the other hand, renders fetuin-B a potential key-player in proteolytic networks controlling angiogenesis, immune-defense, extracellular-matrix-assembly and general cell-signaling, with implications for inflammation, fibrosis, neurodegenerative disorders and cancer.