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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.

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.