Blood vessel functionality is crucial for efficient tumor-targeted drug delivery. Heterogeneous distribution and perfusion of angiogenic blood vessels contribute to suboptimal accumulation of (nano-) therapeutics in tumors and metastases. To attenuate pathological angiogenesis, an L-RNA aptamer inhibiting the CC motif chemokine ligand 2 (CCL2) was administered to mice bearing orthotopic 4T1 triple-negative breast cancer tumors. The effect of CCL2 inhibition on tumor blood vessel functionality and tumor-targeted drug delivery was evaluated via multimodal and multiscale optical imaging, employing fluorophore-labeled polymeric (10 nm) and liposomal (100 nm) nanocarriers. Anti-CCL2 treatment induced a dose-dependent anti-angiogenic effect, reflected by a decreased relative blood volume, increased blood vessel maturity and functionality, and reduced macrophage infiltration, accompanied by a shift in the polarization of tumor-associated macrophages (TAM) towards a less M2-like and more M1-like phenotype. In line with this, CCL2 inhibitor treatment improved the delivery of polymers and liposomes to tumors, and enhanced the antitumor efficacy of free and liposomal doxorubicin. Together, these findings demonstrate that blocking the CCL2-CCR2 axis modulates TAM infiltration and polarization, resulting in vascular normalization and improved tumor-targeted drug delivery.

Multiple mechanisms promote tumor prosperity, which does not only depend on cell-autonomous, inherent abnormal characteristics of the malignant cells that facilitate rapid cell division and tumor expansion. The neoplastic tissue is embedded in a supportive and dynamic tumor microenvironment (TME) that nurtures and protects the malignant cells, maintaining and perpetuating malignant cell expansion. The TME consists of different elements, such as atypical vasculature, various innate and adaptive immune cells with immunosuppressive or pro-inflammatory properties, altered extracellular matrix (ECM), activated stromal cells, and a wide range of secreted/stroma-tethered bioactive molecules that contribute to malignancy, directly or indirectly. In this review, we describe the various TME components and provide examples of anti-cancer therapies and novel drugs under development that aim to target these components rather than the intrinsic processes within the malignant cells. Combinatory TME-modulating therapeutic strategies may be required to overcome the resistance to current treatment options and prevent tumor recurrence.

BACKGROUND:Immunotherapy in microsatellite stable colorectal or pancreatic cancer has not shown promising results. It has been hypothesized that targeting immunosuppressive molecules like SDF1-alpha/CXCL12 could contribute to immunotherapy and animal models showed promising results on T cell activation and migration in combination with immune checkpoint inhibition. METHODS:Here, we describe the successful application of anti-CXCL12 (NOX-A12) in patients with advanced stage pretreated metastatic colorectal and pancreatic cancer (OPERA trial). The treatment consisted of 2 weeks of anti-CXCL12 monotherapy with NOX-A12 followed by combination therapy with pembrolizumab (n=20 patients) until progression or intolerable toxicity had occurred. RESULTS:The treatment was safe and well tolerated with 83.8% grade I/II, 15.5% grade III and 0.7% grade V adverse events. Of note, for a majority of patients, time on trial treatment was prolonged compared with their last standard treatment preceding trial participation. Systematic serial biopsies revealed distinct patterns of modulation. Tissue and clinical responses were associated with Th1-like tissue reactivity upon CXCL12 inhibition. A downregulation of a cytokine cassette of interleukin (IL)-2/IL-16/CXCL-10 was associated with tumor resistance and furthermore linked to a rare, CXCL12-associated CD14+CD15+promonocytic population. T cells showed aggregation and directed movement towards the tumor cells in responding tissues. Serum analyses detected homogeneous immunomodulatory patterns in all patients, regardless of tissue responses. CONCLUSIONS:We demonstrate that the combination of CXCL12 inhibition and checkpoint inhibition is safe and grants further exploration of synergistic combinatorial strategies.

Abstract Background: NOX-A12 is an inhibitor of the chemokine CXCL12 which prevents engagement with its receptors CXCR4 and CXCR7 and blocks the formation of chemotactic CXCL12 concentration gradients in tissue. The Opera study (NCT03168139) is a Phase 1/2 study to evaluate pharmacodynamic effects and safety of monotherapy with NOX-A12 as well as safety and efficacy of a combination with pembrolizumab in metastatic microsatellite-stable colorectal and pancreatic cancer where PD-1 inhibition alone has not shown clinical benefit. Methods: Patients received 300 mg NOX-A12 twice weekly during a 2-week monotherapy phase. Biopsies were taken from liver metastases before and after NOX-A12 monotherapy for analysis of immune cell infiltration and cytokine signature. In a combination phase, patients received repeated 21-day cycles of 300 mg NOX-A12 and 200 mg pembrolizumab until progression or intolerable toxicity. Results: 11 of the patients had CRC and 9 PaC. All patients were heavily pretreated with 5 (colorectal cancer) and 3 median lines (pancreatic cancer) of prior treatment. Best responses to last prior treatment was progressive disease for 95% of patients. The AE profile was comparable with the pembrolizumab profile or typical for the underlying diseases. Two weeks of NOX-A12 monotherapy induced Th1 cytokines (IFNγ, IL-2, IL-16) in approx. half of the patients. Comparison of tumor biopsies from before and after NOX-A12 monotherapy shows trends towards (i) agglomeration of T cells and (ii) migration of T cells towards cancer cells in the patient sub-group where NOX-A12 induced a Th1-type cytokine response. These trends are consistent with preclinical data (Feig 2013, PNAS 110:20212; Zboralski 2017, Cancer Immunol Res 5:950), suggesting that neutralization of CXCL12 signaling - either through one or both receptors - increased infiltration of effector immune cells into tumor tissue and resulted in a more effective immune response. Although there were no objective responses, 25% of patients achieved stable disease; 7 patients showed prolonged time on treatment vs. the prior line of therapy. Median progression-free survival was 1.87 months, overall survival was 42% at 6 months and 22% at 12 months. Conclusions: A trend towards agglomeration of T cells within tumors was observed in about half of the patients where NOX-A12 had induced a Th1-type cytokine response. This agglomeration was accompanied by reduced distances between T cells and cancer cells, suggesting increased infiltration of effector immune cells into tumor tissue. Treatment with NOX-A12 plus pembrolizumab in the combination therapy part of the study resulted in stable disease in 25% of patients, and prolonged time on treatment vs. prior therapy for 35% of patients. The safety profile of the combination therapy was consistent with that of pembrolizumab in advanced cancer patients. Citation Format: Niels Halama, Anja Williams, Ulrike Prüfer, Anna Frömming, Diana Beyer, Dirk Eulberg, Jarl Ulf Jungnelius, Aram Mangasarian. Phase 1/2 study with CXCL12 inhibitor NOX-A12 and pembrolizumab in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT117.

BACKGROUND. NOX-A12 is an inhibitor of the chemokine CXCL12 for treatment of solid tumors. Binding of CXCL12 by NOX-A12 prevents receptor engagement and blocks the ability of CXCL12 to form a chemotactic concentration gradient. The Opera study (NCT03168139) is a Phase I/II study to evaluate pharmacodynamic effects and safety of monotherapy with NOX-A12 as well as safety and efficacy of a combination of NOX-A12 with pembrolizumab in metastatic microsatellite-stable colorectal (CRC) and pancreatic (PaC) cancer. METHODS. Patients received 300 mg NOX-A12 twice weekly during the two-week monotherapy phase. Biopsies were taken from liver metastases before treatment and after NOX-A12 monotherapy for analysis of immune cell infiltration and cytokine signature. In the combination phase, patients received repeated 21-day cycles of 300 mg NOX-A12 and 200 mg pembrolizumab until progression or intolerable toxicity. RESULTS. Recruitment and treatment of all patients is completed, follow-up is ongoing. Eleven of the patients had CRC and 9 PaC. Fifteen of the patients (75%) were male, with a median age of 62 (CRC) and 68 years (PaC). All patients were heavily pretreated with a median of 5 (CRC) and 3 lines (PaC) of prior treatment. Best responses to last prior treatment was progressive disease for all except one patient. The AE profile was comparable with the pembrolizumab profile or typical for the underlying diseases. Biopsies obtained at baseline showed that the mean T cell density at the invasive margin was 327 cells/mm², clearly below the 600 cells/mm² that are predictive for a good prognosis. However, 5 out of 20 patients (25%) achieved a stable disease, thereof 3 CRC and 2 PaC patients; 7 of the patients showed prolonged time on treatment versus the preceding line of therapy. Updated figures for progression-free survival and overall survival for the whole patient cohort will be presented. Additional characterization of immunosuppressive cells in tumor biopsies and the periphery is ongoing. CONCLUSIONS. In patients with microsatellite-stable metastatic pancreatic and colorectal cancer with impaired immune systems and a high tumor load that have failed multiple prior lines of therapy, NOX-A12 plus pembrolizumab shows induction of immune response, stable disease in 25% of patients, and prolonged time on treatment vs. prior therapy for 35% of patients. The safety profile of the combination therapy was consistent with that of pembrolizumab in advanced cancer patients. Citation Format: Niels Halama, Ulrike Pruefer, Anna Froemming, Diana Beyer, Dirk Eulberg, Jarl Ulf Jungnelius, Aram Mangasarian. Clinical outcome and safety in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer treated with the CXCL12 inhibitor NOX-A12 in combination with PD-1 checkpoint inhibitor pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT092.

The blockade of pro-inflammatory mediators is a successful approach to improve the engraftment after islet transplantation. L-aptamers are chemically synthesized, nonimmunogenic bio-stable oligonucleotides that bind and inhibit target molecules conceptually similar to antibodies. We aimed to evaluate if blockade-aptamer-based inhibitors of C-C Motif Chemokine Ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and C-X-C Motif Chemokine Ligand 12/stromal cell-derived factor-1 (CXCL12/SDF-1) are able to favor islet survival in mouse models for islet transplantation and for type 1 diabetes. We evaluated the efficacy of the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12 on islet survival in a syngeneic mouse model of intraportal islet transplantation and in a multiple low doses of streptozotocin (MLD-STZ) diabetes induction model. Moreover, we characterized intrahepatic infiltrated leukocytes by flow cytometry before and 3 days after islet infusion in presence or absence of these inhibitors. The administration for 14 days of mNOX-E36 and NOX-A12 significantly improved islet engraftment, either compound alone or in combination. Intrahepatic islet transplantation recruited CD45 + leucocytes and more specifically CD45+/CD11b+ mono/macrophages; mNOX-E36 and NOX-A12 treatments significantly decreased the recruitment of inflammatory monocytes, CD11b + /Ly6C high /CCR2 + and CD11b + /Ly6C high /CXCR4 + cells, respectively. Additionally, both L-aptamers significantly attenuated diabetes progression in the MLD-STZ model. In conclusion, CCL2/MCP-1 and CXCL12/SDF-1 blockade by L-aptamers is an efficient strategy to improve islet engraftment and survival.

Monocytes and monocyte-derived cells are important players in the initiation, progression, and resolution of inflammatory skin reactions. As inflammation is a prerequisite for fibrosis development, we focused on the role of monocytes in cutaneous fibrosis, the clinical hallmark of patients suffering from systemic sclerosis. Investigating the function of monocytes in reactive oxygen species-induced dermal fibrosis, we observed that early monocyte depletion partially reduced disease severity. Low numbers of inflammatory Ly6C(high) monocytes, as well as inhibition of CCR2 and CCL2 in wild type animals by a specific L-RNA aptamer, mitigated disease parameters, indicating a pivotal role for CCR2(+) inflammatory monocytes and the CCR2/CCL2 axis in fibrosis development. Of note, mice lacking splenic reservoirs failed to recruit monocytes to the skin and developed less fibrosis. Furthermore, enforced monocyte conversion into noninflammatory, patrolling Ly6C(low) monocytes by a nuclear receptor Nur77-agonist also resulted in significantly impaired cutaneous inflammation and dermal fibrosis. Most evident, pronounced monocyte conversion in interferon stimulated gene 12-deficient mice with pronounced nuclear Nur77 signaling completely protected from dermal fibrosis. Our study shows that inflammatory monocytes that are recruited from splenic reservoirs play a key role in the development of skin fibrosis and can be therapeutically challenged by forced conversion via the Nur77/interferon stimulated gene 12 axis.

e14143 Background: NOX-A12 is an inhibitor of the chemokine CXCL12 which prevents receptor engagement and blocks the formation of chemotactic CXCL12 concentration gradients. The OPERA study (NCT03168139) is a Phase 1/2 study to evaluate pharmacodynamic effects and safety of monotherapy with NOX-A12 as well as safety and efficacy of a combination with pembrolizumab in metastatic microsatellite-stable (MSS) colorectal (CRC) and pancreatic (PaC) cancer where PD-1 inhibition alone has not shown clinical benefit. Methods: Patients received 300 mg NOX-A12 twice weekly during a 2-week monotherapy phase. Biopsies were taken from liver metastases before and after NOX-A12 monotherapy for analysis of immune cell infiltration and cytokine signature. In a combination phase, patients received repeated 21-day cycles of 300 mg NOX-A12 and 200 mg pembrolizumab until progression or intolerable toxicity. Results: 11 of the patients had CRC and 9 PaC. All patients were heavily pretreated with 5 (CRC) and 3 median lines (PaC) of prior treatment. Best responses to last prior treatment was progressive disease for 95% of patients. The AE profile was comparable with the pembrolizumab profile or typical for the underlying diseases. At baseline, mean T cell density at the invasive margin was 327 cells/mm², clearly below the 600 cells/mm² that are predictive for a good prognosis. 2 weeks of NOX-A12 monotherapy induced Th1 cytokines (IFNγ, IL-2, IL-16) in approx. half of the patients. Although there were no objective responses, 25% of patients achieved stable disease; 7 patients showed prolonged time on treatment vs. the prior line of therapy. Updated figures for progression-free survival and overall survival for the whole patient cohort will be presented. Conclusions: In patients with MSS metastatic PaC and CRC cancer with impaired immune systems and a high tumor load that have failed multiple prior lines of therapy, NOX-A12 plus pembrolizumab shows induction of immune response, stable disease in 25% of patients, and prolonged time on treatment vs. prior therapy for 35% of patients. The safety profile of the combination therapy was consistent with that of pembrolizumab in advanced cancer patients. Clinical trial information: NCT03168139.

Insufficient podocyte regeneration after injury is a central pathomechanism of glomerulosclerosis and chronic kidney disease. Podocytes constitutively secrete the chemokine CXCL12, which is known to regulate homing and activation of stem cells; hence we hypothesized a similar effect of CXCL12 on podocyte progenitors. CXCL12 blockade increased podocyte numbers and attenuated proteinuria in mice with Adriamycin-induced nephropathy. Similar studies in lineage-tracing mice revealed enhanced de novo podocyte formation from parietal epithelial cells in the setting of CXCL12 blockade. Super-resolution microscopy documented full integration of these progenitor-derived podocytes into the glomerular filtration barrier, interdigitating with tertiary foot processes of neighboring podocytes. Quantitative 3D analysis revealed that conventional 2D analysis underestimated the numbers of progenitor-derived podocytes. The 3D analysis also demonstrated differences between juxtamedullary and cortical nephrons in both progenitor endowment and Adriamycin-induced podocyte loss, with more robust podocyte regeneration in cortical nephrons with CXCL12 blockade. Finally, we found that delayed CXCL12 inhibition still had protective effects. In vitro studies found that CXCL12 inhibition uncoupled Notch signaling in podocyte progenitors. These data suggest that CXCL12-driven podocyte-progenitor feedback maintains progenitor quiescence during homeostasis, but also limits their intrinsic capacity to regenerate lost podocytes, especially in cortical nephrons. CXCL12 inhibition could be an innovative therapeutic strategy in glomerular disorders.

Abstract Background: NOX-A12 (olaptesed pegol) is a novel inhibitor of the chemokine CXCL12, ligand for CXCR4 and CXCR7, for treatment of solid tumors. Binding of the CXCL12 ligand by NOX-A12 prevents receptor engagement and also blocks the ability of CXCL12 to create a chemotactic concentration gradient by neutralization of the anchor domain. CXCL12 in complex with NOX-A12 has an increased half-life relative to unbound CXCL12, and as such increased CXCL12 levels are a marker of inhibition. The OPERA study (NCT03168139) is a phase 1/2 open-label clinical study to evaluate pharmacodynamic effects and safety of monotherapy with NOX-A12 and safety and efficacy of a combination of NOX-A12 with pembrolizumab in advanced MSS, metastatic colorectal and pancreatic cancer with liver metastasis. The study comprises two weeks of NOX-A12 monotherapy followed by repeated 21-day cycles of NOX-A12 plus pembrolizumab. Here we present pharmacodynamic biomarker data from for monotherapy phase with NOX-A12 as well as safety for the combination with pembrolizumab. Methods: Patients received 300 mg NOX-A12 by i.v. infusion on days 1, 4, 8, and 11 of the monotherapy phase. Needle biopsies were taken from suitable liver metastases before treatment and on day 14, peripheral blood was drawn at the same time points. Collected tumor samples were assessed for immune cell infiltration by IHC and cytokine signature using multiplex protein analysis. Results: At the time of abstract submission, 19 out of 20 patients have been recruited, 10 with colorectal and 9 with pancreatic cancer. Serial biopsies at baseline and end of monotherapy suitable for immunohistochemistry (IHC) and cytokine analysis were obtained from 12 out of 19 patients. CXCL12 levels were found to be increased in blood, but also in post-treatment tumor biopsies, suggesting penetration of NOX-A12 into the tumor. The comparison of cytokine levels in the currently available baseline tumor biopsies with those taken after two weeks of NOX-A12 monotherapy revealed changes in markers that are consistent with a Th1-like immune response in multiple patients. In particular IFN-γ, IL-2 and TNF-β were increased, accompanied by an increase of CXCL9 and/or CXCL10 indicating simultaneous T-cell attraction, which indicates a tissue response to treatment with NOX-A12. However, there were also increased levels of IL-1α, IL-1β and IL-6 in a number of patients, indicating potential myeloid counter regulation. IHC analysis is ongoing, and results for all patients will be presented. Treatment with NOX-A12 monotherapy and in combination with pembrolizumab was generally safe and well tolerated (139 AEs in total, thereof 45% grade 1; 37% grade 2; 17% grade 3; no grade 4 and 1 % grade 5). Conclusions: NOX-A12 penetrates into the tumor where it binds and neutralizes CXCL12. Changes in the cytokine signature suggest that NOX-A12 modulates the tumor microenvironment and induces an immune-stimulatory Th1-like signature in multiple patients. The safety profile of NOX-A12 combined with pembrolizumab is consistent with that of pembrolizumab in advanced cancer patients. Citation Format: Niels Halama, Ulrike Prüfer, Anna Froemming, Diana Beyer, Dirk Eulberg, Jarl Ulf Jungnelius, Aram Mangasarian. Evaluation of tumor biomarkers in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer treated with the CXCL12 inhibitor NOX-A12 and preliminary safety in combination with PD-1 checkpoint inhibitor pembrolizumab [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B206.

BACKGROUND & AIMS:Hepatocellular carcinoma (HCC) typically arises in fibrotic or cirrhotic livers, which are characterized by pathogenic angiogenesis. Myeloid immune cells, specifically tumor-associated macrophages (TAMs), may represent potential novel therapeutic targets in HCC, complementing current ablative or immune therapies. However, the detailed functions of TAM subsets in hepatocarcinogenesis have remained obscure. METHODS:TAM subsets were analyzed in-depth in human HCC samples and a combined fibrosis-HCC mouse model, established by i.p. injection with diethylnitrosamine after birth and repetitive carbon tetrachloride (CCl4) treatment for 16 weeks. Based on comprehensively phenotyping TAM subsets (fluorescence-activated cell sorter, transcriptomics) in mice, the function of CCR2+ TAM was assessed by a pharmacologic chemokine inhibitor. Angiogenesis was evaluated by contrast-enhanced micro-computed tomography and histology. RESULTS:We show that human CCR2+ TAM accumulate at the highly vascularized HCC border and express the inflammatory marker S100A9, whereas CD163+ immune-suppressive TAM accrue in the HCC center. In the fibrosis-cancer mouse model, we identified 3 major hepatic myeloid cell populations with distinct messenger RNA profiles, of which CCR2+ TAM particularly showed activated inflammatory and angiogenic pathways. Inhibiting CCR2+ TAM infiltration using a pharmacologic chemokine CCL2 antagonist in the fibrosis-HCC model significantly reduced pathogenic vascularization and hepatic blood volume, alongside attenuated tumor volume. CONCLUSIONS:The HCC microenvironment in human patients and mice is characterized by functionally distinct macrophage populations, of which the CCR2+ inflammatory TAM subset has pro-angiogenic properties. Understanding the functional differentiation of myeloid cell subsets in chronically inflamed liver may provide novel opportunities for modulating hepatic macrophages to inhibit tumor-promoting pathogenic angiogenesis.

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.

Inhibition of monocyte chemotactic protein-1 (MCP-1) with the Spiegelmer emapticap pegol (NOX-E36) shows long-lasting albuminuria-reducing effects in diabetic nephropathy. MCP-1 regulates inflammatory cell recruitment and differentiation of macrophages. Because the endothelial glycocalyx is also reduced in diabetic nephropathy, we hypothesized that MCP-1 inhibition restores glomerular barrier function through influencing macrophage cathepsin L secretion, thus reducing activation of the glycocalyx-degrading enzyme heparanase. Four weeks of treatment of diabetic Apoe knockout mice with the mouse-specific NOX-E36 attenuated albuminuria without any change in systemic hemodynamics, despite persistent loss of podocyte function. MCP-1 inhibition, however, increased glomerular endothelial glycocalyx coverage, with preservation of heparan sulfate. Mechanistically, both glomerular cathepsin L and heparanase expression were reduced. MCP-1 inhibition resulted in reduced CCR2-expressing Ly6Chi monocytes in the peripheral blood, without affecting overall number of kidney macrophages at the tissue level. However, the CD206+/Mac3+ cell ratio, as an index of presence of anti-inflammatory macrophages, increased in diabetic mice after treatment. Functional analysis of isolated renal macrophages showed increased release of IL-10, whereas tumor necrosis factor and cathepsin L release was reduced, further confirming polarization of tissue macrophages toward an anti-inflammatory phenotype during mouse-specific NOX-E36 treatment. We show that MCP-1 inhibition restores glomerular endothelial glycocalyx and barrier function and reduces tissue inflammation in the presence of ongoing diabetic injury, suggesting a therapeutic potential for NOX-E36 in diabetic nephropathy.

Resistance to targeted tyrosine kinase inhibitors (TKI) remains a challenge for the treatment of myeloid leukemias. Following treatment with TKIs, the bone marrow microenvironment has been found to harbor a small pool of surviving leukemic CD34+ progenitor cells. The long-term survival of these leukemic cells has been attributed, at least in part, to the protective effects of bone marrow stroma. We found that the NOX-A12 'Spiegelmer', an L-enantiomeric RNA oligonucleotide that inhibits SDF-1α, showed in vitro and in vivo activity against BCR-ABL- and FLT3-ITD-dependent leukemia cells. NOX-A12 was sufficient to suppress SDF-1-induced migration in vitro. The combination of NOX-A12 with TKIs reduced cell migration in the same in vitro model of SDF-1-induced chemotaxis to a greater extent than either drug alone, suggesting positive cooperativity as a result of the SDF-1 blocking function of NOX-A12 and cytotoxicity resulting from targeted oncogenic kinase inhibition. These results are consistent with our in vivo findings using a functional pre-clinical mouse model of chronic myeloid leukemia (CML), whereby we demonstrated the ability of NOX-A12, combined with the ABL kinase inhibitor, nilotinib, to reduce the leukemia burden in mice to a greater extent than either agent alone. Overall, the data support the idea of using SDF-1 inhibition in combination with targeted kinase inhibition to override drug resistance in oncogene-driven leukemia to significantly diminish or eradicate residual leukemic disease.

Background: Emapticap pegol (NOX-E36) is a Spiegelmer((R)) VR that specifically binds and inhibits the pro-inflammatory chemokine C-C motif-ligand 2 (CCL2) (also called monocytechemotactic protein 1). The objective of this exploratory study was to evaluate the safety and tolerability as well as the renoprotective and anti-diabetic potential of emapticap in type 2 diabetic patients with albuminuria.Methods: A randomized, double-blind, placebo-controlled Phase IIa study was initiated in 75 albuminuric type 2 diabetics. Emapticap at 0.5 mg/kg and placebo were administered subcutaneously twice weekly for 12 weeks to 50 and 25 patients, respectively, followed by a treatment-free phase of 12 weeks.Results: Twice weekly subcutaneous treatment with emapticap over 3 months was generally safe and well tolerated and reduced the urinary albumin/creatinine ratio (ACR) from baseline to Week 12 by 29% (P < 0.05); versus placebo a non-significant ACR reduction of 15% was observed (P = 0.221). The maximum difference, 26% (P = 0.064) between emapticap and placebo, was seen 8 weeks after discontinuation of treatment. At Week 12, the HbA1c changed by -0.31% in the emapticap versus +0.05% in the placebo group (P = 0.146). The maximum difference for HbA1c was observed 4 weeks after the last dose with -0.35% for emapticap versus +0.12% for placebo (P = 0.026). No relevant change in blood pressure or estimated glomerular filtration rate was seen between the treatment groups throughout the study. A post hoc analysis with exclusion of patients with major protocol violations, dual RAS blockade or haematuria increased the ACR difference between the two treatment arms to 32% at Week 12 (P = 0.014) and 39% at Week 20 (P = 0.010).Conclusions: Inhibition of the CCL2/CCL2 receptor axis with emapticap pegol was generally safe and well tolerated. Beneficial effects on ACR and HbA1c were observed in this exploratory study, which were maintained after cessation of treatment. Taken together, emapticap may have disease-modifying effects that warrant further investigation in adequately powered confirmatory studies.

Abstract Immune checkpoint inhibition promotes T cell-mediated killing of cancer cells and can induce striking responses, but objective control of tumor growth is observed in only 10-30% of patients with cancer types that generally respond to this treatment (Fearon 2014, Cancer Immunol Res 2:187). A possible cause for this limitation of checkpoint inhibition may be an immune-privileged tumor microenvironment (TME) which excludes the cytotoxic T cells from the vicinity of cancer cells. The chemokine CXCL12 has recently been described as an important T cell exclusion factor in the TME-driven immune suppression. In this study we aimed to investigate whether CXCL12 inhibition by the clinical stage L-aptamer (Spiegelmer®) NOX-A12 (olaptesed pegol) is able to enhance T cell infiltration in 3D tumor-stroma spheroids, thereby facilitating effective immunotherapy. We established 3D multicellular microtissues that mimic a solid tumor with a CXCL12-abundant TME. For this purpose, CXCL12-expressing murine stromal MS-5 cells were co-cultured with solid human cancer cell lines in ultra-low attachment plates for three days. Primary human T cells isolated from healthy donors were added to the spheroids in the presence of various concentrations of NOX-A12. The next day, spheroids were washed and dissociated for T cell quantification by flow cytometry. T cell localization in the 3D microtissues was assessed by immunohistochemistry (IHC). In order to examine T cell activation in the spheroids, a bioluminescent reporter-based PD-1/PD-L1 blockade bioassay (Promega) was adapted to the 3D format: Jurkat-PD-1/Luc T cells were incubated with anti-PD-1 and added to NOX-A12-treated spheroids (CHO-PD-L1 + MS-5). We found that NOX-A12 increases the amount of T cells in tumor-stroma spheroids in a dose-dependent manner; flow cytometry analyses revealed a 2-3 fold increase in spheroid T cell infiltration at 10 nM NOX-A12 in all examined 3D co-culture types. Enhanced T cell infiltration in the presence of NOX-A12 was corroborated by IHC. In line with this, we found increased infiltration and activation of Jurkat-PD-1/luc T cells in the MS-5/CHO-PD-L1 spheroids treated with NOX-A12. Importantly, NOX-A12 synergized with anti-PD1-induced T cell activation. Taken together, in heterotypic 3D models that mimic the complexity of the TME, the CXCL12 antagonist NOX-A12 improved T cell-based tumor immunotherapy by increasing T cell infiltration. By modulating the CXCL12 gradients within the complex 3D structure, NOX-A12 appears to break the immune-privilege of the TME, thereby paving the way for T cell migration into the tumor. These data provide a rationale for the combination of NOX-A12 with checkpoint inhibitors as well as other T cell-based therapies in patients with solid cancer. Citation Format: Dirk Zboralski, Lisa Bauer, Dirk Eulberg, Axel Vater. CXCL12 inhibition with NOX-A12 (olaptesed pegol) increases T-cell infiltration in tumor-stroma spheroids and synergizes with PD-1 immune checkpoint blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1473.

Acetaminophen (APAP, paracetamol) poisoning is a leading cause of acute liver failure (ALF) in humans and induces hepatocyte necrosis, followed by activation of the innate immune system, further aggravating liver injury. The role of infiltrating monocytes during the early phase of ALF is still ambiguous. Upon experimental APAP overdose in mice, monocyte‐derived macrophages (MoMFs) massively accumulated in injured liver within 12‐24 hours, whereas the number of tissue‐resident macrophages (Kupffer cells) decreased. Influx of MoMFs is dependent on the chemokine receptor, chemokine (C‐C motif) receptor 2 (CCR2), given that Ccr2−/− mice display reduced infiltration of monocytes and attenuated liver injury post‐APAP overdose at early time points. As evidenced by intravital multiphoton microscopy of Ccr2 reporter mice, CCR2+ monocytes infiltrate liver as early as 8‐12 hours post‐APAP overdose and form dense cellular clusters around necrotic areas. CCR2+ MoMFs express a distinct pattern of inflammatory, but also repair‐associated, genes in injured livers. Adoptive transfer experiments revealed that MoMFs primarily exert proinflammatory functions early post‐APAP, thereby aggravating liver injury. Consequently, early pharmacological inhibition of either chemokine (C‐C motif) ligand (CCL2; by the inhibitor, mNOX‐E36) or CCR2 (by the orally available dual CCR2/CCR5 inhibitor, cenicriviroc) reduces monocyte infiltration and APAP‐induced liver injury (AILI) in mice. Importantly, neither the early nor continuous inhibition of CCR2 hinder repair processes during resolution from injury. In line with this, human livers of ALF patients requiring liver transplantation reveal increased CD68+ hepatic macrophage numbers with massive infiltrates of periportal CCR2+ macrophages that display a proinflammatory polarization. Conclusion: Infiltrating monocyte‐derived macrophages aggravate APAP hepatotoxicity, and the pharmacological inhibition of either CCL2 or CCR2 might bear therapeutic potential by reducing the inflammatory reaction during the early phase of AILI. (Hepatology 2016;64:1667‐1682)

Effective cancer immunotherapy requires physical contact between cytotoxic immune cells and malignant cells which is restricted by the tumour microenvironment (TME). The chemokine CXCL12 has recently been described as an important T cell exclusion factor in the TME-driven immune suppression. In this study we aimed to investigate whether CXCL12 inhibition by the clinical stage L-aptamer (Spiegelmer®) NOX-A12 is able to enhance immune cell infiltration into 3D tumour-stroma spheroids. We established 3D multicellular spheroids that mimic a solid tumour with a CXCL12-expressing TME. For this purpose, CXCL12-secreting murine stromal MS-5 cells were co-cultured with human cancer cell lines in ultra-low attachment plates. Peripheral blood mononuclear cells from healthy donors were added to the spheroids in the presence of various concentrations of NOX-A12. The next day, spheroids were dissociated for analysis of infiltrated immune cells by flow cytometry. In parallel, cellular distribution within the spheroids was assessed by immunohistochemistry. A reporter-based T cell activation assay was adapted to the 3D format in order to examine the combination of NOX-A12 with immune checkpoint blockade. We found that CXCL12 inhibition with NOX-A12 enhanced infiltration of CD8+ T cells, CD4+ T cells and NK cells, and to a lesser extent of Treg and B cells into the homogeneous, CXCL12-expressing tumour-stroma spheroids. Monocyte infiltration was not increased by NOX-A12. By facilitating physical contact of T cells with matching tumour cells, NOX-A12 also enhanced activation of T cells and synergized with PD-1 checkpoint inhibition. Mechanistically, NOX-A12 appears to generate CXCL12 gradients within the densely packed in vitro tumour structure and may thereby break the immune privilege of the TME in vivo. Furthermore, a lower monocyte-to-lymphocyte ratio, as found in NOX-A12 treated spheroids, has been recognized as an indicator for better prognosis in various cancer types. These data provide a rationale for the combination of NOX-A12 with checkpoint inhibitors as well as other T and NK cell-based therapies in cancer patients, such as CAR-T and CAR-NK.