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BackgroundAutotaxin (ATX) is a secreted glycoprotein that hydrolyzes lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). The expression of both ATX and LPA is elevated in most solid tumors and plasma. LPA signaling directly modulates tumor cell function and contributes to the development of the fibrotic tumor microenvironment, a mechanism by which tumors evade host immunity and impairs response to therapy. IOA-289 is a potent, orally available autotaxin inhibitor which is being developed as a novel treatment of solid tumours burdened with a high degree of fibrosis.MethodsInhibition of ATX activity in human plasma was determined by measuring reduction in LPA species as quantified by LC-MS/MS. In vitro activity on biomarkers of fibrosis was assessed using the BioMAP screen and fibroblast cell cultures. T cell migration was measured using 48-well chemotaxis chambers. PK/PD studies were performed following a single oral dose of IOA-289 in mice, and plasma LPA was used as a PD biomarker. In vivo efficacy was studied in two models of breast cancer, 4T1 and E0771. Bioinformatics used TCGA and GTEX publicly available datasets.ResultsIOA-289 inhibits plasma LPA18:2 with an IC50 of 36nM, with similar results for other LPA species. IOA-289 inhibited fibrosis relevant factors in the BioMAP phenotypic screen, including sIL-6, MCP-1, αSMA, collagen-III, and sVEGF. In further studies, IOA-289 inhibited the secretion of PAI-1 and IL-6 by stimulated fibroblasts. LPA and cancer cell conditioned media inhibited T cell chemotaxis in vitro and the effect was overcome in the presence of IOA-289. The efficacious human dose of IOA-289 was determined following PK/PD studies using plasma LPA as a biomarker of response to ATX inhibition. In vivo studies showed that IOA-289 inhibited metastasis of 4T1 cells, enhanced the infiltration of T cells into 4T1 s.c. implanted tumors and prevented the growth of primary, orthotopically implanted E0771 tumors. Bioinformatics analysis demonstrated elevated ATX expression in pancreatic cancer (PDAC), and PDAC patient plasma showed a correlation of ATX levels with CA-19-9.ConclusionsThe ATX/LPA pathway represents a novel target for anti-cancer therapy with actions on the tumor, immune cell and stromal environment. IOA-289 is a highly potent and selective inhibitor of ATX with demonstrated monotherapy activity in cancer models. Based on the mechanism of action we are investigating combinations of IOA-289 with chemotherapy, immunotherapy and novel agents in ongoing preclinical studies. An acceptable safety and PK profile support the clinical development of IOA-289 which is currently in a phase I clinical trial.Ethics ApprovalThe 4T1 study was approved by The University Claude Bernard Lyon 1 Ethics Board; approval number DR2014-38 (vM). The E0771 study was reviewed and approved by the Institutional Animal Care and Use Committee of the contract research organization (Covance, Ann Arbor, MI, USA), an AAALAC International accredited program.

The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.

The antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) inhibits cell entry of a number of viruses, and genetic diversity within IFITM3 determines susceptibility to viral disease in humans. Here, we used the murine CMV (MCMV) model of infection to determine that IFITM3 limits herpesvirus-associated pathogenesis without directly preventing virus replication. Instead, IFITM3 promoted antiviral cellular immunity through the restriction of virus-induced lymphopenia, apoptosis-independent NK cell death, and loss of T cells. Viral disease in Ifitm3–/– mice was accompanied by elevated production of cytokines, most notably IL-6. IFITM3 inhibited IL-6 production by myeloid cells in response to replicating and nonreplicating virus as well as following stimulation with the TLR ligands Poly(I:C) and CpG. Although IL-6 promoted virus-specific T cell responses, uncontrolled IL-6 expression in Ifitm3–/– mice triggered the loss of NK cells and subsequently impaired control of MCMV replication. Thus, IFITM3 represents a checkpoint regulator of antiviral immunity that controls cytokine production to restrict viral pathogenesis. These data suggest the utility of cytokine-targeting strategies in the treatment of virus-infected individuals with impaired IFITM3 activity.

To improve our understanding of properties that confer successful inhibition of chemokines in vivo, we analyzed anti-murine CXCL10 monoclonal antibodies (mAb) having different characteristics. 1B6 displayed potent inhibition of cell recruitment in vitro with an IC50 of 0.5 nm but demonstrated little efficacy in various animal models of human disease. On the contrary, 1F11 showed efficacy in several models of inflammation yet was less potent at inhibiting chemotaxis in vitro with an IC50 of 21 nm. Furthermore, we observed that 1B6 displayed a rapid dose-dependent clearance (t½ 10–60 h) in contrast to 1F11, which presented a dose-proportional pharmacokinetic profile and a half-life of 12 days. Moreover, 1B6 recognized glycosaminoglycan (GAG)-bound CXCL10, resulting in target-mediated clearance, which was corroborated using CXCL10-deficient mice. In contrast to 1B6, 1F11 inhibited the interaction of CXCL10 with GAGs, did not recognize GAG-bound CXCL10, and did not display target-mediated drug disposition. Confirming previous animal studies, 1B6 was poor at reversing glycemia in a model of type 1 diabetes, whereas 1F11 induced early and prolonged control of diabetes. Furthermore, when using 1A4, a subsequently generated anti-mCXCL10 mAb that shares the property with 1F11 of being unable to recognize CXCL10 immobilized on GAG, we observed a similar superior control of diabetes as compared with 1B6. We therefore concluded that targeting chemokines with antibodies such as 1B6 that recognize the more abundant GAG-bound form of the chemokine may not be the optimal strategy to achieve disease control.

Life-threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon γ (IFNγ) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection-driven model of sHLH in mice, we observed that the tissue-derived levels of IFNγ are actually 500- to 2000-fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFNγ gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFNγ activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFNγ-biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFNγ activity in patients with sHLH.

The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time-courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gα(i) protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists.

Despite their key role in inflammation, the apparent redundancy in the chemokine system is often cited as an argument against probing chemokines as therapeutic targets for inflammation. However, this in vitro redundancy frequently does not translate to the in vivo situation, as exemplified by the use of specific receptor antagonists, ligand neutralizing or receptor blocking antibodies and gene-deleted mice in models of human disease. Specificity may be conferred onto the chemokine system by fine-tuning of responses both temporally and spatially through their highly specific interactions with glycosaminoglycans (GAGs). In this survey, we present evidence for specificity in the interaction and introduce emerging technologies that enable detailed assessment of protein-GAG interactions. Finally, we address the issue of exploitation of this interaction for therapeutic advantage.

Inappropriate cell recruitment is a hallmark of all autoimmune, allergic and inflammatory diseases. The prevention of inflammation by interfering with cellular recruitment through the neutralization of cytokines and adhesion molecules has proven to be successful in the clinic. Chemokines are important potential targets owing to their central role in the cell recruitment process. Chemokines are unique among cytokines because they signal through seven transmembrane receptors, thus enabling the identification of small molecule inhibitors through high throughput screening. The object of this Review is to discuss the validity and feasibility of targeting several points of therapeutic intervention offered by the chemokine system and to assess the state of play within the field to date.

Chemokines control inflammatory leukocyte recruitment. The propensity of chemokines such as CC chemokine ligand 5 (CCL5)/RANTES (regulated on activation, normal T cell expressed and secreted) to bind to glycosaminoglycans and to form higher order oligomers has been shown to be essential for its in vivo activity. However, the specific functional relevance of RANTES oligomerization for distinct steps of leukocyte recruitment on inflamed endothelium mediated by the RANTES receptors CC chemokine receptor 1 (CCR1) and CCR5 remains undefined. We studied RANTES mutants with deficient oligomerization in an assay in which recruitment of monocytes and CD45RO+ CD4+ T cells is triggered by RANTES immobilized on activated endothelium under flow conditions. Notably, the formation of higher order RANTES oligomers was crucial for CCR1-mediated arrest but not for CCR5-mediated spreading/transmigration in flow or transendothelial chemotaxis of leukocytes. Efficient leukocyte arrest in flow but not transmigration may thus require the presentation of RANTES oligomers to bridge surface-bound RANTES and CCR1.

Upregulation of the immune checkpoint receptor, CD47, on cancer cells promotes immune evasion and is correlated with poor clinical outcome. CD47 is therefore an attractive immuno-oncology target but also a challenging one, given its ubiquitous distribution in healthy tissues. Bispecific antibodies (biAbs) offer superior selectivity as compared to mAbs, as they combine two antigen specificities in one molecule allowing the simultaneous targeting of two cell surface receptors. We used such a dual targeting design to create NI-1701, a biAb that pairs an anti CD47 arm with a high-affinity arm specific for CD19, a clinically validated target expressed by B leukemias and lymphomas. The target cell selectivity of NI-1701 relies principally on the binding affinity of the biAbs anti-CD19 arm. Thus, NI-1701 binds weakly to CD19-negative healthy cells expressing physiological levels of CD47, such as erythrocytes, platelets or T cells. In contrast, NI-1701 binds strongly to CD19-positive cells and blocks CD47 upon concurrent engagement of the two targets at the cell surface. As shown by numerous experiments involving CD19-positive human cancer cell lines and patients cells, NI-1701 effectively kills B cell tumors through antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cell-mediated cytotoxicity (ADCC). Furthermore, NI-1701 controls the growth of sub-cutaneous Raji cell tumors, in a way that was dependent on the co-ligation of both CD19 and CD47 antigens. Examination of the excised tumors revealed that NI-1701 actively reshaped the tumors microenvironment by enhancing the phagocytic activity of macrophages and by reducing the proportion of CD11b+Gr1+myeloid-derived suppressor cells (MDSCs) infiltrating the xenograft tumors. In disseminated mouse models of B-ALL, using both leukemia cell lines and patient-derived xenografts (PDX), NI-1701 was able to reduce tumor burden in peripheral blood and to block the spread of tumor cells to the bone marrow. The therapeutic potential of NI-1701 was also expanded to Diffuse Large B-Cell Lymphoma (DLBCL) using a PDX model, in which the tumor burden was abrogated with significantly higher efficacy than the BTK inhibitor, ibrutinib. In vitro and in vivo studies demonstrated a favorable pharmacokinetic (PK) and tolerability profile of NI-1701. Single and multiple dose studies in non-human primates showed typical IgG PK and no effects on hematological parameters (e.g., red blood cell and platelet counts) up to 100mg/kg, the highest dose tested. Accordingly, in vitro safety testing with human blood showed no evidence of platelet activation or aggregation, hemagglutination or hemolysis event at high antibody concentrations. We also show that NI-1701 target cell selectivity is important for the preservation of tumor cell killing efficacy in the presence of CD47 antigen sink. ADCP and ADCC experiments performed with an excess of bystander CD47-positive cells demonstrate that NI-1701-induced tumor cell killing is not affected by the presence of such antigen sink, in contrast to anti-CD47 mAbs, which loose potency in this situation. We conclude that the dual targeting biAb approach allows a safe yet effective blockade of CD47 due to selectivity for a B cell associated antigen, resulting in impressive tumor cell killing in a range of preclinical models. Thus, dual-targeting biAb open the way to the safe and efficacious therapeutic neutralization of CD47, an immune checkpoint receptor hijacked by cancer cells. NI-1701 is in preclinical enabling studies in preparation for a Phase I clinical study in patients with B cell malignancies, planned for 2017. Citation Format: Krzysztof Masternak, Xavier Chauchet, Vanessa Buatois, Susana Salgado-Pires, Limin Shang, Zoë Johnson, Elie Dheilly, Valéry Moine, Walter G. Ferlin, Marie H. Kosco-Vilbois, Nicolas Fischer. NI-1701, a bispecific antibody for selective neutralization of CD47 in B cell malignancies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B37.

Up-regulation of CD47 in hematological and solid cancers correlates with poor clinical prognosis. CD47 interaction with SIRPα provides a ‘don't eat me’ signal that allows healthy cells to limit elimination by immune cells, in particular macrophages. Although tumor-associated macrophages (TAMs) are often considered pro-tumorigenic, several studies report a high phagocytic potential and tumoricidal function in the presence of therapeutic antibodies (Ab). Therefore, targeting the CD47-SIRPα pathway in the tumor microenvironment is an attractive approach to maximize the tumor killing potential of TAMs to boost tumor destruction. However, clinical development of monoclonal Abs to CD47 is likely to be hindered by the ubiquitous expression of CD47 leading to rapid drug elimination and toxicity including anemia. To address these concerns, we have created NI-1701, a bispecific Ab that drives efficacious binding only to CD19+B cells by pairing a high affinity anti-CD19 targeting arm to an anti-CD47 arm of optimized affinity.. In addition to in vitro data demonstrating that the bispecific Ab, NI-1701, effectively kills CD19+ human tumor B cells through ADCP (antibody-dependent cellular phagocytosis) and antibody-dependent cell-mediated cytotoxicity (ADCC), we have observed significant tumor killing in vivo, as either a monotherapy or in a combination approach. NI-1701 controlled sub-cutaneously implanted Raji cell tumor growth in NOD/SCID mice in a manner dependent on the co-ligation of both CD19 and CD47. Examination of the excised tumors revealed that NI-1701 reshaped the tumor microenvironment by enhancing the tumoricidal activity of macrophages (i.e., more macrophages engulfing tumor cells), by promoting an antitumor M1-like phenotype, and reducing the proportion of CD11b+Gr1+myeloid-derived suppressor cells (MDSCs). Extending these findings to a disseminated in vivo model, NI-1701 eliminated tumor cells from the peripheral blood, bone marrow and liver in mice transplanted either with the B-Acute Lymphocytic Leukemia (B-ALL) cell line NALM-6 or with primary cells from B-ALL patients. Furthermore, NI-1701 also abrogated tumor growth more efficiently than the BTK inhibitor ibrutinib in a Diffuse Large B-Cell Lymphoma (DLBCL) patient-derived xenograft (PDX) mouse model. As combination therapies are gaining traction as successful treatment strategies in the clinic, we next tested the effect of blocking CD47 biology in combination with clinically validated molecules. Interestingly, in NOD/SCID mice implanted with Raji cells, NI-1701 was shown to be more efficacious at controlling tumor cell growth than Rituximab. A combination of NI-1701 and Rituximab was shown to act synergistically at controlling tumor growth and leading to tumor regression in some mice. Finally, in a syngeneic re-challenge model, using bispecific reagents targeting CD47 blockade to the A20 murine B-cell lymphoma, we observed the induction of a durable and protective anti-tumor response when combined with a single administration of cyclophosphamide. Importantly, in vitro safety studies demonstrate a favorable binding profile of NI-1701 to B cells compared with erythrocytes, no evidence of platelet activation or aggregation and no haemagglutination at and above anticipated therapeutic concentrations. Single and multiple dose studies in non-human primates demonstrated favorable elimination kinetics and no effects on hematological parameters (e.g., red blood cell and platelet counts) up to 100mg/kg, the highest dose tested. Taken together, we describe a novel bispecific approach that balances a safe yet effective blockade of CD47 with a high selectivity for a B cell associated antigen resulting in impressive tumor cell killing in a range of preclinical models. The effects on both the reshaping of the tumor microenvironment and the induction of long term tumor immunity provide further evidence that manipulation of myeloid lineage cells (e.g., macrophages and dendritic cells) is a promising approach for the next frontier in immune-oncology treatment strategies. NI-1701 is in preclinical enabling studies in preparation for a Phase I clinical study in patients with CD19+ B cell malignancies, planned for early 2017. Disclosures Ferlin: Novimmune S.A.: Employment, Equity Ownership. Chauchet:Novimmune S.A.: Employment. Buatois:Novimmune S.A.: Employment. Salgado-Pires:Novimmune S.A.: Employment. Shang:Novimmune S.A.: Employment. Dheilly:Novimmune S.A.: Employment. Masternak:Novimmune S.A.: Employment. Johnson:Novimmune S.A.: Employment. DiPersio:Incyte Corporation: Research Funding. Kosco-Vilbois:Novimmune S.A.: Employment. Fischer:Novimmune S.A.: Employment.

The inhibitory “don't eat me” signal of phagocytosis, CD47, is commonly overexpressed in cancer cells, a feature generally associated with poor prognosis. CD47 overexpression in cancer is believed to promote immune evasion by allowing tumor cells to “hide” from innate immune phagocytes like macrophages or dendritic cells. CD47 is therefore a new type of immune checkpoint and an attractive target for cancer immunotherapy. However, as CD47 is also universally expressed on healthy cells, clinical development of anti-CD47 monoclonal antibodies is inevitably limited by toxicity and/or pharmacokinetic issues. To overcome these liabilities, we engineered dual-targeting bispecific antibodies (biAbs) for selective blockade of CD47 in malignant cells. By tethering the biAbs strongly to cells expressing a tumor-associated antigen (TAA), such as CD19 or mesothelin, CD47 is blocked selectively on the target cell. In contrast, as these biAbs will lose the avidity effect with TAA-negative cells, they will bind with very low affinity to healthy cells which express CD47. In this manner, dual-targeting should help to sidestep safety and pharmacokinetic “sink” problems resulting from ubiquitous CD47 expression. Studies in non-human primates performed with the CD47/CD19 therapeutic candidate NI-1701 confirmed this prediction, demonstrating normal IgG1 pharmacokinetics and absence of toxicity, even at high antibody doses (100 mg/kg per week). Hence, the mechanism of action of CD47/TAA dual-targeting antibodies is heavily contingent upon target co-engagement. In vitro, CD19-positive or mesothelin-positive cancer cells are efficiently killed through antibody dependent cellular phagocytosis (ADCP) and/or antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of effector cells, such as macrophages or natural killer cells, and the corresponding dual-targeting CD47/TAA antibodies. Their enhanced ability to induce tumor cell phagocytosis was also demonstrated in vivo, in xenograft models: Mice implanted with subcutaneous human B cell lymphoma xenografts controlled tumor growth following therapy with NI-1701, contrary to mice treated with an anti-CD19 mAb. Importantly, tumor microenvironment (TME) studies revealed that mouse macrophages infiltrating human tumors engulfed tumor cells more frequently—and at a significantly higher rate—in animals treated with NI-1701 as compared to controls. Moreover, the observed superior phagocytic activity of tumor-infiltrating macrophages was associated with a reduction of granulocytic myeloid-derived suppressor cell infiltrates, suggesting that NI-1701 may favor the establishment of a tumor-hostile, immunostimulatory TME. We conclude that dual-targeting CD47/TAA bispecific antibodies may open the way to the safe and efficacious therapeutic neutralization of CD47, the universal ‘don't eat me’ signal hijacked by cancer cells. Citation Format: Krzysztof Masternak, Valéry Moine, Lucile Broyer, Xavier Chauchet, Vanessa Buatois, Elie Dheilly, Stefano Majocchi, Giovanni Magistrelli, Yves Poitevin, Ulla Ravn, Eric Hatterer, Susana Salgado Pires, Limin Shang, Zoë Johnson, Walter Ferlin, Marie Kosco-Vilbois, Nicolas Fischer. Neutralization of CD47 in cancer cells with bispecific antibodies harnesses the phagocytic potential of tumor-infiltrating macrophages. [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 1495.

Chemokines are chemoattractant proteins that are involved in basal trafficking, as well as recruitment and activation of cells in inflammatory processes. They are widely believed to act through a haptotactic mechanism, whereby immobilised gradients of chemokines are established on the endothelial cell surface and in the extracellular matrix [1]. Chemokines are known to bind to glycosaminoglycans (GAGs) in vitro and in vivo [2-4]. This GAG binding has been shown to promote oligomerisation [4], which has been hypothesised to increase local concentrations of chemokine in vivo [5]. Additionally, oligomerisation could enhance presentation to receptors [4, 6]. We recently demonstrated the essential role of the chemokine/GAG interaction with respect to biological activity in vivo [7], using a RANTES/CCL5 variant where the basic amino acids on the 40s loop were mutated to alanine, considerably reducing the capacity for heparin binding [8]. The aim ofthe present study was to investigate the potential of [44AANA47]-RANTES to block chemokine mediated inflammation in vivo.

Chemokine function in vivo depends on the presentation by structures of the extracellular matrix or on endothelial surfaces. CCL5 contains two clusters of basic amino acid residues ( 44 RKNR 47 and 55 KKWVR 59 ) implicated in presentation of the protein. While 44 RKNR 47 has been shown to moderate CCL5 binding to glycosaminoglycans (GAGs), no direct role for the basic residues in the so called 50s loop ( 55 KKWVR 59 ) as a presentation structure has been published to date. In ex vivo studies both regions were found to be necessary for direct tissue binding suggesting a role for 55 KKWVR 59 . In vitro T lymphocyte and monocyte induced firm adhesion under flow, as well as leukocyte recruitment to the peritoneal cavity in vivo was reduced in the 50s mutant. The binding of the 50s mutant to endothelial cells was significantly reduced as compared to the wild type protein demonstrated by ELISA. The 50s mutant had little impact on GAG binding in vitro . These data suggest that functional CCL5 presentation is mediated through both the 40s as well as the 50s loop with differential functions of the two loops of clusters of basic residues.

The antiviral restriction factor interferon-induced transmembrane protein 3 (IFITM3) inhibits endocytosis-dependent entry of numerous viruses and determines susceptible to viral disease in humans. Using the murine cytomegalovirus (MCMV) model, we discovered that regulation of pro-inflammatory cytokine production is a critical function of IFITM3. IFITM3 prevented MCMV-induced weight loss and death without directly restricting MCMV replication. Instead, susceptibility of IFITM3−/− mice was attributed to an enhanced MCMV-induced lymphopenia, activation-induced NK cell death and impaired T cell responses, resulting in uncontrolled virus replication. Exacerbated pathology in IFITM3−/− mice was associated with increased expression of the pro-inflammatory cytokine interleukin-6 (IL-6), and IFITM3 exerted cell-intrinsic inhibition of MCMV-induced IL-6 expression by myeloid cells. Although IL-6 is required for the generation of antiviral adaptive immunity, inhibiting anti-IL-6R reversed MCMV-induced pathology and control of early MCMV replication. These data suggest that IFITM3 restricts viral pathogenesis, at least in part, via regulation of pro-inflammatory cytokine production.

CD47 is a ubiquitous cell surface glycoprotein that serves as a negative regulator of numerous innate immune functions such as phagocytosis, neutrophil inflammatory responses or dendritic cell maturation and activation. In particular, the interaction of CD47 with Signal Regulatory Protein Alpha (SIRP alpha) on myeloid cells is a mechanism of self-recognition, a ‘don't eat me’ signal used by healthy cells to impede their elimination by phagocytes. Cancer cells often up-regulate CD47 expression, which helps them to evade immune surveillance and killing. What is more, increased CD47 levels are generally predictive of poor clinical outcome. Inhibiting CD47-SIRP alpha interaction represents therefore an attractive, generally applicable therapeutic strategy. Yet, achieving that goal with an anti-CD47 monoclonal antibody (Mab) may be challenging in practice, given the ubiquitous expression of the target. The “drug sink” represented by erythrocytes, platelets and other CD47-expressing cells may lead to a rapid elimination of an anti-CD47 Mab through target-mediated drug disposition; another likely consequence of ubiquitous CD47 expression are on-target toxicities like, e.g., anemia. To overcome these limitations, we have developed dual-targeting bispecific antibodies to CD47 and different Tumor-Associated Antigens (TAAs). Dual targeting CD47/TAA bispecific antibodies bind preferentially to TAA-expressing cancer cells and enable selective CD47 neutralization at the same time sparing healthy TAA-negative cells. Various TAAs are currently being pursued at Novimmune (e.g., CD19, mesothelin, or glypican 3). Such dual targeting CD47/TAA bispecific antibodies have the kappa/lambda body format i.e., they are fully human bispecific IgGs composed of a high-affinity anti-TAA arm, a CD47-neutralizing arm, and an unmodified IgG1 Fc. So far, only CD47/CD19 kappa/lambda bodies have been thoroughly characterized in vitro and in vivo. Various human B cell leukemia and lymphoma lines were used to demonstrate selective, CD19-dependent binding of these CD47/CD19 kappa/lambda bodies. As anticipated, the neutralization of the CD47-SIRP alpha interaction was also CD19-dependent, and led to the enhancement of Fc-mediated cancer cell killing through ADCP (antibody mediated cellular phagocytosis) or ADCC (antibody mediated cellular cytotoxicity). Efficient killing of CD19-positive cancer cells was also observed in vivo using a localized tumor xenograft model with NOD/SCID mice implanted with Raji Burkitt lymphoma cells. A dose dependent inhibition of tumor growth or tumor regression was observed, depending on the potency of the CD47-neutralizing arm used to generate the kappa/lambda body. In parallel, a single dose PK study in cynomolgus demonstrated favorable elimination kinetics, comparable to human IgG Mab. Moreover, no effects on hematological parameters were apparent at either of the doses tested (0.5 and 10 mg/kg). The example of CD47/CD19 kappa/lambda bodies illustrates the power of the dual-targeting approach: A favorable safety profile and pharmacokinetics could be achieved in parallel to potent inhibition of the CD47-SIRP alpha interaction and effective cancer cell killing. Antagonizing CD47-SIRP alpha interaction with bispecific antibodies is therefore an attractive immune-potentiating strategy. Because of its distinct mechanism of action and the ability to that harnesses the innate immunity to fight cancer, CD47/TAA bispecific antibodies would be good candidates for combination therapies with immune checkpoint inhibitors or other immunotherapeutics. Citation Format: Krzysztof Masternak, Zoe Johnson, Vanessa Buatois, Francois Rousseau, Giovanni Magistrelli, Valery Moine, Ulla Ravn, Franck Gueneau, Lucile Broyer, Susana Salgado Pires, Maureen Deehan, Nicolas Fischer, Walter Ferlin, Marie Kosco-Vilbois. Antagonizing CD47-SIRP alpha interaction with a bispecific antibody: A novel cancer immunotherapy approach. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B54.

One of the important steps in developing protein therapeutics is the determination of their preliminary PK in vivo. These data are essential to design optimal dosing in animal models prior to progressing to clinical trials in man. The quantitative detection of protein therapeutics in serum is traditionally performed by ELISA, which has the prerequisite of the availability of the appropriate monoclonal antibodies. We have developed an alternative method using polyclonal antibodies immobilized on ProteinChip Arrays and SELDI-TOF mass spectrometry. This method has an advantage over ELISA since it provides simultaneously information on the clearance rate of the protein and it's in vivo processing. We compared these two methods using a RANTES variant, [(44)AANA(47)]-RANTES as the test protein in this study. Using SELDI-TOF mass spectrometry, we were able to establish that the protein is readily oxidized in serum, and moreover is processed in vivo to produce a truncated 3-68 protein, and undergoes a further cleavage to produce the 4-68 protein. These modifications are not identified by ELISA, whilst the serum exposure profiles determined by the two methods show essentially similar protein concentration values.

RANTES (regulated upon activation normally T-cell expressed and secreted) is a member of the subfamily of cytokines called chemokines, the name given to chemoattractant cytokines in 1994. The family consists of small basic proteins whose role is to provide the directional signal for leukocyte migration. The chemokine family are distinct from other cytokines in that they act on G-protein-coupled heptahelical transmembrane spanning receptors. Initially, chemokines were named according to their function but due to the confusion in the nomenclature which arose from the simultaneous identification by different laboratories of many chemokines in the mid to late 1990s, a systematic nomenclature was introduced in 2001. In the new nomenclature the chemokines were named according to their genomic localization, and RANTES was renamed CCL5. RANTES probably has the broadest range of target cells of known chemokines due to its multiple receptor usage and thus is implicated in the recruitment of memory T cells, monocytes, eosinophils, and basophils, thus making it one of the key players in respiratory disease.

Chemokines are small chemoattractant cytokines that control a wide variety of biological and pathological processes, ranging from immunosurveillance to inflammation, and from viral infection to cancer. Genetic and pharmacological studies have shown that chemokines are responsible for the excessive recruitment of leucocytes to inflammatory sites and damaged tissue. In the present paper, we discuss the rationale behind interfering with the chemokine system and introduce various points for therapeutic intervention using either protein-based or small-molecule inhibitors. Unlike other cytokines, chemokines signal via seven-transmembrane GPCRs (G-protein-coupled receptors), which are favoured targets by the pharmaceutical industry, and, as such, they are the first cytokines for which small-molecule-receptor antagonists have been developed. In addition to the high-affinity receptor interaction, chemokines have an in vivo requirement to bind to GAGs (glycosaminoglycans) in order to mediate directional cell migration. Prevention of the GAG interaction has been shown to be a viable therapeutic strategy. Targeting chemokine intracellular signalling pathways offers an alternative small-molecule approach. One of the key signalling targets downstream of a variety of chemokine receptors identified to date is PI3Kγ (phosphoinositide 3-kinase γ), a member of the class I PI3K family. Thus the chemokine system offers many potential entry points for innovative anti-inflammatory therapies for autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis and allergic contact dermatitis.

Aims The paradigm in IL-6 biology is that classical ( cis) IL-6 signaling, mediated by IL-6 binding to the membrane form of IL-6 receptor (mIL-6R), is crucial for homeostatic functions. Conversely, the complex of the soluble form of IL-6R (sIL-6R) and IL-6 binding to cell-membrane expressed gp130, known as IL-6 trans -signaling, is thought to mediate local inflammation. In both cases, IL-6 signaling is believed to be driven through the formation of a hexameric complex consisting of 2 trimers of IL-6/IL-6R/gp130. Our aim was to challenge this paradigm and evaluate whether under certain conditions trans -signaling drives the production of acute phase proteins. These experiments would thus also provide insights into IL-6 signaling complex formation, exploring if a trimeric versus a tetrameric complex is sufficient to drive IL-6 responses. Methods & Results 25F10 is an anti-mouse IL-6R monoclonal antibody (mAb) that binds to both sIL-6R and mIL-6R yet the unique property of inhibiting only trans signaling events. Using Biacore, 25F10 does not prevent the binding of IL-6 to the IL-6R or the recruitment of gp130 to the receptor/ligand complex. Mapping the epitope of 25F10 through mutation of the mouse IL-6R, revealed that 25F10 binds IL-6R at site IIb, potentially preventing the formation of the hexameric signalling complex. Data obtained from in vivo blockade of IL-6 trans -signaling in mouse models of inflammation show that under certain inflammatory conditions 25F10 blocks the production of acute phase proteins, i.e., serum amyloid A, previously believed to be produced via classical IL-6 signaling only. Conclusions These data challenge the existing paradigm that only classical ( cis ) signaling can induce the production of acute phase proteins. In addition, our data suggest new insights into the biology of the IL-6 signaling complex formation be exploiting the unique mechanism of action of 25F10.

A hallmark of autoimmunity and other chronic diseases is the overexpression of chemokines resulting in a detrimental local accumulation of proinflammatory immune cells. Chemokines play a pivotal role in cellular recruitment through interactions with both cell surface receptors and glycosaminoglycans (GAGs). Anti-inflammatory strategies aimed at neutralizing the chemokine system have to-date targeted inhibition of the receptor-ligand interaction with receptor antagonists. In this study, we describe a novel strategy to modulate the inflammatory process in vivo through mutation of the essential heparin-binding site of a proinflammatory chemokine, which abrogates the ability of the protein to form higher-order oligomers, but retains receptor activation. Using well-established protocols to induce inflammatory cell recruitment into the peritoneal cavity, bronchoalveolar air spaces, and CNS in mice, this non-GAG binding variant of RANTES/CCL5 designated [44AANA47]-RANTES demonstrated potent inhibitory capacity. Through a combination of techniques in vitro and in vivo, [44AANA47]-RANTES appears to act as a dominant-negative inhibitor for endogenous RANTES, thereby impairing cellular recruitment, not through a mechanism of desensitization. [44AANA47]-RANTES is unable to form higher-order oligomers (necessary for the biological activity of RANTES in vivo) and importantly forms nonfunctional heterodimers with the parent chemokine, RANTES. Therefore, although retaining receptor-binding capacity, altering the GAG-associated interactive site of a proinflammatory chemokine renders it a dominant-negative inhibitor, suggesting a powerful novel approach to generate disease-modifying anti-inflammatory reagents.