Kai Hoehlig

A novel C5a-neutralizing mirror-image (l-)aptamer prevents organ failure and improves survival in experimental sepsis.

Complement factor C5a is a potent proinflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Here, we describe the discovery of NOX-D20, a PEGylated biostable mirror-image mixed (l-)RNA/DNA aptamer (Spiegelmer) that binds to mouse and human C5a with picomolar affinity. In vitro, NOX-D20 inhibited C5a-induced chemotaxis of a CD88-expressing cell line and efficiently antagonized the activation of primary human polymorphonuclear leukocytes (PMN) by C5a. Binding of NOX-D20 to the C5a moiety of human C5 did not interfere with the formation of the terminal membrane attack complex (MAC). In sepsis, for which a specific interventional therapy is currently lacking, complement activation and elevated levels of C5a are suggested to contribute to multiorgan failure and mortality. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), NOX-D20 attenuated inflammation and organ damage, prevented the breakdown of the vascular endothelial barrier, and improved survival. Our study suggests NOX-D20 as a new therapeutic candidate for the treatment of sepsis.

Structural basis for the targeting of complement anaphylatoxin C5a using a mixed L-RNA/L-DNA aptamer

L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases. The anaphylatoxin C5a, a potent inflammatory mediator generated during complement activation that has been implicated with organ damage, can be efficiently targeted by Spiegelmers. Here, we present the first crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg. The structures reveal a complex 3D architecture for the L-aptamer that wraps around C5a, including an intramolecular G-quadruplex stabilized by a central Ca2+ ion. Functional validation of the observed L-aptamer:C5a binding mode through mutational studies also rationalizes the specificity of NOX-D20 for mouse and human C5a against macaque and rat C5a. Finally, our structural model provides the molecular basis for the Spiegelmer affinity improvement through positional L-ribonucleotide to L-deoxyribonucleotide exchanges and for its inhibition of the C5a:C5aR interaction.

A Combined PD-1/C5a Blockade Synergistically Protects Against Lung Cancer Growth and Metastasis

Disruption of the programmed cell death protein 1 (PD-1) pathway with immune checkpoint inhibitors represents a major breakthrough in the treatment of non-small cell lung cancer. We hypothesized that combined inhibition of C5a/C5aR1 and PD-1 signaling may have a synergistic antitumor effect. The RMP1-14 antibody was used to block PD-1, and an L-aptamer was used to inhibit signaling of complement C5a with its receptors. Using syngeneic models of lung cancer, we demonstrate that the combination of C5a and PD-1 blockade markedly reduces tumor growth and metastasis and leads to prolonged survival. This effect is accompanied by a negative association between the frequency of CD8 T cells and myeloid-derived suppressor cells within tumors, which may result in a more complete reversal of CD8 T-cell exhaustion. Our study provides support for the clinical evaluation of anti-PD-1 and anti-C5a drugs as a novel combination therapeutic strategy for lung cancer.Significance: Using a variety of preclinical models of lung cancer, we demonstrate that the blockade of C5a results in a substantial improvement in the efficacy of anti-PD-1 antibodies against lung cancer growth and metastasis. This study provides the preclinical rationale for the combined blockade of PD-1/PD-L1 and C5a to restore antitumor immune responses, inhibit tumor cell growth, and improve outcomes of patients with lung cancer. Cancer Discov; 7(7); 694-703. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 653.

Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis.

The sphingolipid S1P (sphingosine 1-phosphate) is known to be involved in a number of pathophysiological conditions such as cancer, autoimmune diseases and fibrosis. It acts extracellularly through a set of five G-protein-coupled receptors, but its intracellular actions are also well documented. Employing in vitro selection techniques, we identified an L-aptamer (Spiegelmer®) to S1P designated NOX-S93. The binding affinity of NOX-S93 to S1P had a Kd value of 4.3 nM. The Spiegelmer® shows equal binding to dihydro-S1P, but no cross-reactivity to the related lipids sphingosine, lysophosphatidic acid, ceramide, ceramide-1-phosphate or sphingosine phosphocholine. In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated β-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range. The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells. These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.

Increasing Tumor-Infiltrating T Cells through Inhibition of CXCL12 with NOX-A12 Synergizes with PD-1 Blockade

Immune checkpoint inhibitors benefit only some patients, perhaps due to exclusion of CTLs by the tumor microenvironment through CXCL12. Treatment with the CXCL12 inhibitor NOX-A12 enhanced infiltration of immune cells and overcame resistance to anti–PD-1 in a murine model. Immune checkpoint inhibitors promote T cell–mediated killing of cancer cells; however, only a subset of patients benefit from the treatment. A possible reason for this limitation may be that the tumor microenvironment (TME) is immune privileged, which may exclude cytotoxic T cells from the vicinity of cancer cells. The chemokine CXCL12 is key to the TME-driven immune suppression. In this study, we investigated the potential of CXCL12 inhibition by use of the clinical-stage l-RNA-aptamer NOX-A12 (olaptesed pegol) to increase the number of tumor-infiltrating lymphocytes. We used heterotypic tumor–stroma spheroids that mimic a solid tumor with a CXCL12-abundant TME. NOX-A12 enhanced the infiltration of T and NK cells in a dose-dependent manner. NOX-A12 and PD-1 checkpoint inhibition synergistically activated T cells in the spheroids, indicating that the agents complement each other. The findings were validated in vivo in a syngeneic murine model of colorectal cancer in which the addition of NOX-A12 improved anti–PD-1 therapy. Taken together, our work shows that CXCL12 inhibition can break the immune-privileged status of the TME by paving the way for immune effector cells to enter into the tumor, thereby broadening the applicability of checkpoint inhibitors in cancer patients. Cancer Immunol Res; 5(11); 950–6. ©2017 AACR.

Stereospecificity of Oligonucleotide Interactions Revisited: No Evidence for Heterochiral Hybridization and Ribozyme/DNAzyme Activity

A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.

Signaling of the Complement Cleavage Product Anaphylatoxin C5a Through C5aR (CD88) Contributes to Pharmacological Hematopoietic Stem Cell Mobilization

Several mechanisms have been postulated for orchestrating the mobilization of hematopoietic stem/progenitor cells (HSPCs), and we previously proposed that activation of the complement cascade plays a crucial role in the initiation and execution of the egress of HSPCs from bone marrow (BM) into peripheral blood (PB). In support of this notion, we demonstrated that mice deficient in the mannan-binding lectin (MBL) pathway, which activates the proximal part of the complement cascade, as well as mice deficient in the fifth component of the complement cascade (C5), which is part of the distal part of the complement cascade, are poor mobilizers. To further narrow down on the exact mechanisms and the molecules involved, we performed studies in mice that do not express the receptor C5aR, which binds the C5 cleavage fragments, C5a and C5adesArg. We also employed the plasma stable nucleic acid aptamer AON-D21 that binds and neutralizes C5a and C5adesArg. We present evidence that mice deficient in C5aR or treated with AON-D21 are poor HSPC mobilizers, thereby establishing a critical role for the C5a/C5adesArg–C5aR axis in the mobilization process. While enhancing mobilization is of clinical importance for poor mobilizers, inhibition of the complement cascade could be of therapeutic importance in patients suffering from paroxysmal nocturnal hemoglobinuria (PNH) or acquired hemolytic syndrome (aHUS).

Structural basis for inhibition of complement C5a by an L-RNA aptamer

Complement is a central component of innate immunity providing a first line of defense against invading pathogens. It also bridges the innate and adaptive immunity, initiates the inflammatory response, and participates in immune surveillance. The anaphylatoxin C5a, generated during complement activation, is a potent inflammatory mediator which induces chemotaxis, oxidative burst, histamine release and increased vasodilatation, through G-protein coupled receptor signaling. Although inflammation is an integral part of the healing process following tissue damage and infection, excessive levels of C5a correlate with the onset of various inflammatory disorders including sepsis, rheumatoid arthritis, acute lung injury, ischemia-reperfusion injury, allergy, transplantation and asthma. Therapeutical targeting of the C5a:receptor axis is considered a promising strategy to down-regulate complementmediated inflammation. The L-aptamer NOX-D20, fully composed of non-natural mirror-image nucleotides (a so called Spiegelmer), has been identified as a potent C5a inhibitor. NOX-D20 has already shown encouraging efficacy in an experimental model of sepsis [1]. Here, we present the first crystallographic structure of an active Spiegelmer®, NOX-D20, bound to its physiological target, the mouse C5a anaphylatoxin, determined at 1.8 Å resolution. The structure reveals a complex 3D-architecture for the L-RNA molecule that wraps around C5a, including an intramolecular G-quadruplex stabilized by a Ca2+ ion as validated through anomalous diffraction data. The aptamer:C5a binding mode observed in the structure was validated through mutational studies using SPR. Our structure provides a molecular basis for NOX-D20 inhibitory properties and allows us to rationalize NOX-D20 selectivity towards human and mouse C5a