Figure S4. Binding of DSP502 to A549 and SKOV-3 cells with and without IFN-γ pretreatment.

Figure S1. Characterization of DSP502 by Size Exclusion Chromatography (SEC) and SEC-multi-angle light scattering (MALS).

Figure S5. Expression of DNAM-1 in T and NK cells and CD25 expressions in T and NK cells with or without DSP502 treatment co-cultured with ES-2.WT, ES-2.PD-L1KO, ES-2.PVRKO, or ES-2.PVR/PD-L1KO.

PD-1 immune checkpoint inhibition (ICI) is ineffective in most patients with cancer. However, combination therapy can improve response rates, with the checkpoint TIGIT being a particularly interesting candidate as it is expressed on tumor-infiltrating exhausted T and NK cells. TIGIT's primary ligand, PVR, is overexpressed in many cancers, and both TIGIT and PVR correlate with poor prognosis. To therapeutically exploit this, we developed a novel therapeutic termed dual signaling protein 502 (DSP502). DSP502 is composed of the extracellular domains of TIGIT and PD-1, each fused to human IgG1 Fc containing knob-in-hole mutations. DSP502 was designed to simultaneously block PVR/TIGIT and PD-L1/PD-1 by binding to cancer cell-expressed PVR and PD-L1. Moreover, the human IgG1 domain can recruit FcR-positive effector cells to further reactivate anticancer immunity. Treatment with DSP502 potentiated NK cell activation and boosted the anticancer cytotoxicity of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes from patients with non-small cell lung cancer (NSCLC) and metastatic colorectal cancer toward cancer cells expressing both PD-L1 and PVR. Transcriptomic analysis confirmed NSCLC as a potential target, showing co-expression of TIGIT and PD-1 (PDCD1) on a high percentage of exhausted CD8+ T cells. Notably, treatment with DSP502 not only blocked checkpoint signaling but also preserved surface expression of the co-stimulatory PVR ligand, DNAM-1, on T and NK cells. Finally, DSP502 inhibited tumor growth by potentiating antitumor immunity in xenograft ovarian and lung cancer models. Collectively, these findings demonstrate that DSP502, by blocking PVR and PD-L1 pathways, has dual ICI activity and holds potential therapeutic benefits for cancers such as NSCLC.

CD47 is a cell surface ligand expressed on all nucleated cells. It is a unique immune checkpoint protein acting as “don’t eat me” signal to prevent phagocytosis and is constitutively overexpressed in many tumors. However, the underlying mechanism(s) for CD47 overexpression is not clear. Here, we show that irradiation (IR) as well as various other genotoxic agents induce elevated expression of CD47. This upregulation correlates with the extent of residual double-strand breaks (DSBs) as determined by γH2AX staining. Interestingly, cells lacking mre-11, a component of the MRE11-RAD50-NBS1 (MRN) complex that plays a central role in DSB repair, or cells treated with the mre-11 inhibitor, mirin, fail to elevate the expression of CD47 upon DNA damage. On the other hand, both p53 and NF-κB pathways or cell-cycle arrest do not play a role in CD47 upregualtion upon DNA damage. We further show that CD47 expression is upregulated in livers harvested from mice treated with the DNA-damage inducing agent Diethylnitrosamine (DEN) and in cisplatin-treated mesothelioma tumors. Hence, our results indicate that CD47 is upregulated following DNA damage in a mre-11-dependent manner. Chronic DNA damage response in cancer cells might contribute to constitutive elevated expression of CD47 and promote immune evasion.

Supplementary Table 1 from hsa-miR-29c* Is Linked to the Prognosis of Malignant Pleural Mesothelioma

Supplementary Figures 1-9, Table 1 from hsa-miR-191 Is a Candidate Oncogene Target for Hepatocellular Carcinoma Therapy

Treatment of Diffuse Large B Cell Lymphoma (DLBCL) patients with rituximab and the CHOP treatment regimen is associated with frequent intrinsic and acquired resistance. However, treatment with a CD47 monoclonal antibody in combination with rituximab yielded high objective response rates in patients with relapsed/refractory DLBCL in a phase I trial. Here, we report on a new bispecific and fully human fusion protein comprising the extracellular domains of SIRPα and 4-1BBL, termed DSP107, for the treatment of DLBCL. DSP107 blocks the CD47:SIRPα ‘don’t eat me’ signaling axis on phagocytes and promotes innate anticancer immunity. At the same time, CD47-specific binding of DSP107 enables activation of the costimulatory receptor 4-1BB on activated T cells, thereby, augmenting anticancer T cell immunity. Using macrophages, polymorphonuclear neutrophils (PMNs), and T cells of healthy donors and DLBCL patients, DSP107-mediated reactivation of immune cells against B cell lymphoma cell lines and primary patient-derived blasts was studied with phagocytosis assays, T cell activation and cytotoxicity assays. DSP107 anticancer activity was further evaluated in a DLBCL xenograft mouse model and safety was evaluated in cynomolgus monkey. Treatment with DSP107 alone or in combination with rituximab significantly increased macrophage- and PMN-mediated phagocytosis and trogocytosis, respectively, of DLBCL cell lines and primary patient-derived blasts. Further, prolonged treatment of in vitro macrophage/cancer cell co-cultures with DSP107 and rituximab decreased cancer cell number by up to 85%. DSP107 treatment activated 4-1BB-mediated costimulatory signaling by HT1080.4-1BB reporter cells, which was strictly dependent on the SIRPα-mediated binding of DSP107 to CD47. In mixed cultures with CD47-expressing cancer cells, DSP107 augmented T cell cytotoxicity in vitro in an effector-to-target ratio-dependent manner. In mice with established SUDHL6 xenografts, the treatment with human PBMCs and DSP107 strongly reduced tumor size compared to treatment with PBMCs alone and increased the number of tumor-infiltrated T cells. Finally, DSP107 had an excellent safety profile in cynomolgus monkeys. DSP107 effectively (re)activated innate and adaptive anticancer immune responses and may be of therapeutic use alone and in combination with rituximab for the treatment of DLBCL patients.

Background TIGIT, an inhibitory immune checkpoint, is a target of interest for immuno-oncology combination therapies. TIGIT is part of a complex molecular network containing four receptors (DNAM1, TIGIT, PVRIG and CD96) and two ligands (PVR and PVRL2). Here we describe Dual Signaling Protein 502 (DSP502), a novel, multi-functional IgG1-Fc-fusion protein targeting this molecular pathway in a unique way. DSP502, comprising the extracellular domains of TIGIT and PD1, is designed to simultaneously bind its two respective ligands, PVR and PD-L1, overexpressed on cancer and myeloid cells in the tumor microenvironment. DSP502 binds PVR preventing inhibitory signaling through TIGIT and CD96 and promoting DNAM1 costimulatory signaling on activated T- and NK-cells. DSP502's PD1 arm binds PD-L1 to unleash effector T-cells through checkpoint inhibition. In parallel, DSP502's IgG1-Fc delivers an immune-activating signal via Fc receptors. The net effect is enhanced anti-tumor immunity (figure 1). Methods DSP502 heterodimer was successfully produced in a mammalian expression system. DSP502 was evaluated for binding to its cognate ligands on cells and in ELISA-based assays, with and without competing antibodies. NK and PBMC killing activity were evaluated against human K562 CML cells overexpressing PVR. Simultaneous binding of DSP502 to fluorescently-labeled tumor and NK-cells was evaluated by FACS. In vivo activity of DSP502 was evaluated in a humanized NSG A549 NSCLC xenograft mouse model. Results Both DSP502 arms were shown to bind their cognate ligands in ELISA and on cell surface. DSP502 binding was dependent on the presence of both ligands on cells and was abolished by competing antibodies to the respective targets, demonstrating binding specificity and the 'AND-gate' phenomenon. Overexpression of PVR reduced the sensitivity of K562 cells to NK-cell mediated killing, while DSP502 treatment restored it as measured by target cell killing and granzyme-B secretion. Increased, dose-dependent, complexation of NK- and tumor cells was observed following DSP502 treatment and was abolished by both PVR and FcR antibodies. Treatment with DSP502 markedly inhibited tumor growth of A549-NSCLC xenograft in a humanized NSG mouse model, with all mice being tumor-free at the end of the experiment, compared to control PBMC-injected mice. Conclusions Here we report the design and function of a novel immunotherapeutic fusion protein, DSP502, that offers multiple functionalities that can coordinately and synergistically drive anti-tumor immunity. Beyond targeting PVR and PDL1, DSP502 has the potential to additionally impact the TIGIT pathway through its effects on CD96 and DNAM1. DSP502 is currently in IND-enabling studies and CMC development. Ethics Approval The study was conducted at the Authority of Biological and Preclinical Models, the Hebrew University of Jerusalem, Ein Kerem, Sharet Specific Pathogen-Free (SPF) Unit under the Hebrew university ethic committee board approval (number MD-19-15815-5). Abstract 790 Figure 1

The mainstay of treatment for Diffuse Large B cell Lymphoma (DLBCL) is conventional chemotherapy combined with anti-CD20 monoclonal antibody rituximab (RTX). However, a subset of patients is refractory to treatment and between 20 to 50% of patients will, after experiencing an initial complete response (CR), develop resistance to treatment and relapse with poor prognosis. Therefore, additional therapeutic options are urgently needed. In this respect, combination of RTX treatment with CD47 monoclonal antibodies has yielded high objective response rates in patients with relapsed/refractory DLBCL in recent phase I trials. Interestingly, although CD47-targeting specifically activates the innate immune system, treatment with CD47 antibodies augments antigen-presentation in the context of MHC by macrophages and dendritic cells, thereby, triggering cross-priming of T cells in murine models. This T cell activation was pivotal in vivo efficacy in these murine models. Thus, a clear rationale exists for the development of novel therapeutics that exploit CD47 checkpoint inhibition while simultaneously stimulating anticancer T cell immunity. Here, we report on such an immunotherapeutic, termed Dual Signaling Protein 107 (DSP107), comprising a computationally-designed fusion of human soluble SIRPα and 4-1BBL. DSP107 was designed to bind to CD47 on cancer cells and block the CD47/SIRPα inhibitory signal delivered to phagocytes. Further, DSP107 was designed to bind to 4-1BB, a costimulatory receptor upregulated upon TCR/MHC interaction and a validated surrogate marker for the tumor-reactive subset of T cells in tumor tissue. Since 4-1BB activation by soluble 4-1BBL requires cross-linking, DSP107 will trigger 4-1BB signaling only after binding to CD47. This CD47-mediated surface immobilization of DSP107 enables delivery of the 4-1BBL-4-1BB costimulatory signal to tumor localized T cells. This dual immunomodulatory effect of DSP107 is designed to unleash both innate and adaptive immune responses targeted to the tumor site (Figure 1). Treatment with DSP107 alone or in combination with RTX triggered significant phagocytosis of a panel of DLBCL cancer cell lines as well as primary patient-derived DLBCL cells by macrophages and neutrophils within 3 hours. Further, after longer term incubation of 24h an ~85% reduction in remaining tumor cells was detected upon combined DSP107 and RTX treatment compared to medium control, whereas an increase in apoptosis was detected in the remaining cells. The pro-phagocytic activity of DSP107 was equal to both CD47 antibody as well as SIRPα:Fc. Simultaneously, binding of DSP107 to CD47 enabled 4-1BB costimulatory signaling by reporter cell line HT1080.4-1BB only on CD47-coated plates. Further, in co-cultures of HT1080.4-1BB with CHO.wt and CHO cells ectopically expressing human CD47, 41BB activation was only observed after binding of DSP107 to human CD47. This activation of 4-1BB costimulatory signaling triggered prominent T cell proliferation in mixed cultures of isolated peripheral blood T cells with cancer cells and augmented T cell cytotoxicity in vitro in a concentration and Effector to Target ratio dependent manner. Finally, injection of peripheral blood mononuclear cells (PBMCs) in mice with established SUDHL6 xenografts and simultaneous treatment with DSP107 triggered a strong reduction in tumor size compared to treatment with PBMCs alone. In conclusion, DSP107 clearly inhibits the CD47/SIRPα inhibitory axis and augments phagocytic removal of cancer cells by innate immune cells. Moreover, binding of DSP107 to CD47 enables the 4-1BBL-mediated costimulation of antitumor T cell cytotoxicity. Thus, DSP107 activates both innate and adaptive anticancer immunity and may be of use for the treatment of DLBCL alone or in combination with RTX. Disclosures Cendrowicz: Kahr Medical: Research Funding. Jacob:Kahr Medical: Current Employment. Greenwald:Kahr Medical: Current Employment. Tamir:Kahr Medical: Current Employment. Huls:Kahr Medical: Research Funding. Foley-Comer:Kahr Medical: Current Employment. Pereg:Kahr Medical: Current Employment. Chajut:Kahr Medical: Current Employment. Peled:Kahr Medical: Consultancy. Bremer:Kahr Medical: Consultancy, Research Funding.

Abstract Background: The (re)activation of anticancer innate and adaptive immunity is at the forefront of developments in cancer therapy. Here, we report on a new immunotherapeutic fusion protein, termed Dual Signaling Protein 107 (DSP107). DSP107 was designed to combine activation of innate and adaptive immunity, by both blocking CD47/SIRPα interaction and activating 4-1BB. CD47 is overexpressed on cancer cells and upon binding to SIRPα on phagocytes transmits a “don’t eat me” signal, thereby suppressing innate immunity. 4-1BB is a costimulatory receptor that is transiently upregulated on tumor-infiltrating T-cells and is considered a surrogate marker for the tumor-reactive T-cell population. Activation of 4-1BB using its ligand or by agonistic antibodies reactivates anti-cancer T-cell immunity. In DSP107, the extracellular domains of SIRPα and 41BBL have been fused, yielding a dual function protein. DSP107 is produced as a homotrimer due to the trimerization property of 41BBL, an essential element for activating the 41BB receptor, a member of the TNF super-family of receptors. DSP107 was designed to bind to CD47 on tumor cells, thereby removing the inhibitory signal delivered to phagocytes. Simultaneously, CD47-mediated surface immobilization of DSP107 enables delivery of the 41BBL-4-1BB costimulatory signal to tumor localized T-cells. This dual immunomodulatory effect of DSP107 is designed to unleash both innate and adaptive immune responses targeted to the tumor site. Methods and Results: Trimeric DSP107 was successfully produced in a mammalian expression system. Both sides of DSP107 bound their cognate counterparts in kinetic Blitz binding assays and on human tumor and immune cell surfaces. The binding affinity of DSP107 was 1.6 nM for human CD47 and 0.69 nM for human 4-1BB as determined using BIAcore analysis. DSP107 blocked the interaction of SIRPα with CD47 in an ELISA-based competition assay (EC50 of 0.03 nM). DSP107 induced granulocyte- and macrophage-mediated phagocytosis of several lymphoma, leukemia and carcinoma cell lines in vitro. Further, DSP107 treatment triggered phagocytosis of primary AML cells by autologous macrophages. Co-treatment with DSP107 and therapeutic tumor-targeting antibodies, i.e., rituximab or cetuximab, resulted in enhanced phagocytosis of lymphoma or carcinoma cells, respectively. In a reporter assay measuring IL-8 secretion upon binding to/activation of 4-1BB, DSP107 activated 4-1BB signaling only in the presence of CD47-expressing cells. Further, DSP107 augmented the activation of purified T-cells activated by suboptimal concentrations of αCD3 + IL2 or αCD3/αCD28 Dynabeads in CD47 coated plates, as measured by percentage of CD25 expressing cells (up to 3-fold). When PBMCs were co-cultured with or without CD47-expressing cancer cells and stimulated with suboptimal concentrations of αCD3 + IL2, DSP107 treatment resulted in increased secretion of IFNg up to 2-fold), and increased T-cell proliferation (up to 2-fold). Conclusions: Here we demonstrate the feasibility and functional activity of DSP107, a novel therapeutic protein that combines activation of both the innate and adaptive immune system. Dual targeting, by the two functional sides of DSP107, offers multiple functionalities that act simultaneously and may result in a synergistic effect. DSP107 is now in IND-enabling studies and CMC development. The DSP platform can be designed for selective tumor site or microenvironment targeting and is adaptable to most checkpoint targets. Citation Format: Yosi M. Gozlan, Susan Hilgendorf, Alexandra Aronin, Yehudith Sagiv, Liat Ben-gigi-Tamir, Shira Amsili, Ami Tamir, Iris Pecker, Shirley Greenwald, Ayelet Chajut, Adam Foley-Comer, Yaron Pereg, Amnon Peled, Michal Dranitzki-Elhalel, Edwin Bremer. DSP107—a novel SIRPα-4-1BBL dual signaling protein (DSP) for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A076.

Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144-111 mmHg; TLR7 145-106 mmHg; both P<0.05), and also normalized their elevated urinary protein:creatinine ratios (TLR3 5.68-3.72; TLR7 5.57-3.84; both P<0.05). On gestational day 17, aortic endothelium-dependent relaxation responses improved significantly in TLR3-induced and TLR7-induced hypertensive mice that received PLX-PAD cells on gestational day 14 (TLR3 35-65%; TLR7 37-63%; both P<0.05). In addition, markers of systemic inflammation and placental injury, increased markedly in both groups of TLR-induced hypertensive mice, were reduced by PLX-PAD cells. Importantly, PLX-PAD cell therapy had no effects on these measures in pregnant control mice or on the fetuses. These data demonstrate that PLX-PAD cell therapy can safely reverse pre-eclampsia-like features during pregnancy and have a potential therapeutic role in pre-eclampsia treatment.

Preeclampsia is a human pregnancy-specific disease, defined as the occurrence of hypertension and significant proteinuria after the 20th week of gestation, and affects 2-6% of previously healthy women. Preeclampsia is characterized by a generalized systemic maternal inflammatory response, placental dysfunction and has no effective treatment except abortion or delivery. PLacenta eXpanded (PLX)-PAD are placenta derived mesenchymal-like adherent stromal cells expanded in Pluristem's proprietary bioreactor system using a three-dimensional culture method. PLX-PAD possess immunomodulatory properties as well as pro-angiogenic and anti-fibrotic properties and have demonstrated a therapeutic potential in various animal models. PLX-PAD are suitable for allogeneic administration without HLA-matching due to their low immunogenicity. . The therapeutic effect of PLX-PAD was tested in a mouse model of preeclampsia. Preeclampsia was generated by intra-peritoneal introduction of either TLR3 agonist (poly I:C) or TLR7 agonist (R837) on days 13, 15, and 17 of gestation in C57BL/6 mice followed by PLX-PAD administration on day 14 of gestation. PLX-PAD treatment resulted in progressive reduction of SBP over a 3 day period and normalization of urinary protein/creatinine ratio and aortic endothelium-dependent relaxation responses within 4 days after treatment. PLX-PAD had no significant effects on the number of fetuses or incidence of fetal demise. PLX-PAD also reduced spleen weight/body weight ratios, normalized splenic levels of gamma-delta T cells, decreased plasma IL-6 levels, and restored plasma IL-4 levels in TLR agonist treated mice. Additionally, PLX-PAD treatment decreased fibrin deposition in the placental vasculature and significantly reduced placental HIF-1alpha protein levels. These data suggest that PLX-PAD treatment is a potential therapy for preeclampsia.

PLacental eXpanded (PLX) cells (Pluristem Therapeutics Inc.) are human placenta-derived, mesenchymal-like adherent stromal cells that release proteins in response to the environment of the host. PLX cells are non-immunogenic and have been shown to decrease inflammation and increase angiogenesis in inflammatory and ischemic conditions. Therefore, we tested whether PLX cell treatment could attenuate symptoms of preeclampsia (PE) in mice. We hypothesized that one-time PLX cell treatment would decrease the pregnancy-dependent hypertension, proteinuria, endothelial dysfunction, splenomegaly, inflammation, and placental injury induced by Toll-like receptor (TLR) activation during pregnancy. Pregnant C57BL/6 mice were given ip injections of saline vehicle (P), the TLR3 agonist poly I:C (PPIC), or the TLR7 agonist R837 (PR) on days 13, 15, and 17 of gestation. P, PPIC, and PR mice were also given either plasmalyte A (PLA, vehicle) or PLX cells (1 million) by im injection in the right leg on gestational day 14 (n=8 in each group). PLX cell treatment progressively decreased SBP over 3 days in PPIC and PR mice and had no effect in P control mice (day 17 SBP in mmHg: P+PLA = 100±4, P+PLX = 96±4, PPIC+PLA = 144±3, PPIC+PLX = 111±1, PR+PLA = 145±2, PR+PLX = 106±3; PPIC+PLA and PR+PLA p<0.05 vs. P+PLA). PLX cell treatment also normalized the urinary protein/creatinine ratio and aortic endothelium-dependent relaxation responses in PPIC and PR mice to that of P mice while having no significant effects on the number of fetuses or incidence of fetal demise per litter. Inflammation plays a central role in the development of TLR-induced PE and PLX cell treatment reduced spleen weight/body weight ratios, normalized splenic levels of gamma-delta T cells, decreased plasma IL-6 levels, and restored plasma IL-4 levels in PPIC and PR mice. Additionally, PLX cell treatment reduced fibrin deposition in the placental vasculature and significantly reduced placental HIF-1alpha protein levels. These data demonstrate that one-time PLX cell treatment after PE is induced was able to decrease inflammation, proteinuric hypertension, endothelial dysfunction, and placental injury in mice and may be beneficial in women with PE.