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T cell engagers display meaningful, but limited, clinical activity often associated with toxicity. Activating innate immune cells is considered a safer, yet efficacious, alternative to T cells. We report here the preclinical development of AFVT-2101, a novel bispecific innate cell engager designed to induce tumor killing by high avidity binding to CD16A on innate cells and folate receptor alpha (FRα), a tumor-associated antigen with upregulated expression on certain solid tumor cells. Binding of AFVT-2101 to CD16A and FRα was assessed by ELISA and flow cytometry in the presence of physiological levels of IgG. The antitumor activity of the innate engager was evaluated by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in vitro assays, using purified NK cells or differentiated macrophages as effector cells against tumor cell lines with varying levels of FRα expression. In vivo studies were conducted in transgenic mice expressing human CD16A on myeloid cells and transplanted with FRα-expressing cancer cell lines. AFVT-2101 binds to CD16A and FRα with an apparent avidity of 0.1 nM and 0.05 nM, respectively. Physiological levels of human IgG do not alter binding kinetics. AFVT-2101 induces potent and selective ADCC and ADCP, even on FRαlow cancer cells. Further, AFVT-2101 is more efficacious and potent in both ADCC and ADCP assays than farletuzumab, an Fc-competent monoclonal antibody targeting FRα, which shares the same VH/VL sequence as AFVT-2101. In in vivo experiments, AFVT-2101 is active and significantly inhibits the growth of FRα+ tumors. Ex vivo flow cytometry analyses show a repolarization of the tumor microenvironment (TME) with reduction of the frequency of tumor-associated macrophages upon treatment with AFVT-2101. A reduction of FRα+ tumor cells in groups receiving therapy was also observed. In conclusion, AFVT-2101 binds selectively to CD16A and FRα, even in the presence of physiological levels of IgG, resulting in a potent and selective elimination of FRα+ tumor cells through the engagement of NK cell cytotoxicity (ADCC) and macrophage phagocytosis (ADCP). The high avidity for CD16A likely imparts increased potency and efficacy compared to the FRα-specific antibody farletuzumab. Moreover, the innate immune engager significantly restrains in vivo tumor growth and induces pharmacodynamic changes in line with TME repolarization. AFVT-2101 engages innate immune cells in a target-restricted manner and potentially imparts an efficacious clinical profile with favorable tolerability. Citation Format: Nicolo' Rigamonti, Jan Schmollinger, Peter Sandy, Michael Tomaszowski, Josef Caslavsky, Ahmad Trad, Daniel O’Shannessy, Jana Siegler, Eric J. Gaukel, Daniela Penston, Uwe Reusch, Zoë Johnson. AFVT-2101: A FRα × CD16A bispecific innate cell engager for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1811.

The TGFβ receptor inhibitor galunisertib (GAL) has been demonstrated as an effective strategy for the treatment of pancreatic cancer (PC) patients, in part through the modulation of stromal microenvironment. However, alternative pathways driving stromal paracrine signals might impair its effect. Autotaxin (ATX) is an enzyme that converts lysophosphatidylcholine into its bioactive lipid, lysophosphatidic acid (LPA), which in turn promotes inflammation and fibrosis, and contributes to treatment resistance. IOA-289 is a novel ATX inhibitor in clinical development for the treatment of tumors burdened with a high degree of stromal involvement. Here, we hypothesized that ATX could sustain an adaptive response upon inhibition of TGFβ receptor signaling, impairing GAL antitumor activity.Among ten different murine PC orthotopic models, mice bearing RC416 tumors had the highest plasma levels of TGFβ1 as well as high infiltration of cancer associated fibroblasts (CAFs) and fibrosis as measured by Masson’s staining. In coculture models of RC416 with murine pancreatic stellate cells (mPSC4), we measured a significant overexpression of ATX upon treatment with galunisertib mainly by mPSC4, which showed a skewing toward an inflammatory iCAF phenotype through the upregulation of CXCL1 and LY6C1 and the down-regulation of myCAF markers including ACTA2 and Taglin. In the RC416-mPSC4 coculture model, anti-tumor activity was achieved with a combination of gemcitabine plus GAL plus IOA-289, whereas these treatments were ineffective in single culture conditions. Mice bearing RC416 tumors reached a statistically significantly longer survival following treatment with gemcitabine plus IOA-289 or gemcitabine plus GAL [median overall survival (mOS)= 35 days, p Citation Format: Fabio Sabbadini, Monica Bertolini, Domenico Mangiameli, Serena Contarelli, Zoë Johnson, Michael M. Lahn, Silvia Pietrobono, Davide Melisi. Targeting stromal autotaxin synergizes with TGF beta inhibition in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1064.

CD73, an ecto-5'-nucleotidase, is frequently overexpressed in the tumor microenvironment and can be found on tumor, stromal cells and immune cells. Its function is to catalyze the conversion of adenosine monophosphate (AMP) to adenosine and phosphate, both at the cell surface and as a soluble factor. CD73 has gained interest as a therapeutic target in cancer due to the immunosuppressive role of adenosine in the tumor microenvironment. IOA-237 is a fully human, phage derived monoclonal antibody fully cross-reactive to mouse and human and with high potency against soluble and cell surface CD73. This novel antibody demonstrates in vitro and in vivo characteristics that suggest a best-in-class potential. We demonstrate that IOA-237 is able to significantly reduce tumor outgrowth in multiple mouse models of solid tumors. In the low CD73 expressing CT26 mouse colorectal cancer model, monotherapeutic application of IOA-237 showed tumor outgrowth reduction compared with isotype control. Best-in-class potential was confirmed with head-to-head comparison against a clinical stage anti-CD73 mAb, MEDI9447. Furthermore, inhibition of CD73 by IOA-237 was combined with inhibitors of novel immuno-oncology targets to study complementary modes of action in mouse models of solid tumors. These data indicate the potential of IOA-237 in monotherapeutic and combination therapeutic approaches and warrants further development. Additional in vitro and in vivo efficacy studies are ongoing and non-clinical safety studies are being planned. Citation Format: Marcel A. Deken, Alan M. Carruthers, Karolina Niewola-Staszkowska, Gráinne Gernon, Hilary Sandig, Pritom Shah, Michael M. Lahn, Zoë Johnson. IOA-237 - A potent anti-CD73 mAb demonstrates monotherapeutic and combination efficacy in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1877.

CD73 is a membrane-bound nucleotidase receptor which is frequently overexpressed in the tumour microenvironment and can be found on both tumour and immune cell types. Its function is to catalyse the conversion of adenosine monophosphate (AMP) to adenosine and phosphate and it has been proposed as a therapeutic target in cancer due to adenosines role in tumour immune suppression. Using multiple approaches, a series of novel CD73 antibodies have been characterised for their therapeutic potential and benchmarked against agents currently undergoing clinical evaluation Inhibition of CD73 activity was evaluated using an Amplex Red-based coupled adenosine assay against both human and mouse CD73, and kinetics of antibody binding were determined using BioLayer Interferometry. Cellular assays were then utilised to further evaluate the antibodies in vitro. The ability of the CD73 antibodies to internalise was evaluated using two different methods, a Fab-ZAP killing assay and the IncuCyteTM FabFluor internalisation assay. The antibodies have also undergone functional studies, investigating the ability of the CD73 antibodies to disrupt the production of adenosine in tumour cells. We demonstrate that the novel CD73 antibodies inhibit CD73 function by two different mechanisms; direct inhibition of enzyme activity and modulation of cell surface expression, both of a which have therapeutic potential to disrupt CD73-mediated adenosine production and therefore reduce antitumor immune responses. Ongoing studies are investigating the in vitro and in vivo efficacy of the clone selected for further development as a monotherapy and in combination with a novel small molecule inhibitor in murine models of cancer. Citation Format: Grainne Gernon, Suzanne Grooby, Louise Tonkin, Anja-Christina Bitterwolf, Lorna Stewart, Marcel Deken, Pritom Shah, Katherine Ewings, Zoe Johnson. Characterisation of novel CD73 antibodies as a therapeutic method of adenosine regulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5660.

The inhibition of PI3Kδ preferentially targets regulatory T cells and myeloid derived suppressor cells, breaking tumour-induced immune tolerance and restoring anti-tumour immunity. Bioinformatics and protein expression studies have shown that PIK3CD/PI3Kδ is also highly expressed in certain solid malignancies. IOA-244 is a novel, highly selective PI3Kδ inhibitor, with the unique property of being ATP non-competitive. In addition to the effects of PI3Kδ inhibition on the balance of CD8+ T cells and T regulatory cells in the tumour microenvironment, we have recently demonstrated that IOA-244 effectively controls the growth of tumour cells with high levels of PI3Kδ in vivo in T-cell deficient hosts. Analysis of TCGA datasets shows that certain solid tumours express high levels of PIK3CD transcript, comparable to that of diffuse large B cell lymphoma. Of these indications, cutaneous and uveal melanoma are amongst the highest expressers. To support the clinical development of IOA-244, we demonstrate that patient-derived melanoma cells with a high expression of PIK3CD are susceptible to treatment with IOA-244 in a mouse patient-derived xenograft model. Furthermore, in vitro explant studies using clinical biopsy material demonstrates that PI3Kδ expression can be detected in tumour tissue and that IOA-244 has an anti-proliferative function when added to these samples. Based on these and other supporting data, IOA-244 has received clinical trial approval in Europe and phase I clinical testing is underway in solid tumours with an anticipated high Treg:CD8 ratio and/or high protein expression of PI3Kδ. Citation Format: Amy R. MacQueen, Giuditta Viticchie, Karolina Niewola, Francesca Finotello, Ian Powley, Pritom Shah, Lars van der Veen, Michael Lahn, Zoe Johnson. A novel, highly selective PI3Kδ inhibitor for the treatment of solid malignancies that express high levels of target protein as assessed by immunohistochemistry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 666.

The Class I Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) family is an attractive target class for the treatment of hematological and solid malignancies. In addition to the direct effects of PI3Kδ inhibition on B cell malignancies, inhibiting PI3Kδ preferentially targets regulatory T cells and myeloid derived suppressor cells, thus breaking tumor-induced immune tolerance and restoring anti-tumor immunity. To date, the development of selective inhibitors of the Class I PI3K family has been hampered by the lack of isoform specificity as well as safety issues. IOA-244 is a novel, highly selective, orally bioavailable PI3Kδ inhibitor, with the unique property of being ATP non-competitive; these characteristics make IOA-244 the ideal drug to explore the hypothesis that PI3Kδ inhibition can modulate anti-tumor immunity as a monotherapy and in combination in clinical trials. In mouse models, IOA-244 inhibited tumor growth when combined with either anti-PD-1 or anti-PD-L1. In the CT26 model the composition of the tumour infiltrating lymphocytes showed a marked decrease in the suppressor cell populations, i.e. Tregs and MDSCs, as well as tumor associated macrophages (TAMs). Conversely a concomitant increase in natural killer cells as well as the ratio of cytotoxic CD8+ T cells: Treg cells was observed. Furthermore, in vitro studies using primary human T cells demonstrated a selective and concentration-dependent suppression of Treg cells by IOA-244 whilst leaving CD8+ T cell proliferation intact. In addition to effects on the immune system, we are exploring the potential of IOA-244 to mediate direct anti-cancer effects on solid tumors with a high expression of PIK3CD. IOA-244 induced a concentration-dependent inhibition of p-AKT in MDA-MB-231 cells. Furthermore, we demonstrate strong anti-proliferative activity by IOA-244 on hepatocellular tumor lines in vitro. Activity in these and other cell and patient derived lines is being studied, both in xenograft in vivo studies and in vitro models. Based on GLP toxicology studies, IOA-244 has the potential to be a best-in-class PI3Kδ inhibitor with a safety and pharmacokinetic profile that is amenable for use alone and in combination with immunotherapies for the treatment of solid malignancies. IOA-244 is poised to enter phase I clinical testing in selected cancer indications in the first half of 2019. Citation Format: Katherine Ewings, Amy MacQueen, Pritom Shah, Anna Tsapara, Evangelia Papakonstanti, Lars van der Veen, Michael Lahn, Zoe Johnson. Preclinical development of a novel, highly selective PI3Kδ inhibitor, IOA-244, for the treatment of solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2692.

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.

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