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In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1beta/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.

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.

Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies., Bispecific antibodies allow for novel therapeutic approaches but industrial-scale production and immunogenicity represent significant challenges. Here Fischer et al. describe a unique human bispecific antibody format that exploits differing light chains to overcome these obstacles.

Rhipicephalus sanguineus, the common brown dog tick, produces several chemokine-binding proteins which are secreted into the host in its saliva to modulate the host response during feeding. Two of these demonstrate very restricted selectivity profiles. Here, we describe the characterization of the third, which we named Evasin-4. Evasin-4 was difficult to produce recombinantly using its native signal peptide in HEK cells, but expressed very well using the urokinase-type plasminogen activator signal peptide. Using SPR, Evasin-4 was shown to bind most CC chemokines. Investigation of the neutralization properties by inhibition of chemokine-induced chemotaxis showed that binding and neutralization did not correlate in all cases. Two major anomalies were observed: no binding was observed to CCL2 and CCL13, yet Evasin-4 was able to inhibit chemotaxis induced by these chemokines. Conversely, binding to CCL25 was observed, but Evasin-4 did not inhibit CCL25-induced chemotaxis. Size-exclusion chromatography confirmed that Evasin-4 forms a complex with CCL2 and CCL18. In accordance with the standard properties of unmodified small proteins, Evasin-4 was rapidly cleared following in vivo administration. To enhance the in vivo half-life and optimize its potential as a therapeutic agent, Fc fusions of Evasin-4 were created. Both the N- and C-terminal fusions were shown to retain binding activity, with the C-terminal fusion showing a modest reduction in potency.

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.

Antagonism of the CRTH2 receptor represents a very attractive target for a variety of allergic diseases. Most CRTH2 antagonists known to date possess a carboxylic acid moiety, which is essential for binding. However, potential acid metabolites O-acyl glucuronides might be linked to idiosynchratic toxicity in humans. In this communication, we describe a new series of compounds that lack the carboxylic acid moiety. Compounds with high affinity (K i10 nM) for the receptor have been identified. Subsequent optimization succeeded in reducing the high metabolic clearance of the first compounds in human and rat liver microsomes. At the same time, inhibition of the CYP isoforms was optimized, giving rise to stable compounds with an acceptable CYP inhibition profile (IC50 CYP2C9 and 2C191 μM). Taken together, these data show that compounds devoid of carboxylic acid groups could represent an interesting alternative to current CRTH2 antagonists in development.

New spiroindolinone antagonists of CRTH2 are described. Following identification of insufficient stability in human plasma as an important liability of the lead compounds, replacement of the spirosuccinimide core with a spirohydantoin or spiropyrrolidinone structure has yielded a compound that is fully stable in human plasma and with good potency in a human whole blood assay (IC50 = 69 nM) but shows a much lower oral bioavailability (6–9% in rodents) than the earlier compounds. Successive optimization aimed at restoring an acceptable oral bioavailability has yielded compound (S)-17a, which exhibits both stability in human plasma and a good oral bioavailability in rat (37%) and mouse (39%). This compound is also active in a mouse model of ovalbumin-induced lung inflammation following oral dosing at 30 mg/kg.

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.

Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, (57)KSKQAR(62)) along with Lys(17), significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.

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.

Neutralizing CD47, the ‘don't eat me signal’ hijacked by different tumor types, is a novel generally applicable therapeutic strategy. Because of a distinct mechanism of action and the ability to stimulate the innate anti-tumor immunity, CD47-neutralizing agents are poised as attractive candidates for combination therapies in association with other immunotherapies. However, the development of general CD47 antagonists could be hindered by the ubiquitous and abundant expression of CD47 on virtually all healthy cells. To overcome potential pharmacological and clinical liabilities of a general CD47 antagonist, we have developed bispecific kappa/lambda bodies, which selectively target CD47 in cancer cells. These kappa/lambda bodies: (i) are full-length bispecific IgGs, (ii) bind with high affinity and neutralize the CD47-SIRP alpha interaction in cancer cells expressing a tumor-associated antigen (TAA), and (iii) mediate efficient cell killing of TAA-positive cancer cells in vitro through Fc-dependent mechanisms such as ADCP (antibody mediated cellular phagocytosis) and ADCC (antibody mediated cellular cytotoxicity). We are currently developing two molecules of this type, one targeting CD47 and CD19 (for B cell malignancies), the other targeting CD47 and mesothelin (for various mesothelin-positive solid tumors). The efficacy of the CD47/CD19 kappa/lambda body was demonstrated in vivo, using two B-cell lymphoma xenograft models in NOD/SCID mice. We also performed a pharmacokinetics study in non-human primates with the CD47/CD19 lead candidate, with the objective of assessing the potential “antigen sink” effect related to ubiquitous CD47 expression on erythrocytes, platelets and other cells. Encouragingly, the CD47/CD19 kappa/lambda body administered in a single dose to cynomolgus monkeys, at 0.5 and 10 mg/kg, showed an acceptable pharmacokinetic profile and the absence of hematological toxicities. The example of the CD47/CD19 kappa/lambda body illustrates the power of the dual-targeting approach for addressing a ubiquitous cell surface receptor such as CD47. Citation Format: Krzysztof Masternak, Lucile Broyer, Elie Dheilly, Stefano Majocchi, Valéry Moine, Giovanni Magistrelli, François Rousseau, Ulla Ravn, Franck Gueneau, Pauline Malinge, Sébastien Calloud, Maud Charreton-Galby, Mireille Guerrier, Nessie Costes, Nicolas Bosson, Gérard Didelot, Lucie Bernard, Vanessa Buatois, Laura Cons, Laurence Chatel, Anne Papaioannou, Zoë Johnson, Walter Ferlin, Marie Kosco-Vilbois, Nicolas Fischer. Neutralizing CD47 in cancer cells with dual targeting kappa/lambda bodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2482. doi:10.1158/1538-7445.AM2015-2482

e14016 Background: Many cancers up-regulate the expression of CD47 in order to evade immune surveillance and killing. This ‘don’t eat me’ signal is a molecular means for healthy cells to limit thei...

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.

The biological activity of chemokines requires interactions with cell surface proteoglycans. We have determined the structure of the chemokine RANTES (regulated on activation normal T cell expressed) in the presence of heparin-derived disaccharide analogs by X-ray crystallography. These structures confirm the essential role of the BBXB motif in the interaction between the chemokine and the disaccharide. Unexpected interactions were observed in the 30s loop and at the amino terminus. Mutant RANTES molecules were designed to abrogate these interactions and their biological activity examined in vivo. The K45E mutant within the BBXB motif lost the capacity to bind heparin and the ability to elicit cellular recruitment. The Y3A mutant maintained its capacity to bind heparin but was unable to elicit cellular recruitment. Finally, a tetrasaccharide is the smallest oligosaccharide which effectively abolishes the ability of RANTES to recruit cells in vivo. These crystallographic structures provide a description of the molecular interaction of a chemokine with glycosaminoglycans.

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.

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1beta/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.

During organogenesis, immunosurveillance, and inflammation, chemokines selectively recruit leukocytes by activating seven-transmembrane-spanning receptors. It has been suggested that an important component of this process is the formation of a haptotactic gradient by immobilization of chemokines on cell surface glycosaminoglycans (GAGs). However, this hypothesis has not been experimentally demonstrated in vivo . In the present study we investigated the effect of mutations in the GAG binding sites of three chemokines, monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2, macrophage-inflammatory protein-1β/CCL4, and RANTES/CCL5, on their ability to recruit cells in vivo . These mutant chemokines retain chemotactic activity in vitro , but they are unable to recruit cells when administered intraperitoneally. Additionally, monomeric variants, although fully active in vitro , are devoid of activity in vivo . These data demonstrate that both GAG binding and the ability to form higher-order oligomers are essential for the activity of particular chemokines in vivo , although they are not required for receptor activation in vitro . Thus, quaternary structure of chemokines and their interaction with GAGs may significantly contribute to the localization of leukocytes beyond migration patterns defined by chemokine receptor interactions.

The chemokine superfamily consists of small, basic, heparin-binding proteins that play a pivotal role in basal trafficking as well as in activation and recruitment of leukocytes from the circulation to sites of inflammation. The chemokines are a subset of the cytokine family and are distinguished from other cytokines in that they activate seven-transmembrane (7TM) G protein-coupled receptors. They are a large family, with approximately 50 members identified to date, and for which 19 receptors have been described. The chemokine family is divided structurally into four subfamilies CXC, CC, CX3C and C, based on the position of the amino terminal cysteine residues. The majority of chemokines fall into the CXC or CC groups (also referred to as α and β subclasses respectively), and hence have been the most extensively studied. The known chemo-kine/receptor pairs are depicted in Fig. 1, which also indicates a second division based on the recent advances in chemokine biology—chemokines are either expressed constitutively and control basal trafficking or homing, or are inducible, and are involved in inflammation. Chemokines were generally named according to the function that was identified such as monocyte chemoattractant protein (MCP) or neutrophil activating peptide (NAP) but since many chemokines were concomitantly identified in more than one laboratory, a single sequence was attributed more than one name. Therefore a systematic nomenclature was recently adopted (Zlotnik and Yoshie 2000) and both common and systematic names are shown in Fig. 1.

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.