Krzysztof Masternak

Selective Blockade of the Ubiquitous Checkpoint Receptor CD47 Is Enabled by Dual-Targeting Bispecific Antibodies

CD47 is a ubiquitously expressed immune checkpoint receptor that is often upregulated in cancer. CD47 interacts with its counter-receptor SIRPα on macrophages and other myeloid cells to inhibit cancer cell phagocytosis and drive immune evasion. To overcome tolerability and “antigen sink” issues arising from widespread CD47 expression, we generated dual-targeting bispecific antibodies that selectively block the CD47-SIRPα interaction on malignant cells expressing a specific tumor-associated antigen; e.g., CD19 or mesothelin. These bispecific κλ bodies are fully human, native IgG1 molecules, combining tumor targeting and selective CD47 blockade with immune activating mechanisms mediated by the Fc portion of the antibody. CD47-neutralizing κλ bodies efficiently kill cancer cells in vitro and in vivo but interact only weakly with healthy cells expressing physiological levels of CD47. Accordingly, a κλ body administered to non-human primates showed a typical IgG pharmacokinetic profile and was well tolerated. Importantly, κλ bodies preserve their tumoricidal capabilities in the presence of a CD47 antigen sink. Thus, dual-targeting κλ bodies allow for efficacious yet safe targeting of CD47 in cancer. Such a bispecific design could be applied to limit the extent of neutralization of other ubiquitously expressed therapeutic targets.

Tumor-Directed Blockade of CD47 with Bispecific Antibodies Induces Adaptive Antitumor Immunity

CD47 serves as an anti-phagocytic receptor that is upregulated by cancer to promote immune escape. As such, CD47 is the focus of intense immuno-oncology drug development efforts. However, as CD47 is expressed ubiquitously, clinical development of conventional drugs, e.g., monoclonal antibodies, is confronted with patient safety issues and poor pharmacology due to the widespread CD47 “antigen sink”. A potential solution is tumor-directed blockade of CD47, which can be achieved with bispecific antibodies (biAbs). Using mouse CD47-blocking biAbs in a syngeneic tumor model allowed us to evaluate the efficacy of tumor-directed blockade of CD47 in the presence of the CD47 antigen sink and a functional adaptive immune system. We show here that CD47-targeting biAbs inhibited tumor growth in vivo, promoting durable antitumor responses and stimulating CD8+ T cell activation in vitro. In vivo efficacy of the biAbs could be further enhanced when combined with chemotherapy or PD-1/PD-L1 immune checkpoint blockade. We also show that selectivity and pharmacological properties of the biAb are dependent on the affinity of the anti-CD47 arm. Taken together, our study validates the approach to use CD47-blocking biAbs either as a monotherapy or part of a multi-drug approach to enhance antitumor immunity.

Abstract B54: Antagonizing CD47-SIRP alpha interaction with a bispecific antibody: A novel cancer immunotherapy approach

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 ‘don9t 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.

Remodeling tumor-associated macrophages with a CD47xMesothelin bispecific antibody for efficient elimination of solid tumor cells.

e15126Background: Mesothelin (MSLN) is a cell surface glycoprotein associated with poor prognosis in many cancers, with elevated soluble MSLN (sMSLN) serum levels considered a biomarker of disease ...

Abstract B37: NI-1701, a bispecific antibody for selective neutralization of CD47 in B cell malignancies

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, Zoe Johnson, Elie Dheilly, Valery 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.

Abstract 1495: Neutralization of CD47 in cancer cells with bispecific antibodies harnesses the phagocytic potential of tumor-infiltrating macrophages

The inhibitory “don9t 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 ‘don9t eat me’ signal hijacked by cancer cells. Citation Format: Krzysztof Masternak, Valery Moine, Lucile Broyer, Xavier Chauchet, Vanessa Buatois, Elie Dheilly, Stefano Majocchi, Giovanni Magistrelli, Yves Poitevin, Ulla Ravn, Eric Hatterer, Susana Salgado Pires, Limin Shang, Zoe 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.

Abstract 2482: Neutralizing CD47 in cancer cells with dual targeting kappa/lambda bodies

Neutralizing CD47, the ‘don9t 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, Valery Moine, Giovanni Magistrelli, Francois Rousseau, Ulla Ravn, Franck Gueneau, Pauline Malinge, Sebastien Calloud, Maud Charreton-Galby, Mireille Guerrier, Nessie Costes, Nicolas Bosson, Gerard Didelot, Lucie Bernard, Vanessa Buatois, Laura Cons, Laurence Chatel, Anne Papaioannou, Zoe 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