Table S1 shows the affinity and kinetics of AO-176 and AO-104 to human and cynomolgus monkey CD47 determined by surface plasmon resonance

Figure S1 depicts the sequence and analytical characteristics of AO-176

Figure S2 shows that AO-176 does not bind to mouse CD47

Figure S4 demonstrates the minimal reduction in RBC and hemoglobin levels in cynomolgus monkeys treated with AO-176

Figure S5 demonstrates that AO-176 does not mediate antibody dependent cell cytotoxicity of tumor cells

Figure S3 shows the specificity of AO-176 for human CD47

Table S2 is a summary of AO-176 tumor cell killing in solid and hematologic tumor cell lines

Table S3 is a summary of AO-176 mediated phagocytosis of solid and hematologic tumor cells

Abstract Overexpression of CD47 by tumor cells exploits an immune checkpoint that prevents tumor recognition and destruction by innate immune cells. Binding of tumor CD47 to SIRPα on macrophages and dendritic cells triggers a “don't eat me” signal that inhibits phagocytosis enabling escape from innate immune surveillance. Blockade of the CD47/SIRPα axis enables immune recognition and phagocytic clearance of tumor cells. We have developed a clinical stage CD47 targeting antibody AO-176, that is highly differentiated among agents in this class. AO-176 not only blocks the CD47/SIRPα interaction to induce tumor cell phagocytosis, but also: a) directly induces solid and hematologic tumor cell cytotoxicity and damage-associated molecular patterns (DAMPs); b) preferentially binds tumor versus normal cells, which is correlated with β1-integrin expression and localization; c) negligibly binds RBC; and d) exhibits improved binding at acidic pH as found in tumor microenvironments.Previously we have shown that AO-176 is efficacious in a variety of solid tumor xenograft models as well as in models of multiple myeloma and AML. Here, we show the therapeutic potential of AO-176 in pre-clinical models of lymphoma where CD47 is upregulated and associated with poor prognosis. Using a variety of cell based and in vivo models we show that AO-176 demonstrates increased binding to lymphoma cells at an acidic versus physiologic pH and that this binding and blocking of the do not eat me signal leads to enhanced phagocytosis of lymphoma cells either alone or in combination with rituximab. In addition, we show that AO-176 induces annexin V positivity in lymphoma cells as well as inducing a variety of DAMPs that ultimately may aid in inducing immunogenic cell death of the lymphoma cells. We also demonstrate that AO-176 is a potent tumor growth inhibitor in lymphoma xenograft models and appears to induce immune infiltrates as well as inflammatory cytokines.Taken together, these data show that AO-176 has strong therapeutic potential in lymphoma. AO-176 is currently being evaluated in clinical trials of select solid tumors (NCT03834948) and multiple myeloma (NCT04445701). Citation Format: Benjamin J. Capoccia, Michael J. Donio, John O. Richards, Ronald R. Hiebsch, Robyn J. Puro, Arun K. Kashyap, Daniel S. Pereira. AO-176, a highly differentiated clinical stage anti-CD47 antibody, is efficacious in pre-clinical models of lymphoma [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 954.

The signal regulatory protein α (SIRPα)/CD47 axis has emerged as an important innate immune checkpoint that enables cancer cell escape from macrophage phagocytosis. SIRPα expression is limited to macrophages, dendritic cells, and neutrophils-cells enriched in the tumor microenvironment. In this study, we present novel anti-SIRP Abs, SIRP-1 and SIRP-2, as an approach to targeting the SIRPα/CD47 axis. Both SIRP-1 and SIRP-2 bind human macrophage SIRPα variants 1 and 2, the most common variants in the human population. SIRP-1 and SIRP-2 are differentiated among reported anti-SIRP Abs in that they induce phagocytosis of solid and hematologic tumor cell lines by human monocyte-derived macrophages as single agents. We demonstrate that SIRP-1 and SIRP-2 disrupt SIRPα/CD47 interaction by two distinct mechanisms: SIRP-1 directly blocks SIRPα/CD47 and induces internalization of SIRPα/Ab complexes that reduce macrophage SIRPα surface levels and SIRP-2 acts via disruption of higher-order SIRPα structures on macrophages. Both SIRP-1 and SIRP-2 engage FcγRII, which is required for single-agent phagocytic activity. Although SIRP-1 and SIRP-2 bind SIRPγ with varying affinity, they show no adverse effects on T cell proliferation. Finally, both Abs also enhance phagocytosis when combined with tumor-opsonizing Abs, including a highly differentiated anti-CD47 Ab, AO-176, currently being evaluated in phase 1 clinical trials, NCT03834948 and NCT04445701 SIRP-1 and SIRP-2 are novel, differentiated SIRP Abs that induce in vitro single-agent and combination phagocytosis and show no adverse effects on T cell functionality. These data support their future development, both as single agents and in combination with other anticancer drugs.

Upregulation of CD47, the “don't eat me” signal, on the surface of tumors to evade immune surveillance is a common escape mechanism utilized during hematological malignancy and solid tumor development, progression, and relapse. We recently reported that AO-176, a clinical stage humanized anti-CD47 IgG2 antibody, possesses differentiated characteristics such as preferential binding of tumor cells compared to normal cells, negligible binding to red blood cells, non-ADCC direct tumor killing and elicits immunogenic cell death and DAMP induction, all in addition to single-agent phagocytosis. In vivo, AO-176 has exhibited broad anti-tumor activity in preclinical xenograft models of multiple myeloma (MM), acute myeloid leukemia, T cell acute lymphoblastic leukemia, and Burkitt lymphoma. In this study, the anti-tumor activity of AO-176 in an expanded set of preclinical models of B cell neoplasms was evaluated. We assessed In vivo anti-tumor activity in a diffuse large B cell lymphoma (DLBCL) preclinical xenograft model by inoculating Toledo cells into NSG mice and treating once weekly with either 25 mg/kg AO-176 or human IgG2 isotype control. Treatment with AO-176 resulted in profound tumor shrinkage, achieved complete responses in 8/10 mice, and extended survival for all treated mice through the 46 day dosing period, compared to all isotype control treated tumors reaching endpoint by day 21.

Background CD47 is a cell surface protein expressed on tumors that binds SIRPα on macrophages and dendritic cells resulting in a "don't eat me" signal that allows tumors to evade phagocytosis. The highly differentiated monoclonal antibody, AO-176 directly targets CD47 and blocks this signal. AO-176 is currently being tested in phase 1 clinical trials in solid tumors and multiple myeloma. The purpose of this study was to assess in vivo efficacy of AO-176 in solid tumor models as a single agent and in combination with multiple classes of therapeutics including chemotherapeutics, monoclonal antibodies and T-cell checkpoint inhibitors. Methods CD47 expression levels on solid tumor types were assessed by immunohistochemistry using a tumor tissue microarray. Cell-based binding was performed using flow cytometry under acidic and physiologic pH conditions to characterize the functional activity of AO-176 in the two pH environments representing tumor and normal physiologic environments. In vivo studies were performed using models of solid cancers. Results All 12 solid tumor indications assessed were positive for cell membrane localized CD47 (3.3–98.6 H-scores). Cell-based binding of AO-176 to solid cancer cell lines was significantly greater (1.6–25-fold decrease in EC50, 11–39% increase in Bmax) in acidic conditions as compared to a neutral pH environment, demonstrating improved binding in the lower pH environments associated with solid tumors. AO-176 treatment in solid tumor xenograft models resulted in potent anti-tumor activity as a monotherapy (40–58% TGI) and in combination with paclitaxel in an ovarian model (99% TGI), cisplatin in an ovarian model (84% TGI), cisplatin in a gastric model (76% TGI), and an anti-VEGFR-2 in a gastric model (86% TGI). In vivo efficacy of CD47 blockade alone (~33% TGI) and in combination with anti-PD-1 (74% TGI) and anti-PD-L1 (80% TGI) T-cell checkpoint inhibitors was observed in a syngeneic model of colon cancer using a surrogate anti-CD47 blocking antibody. Conclusions AO-176 is a differentiated anti-CD47 agent that in addition to blocking the don't eat me signal, directly kills cancer cells, shows lower binding to normal cells such as RBCs and demonstrates increased binding activity in acidic conditions as found in the microenvironment of solid tumors. AO-176 also elicits potent anti-tumor activity in xenograft and syngeneic models as a single agent and in combination with chemotherapies, monoclonal antibodies and T-cell checkpoint inhibitors. AO-176 is currently in clinical trials as a single agent and in combination in patients with select solid cancers (NCT03834948) and in multiple myeloma (NCT04445701).

While T cell checkpoint inhibitors are mainstays of cancer immunotherapy, therapies that direct innate immune responses against cancer are lacking. CD47, a "don't eat me" signal, is an innate immune cell checkpoint which binds SIRPα on macrophages and dendritic cells to limit phagocytosis and its upregulation on tumor cells leads to evasion of immune detection and clearance. Therapeutic antibodies have previously been developed to block CD47 and induce phagocytosis of tumor cells, thus validating the pathway. AO-176, a next generation humanized IgG2 anti-CD47 antibody, was developed to block the CD47/SIRPα interaction and induce tumor cell phagocytosis. Moreover, AO-176 directly kills tumor cells through a non-ADCC-dependent mechanism via induction of programmed cell death type III. In addition to these tumor eliminating properties, AO-176 has the potential for a strong safety profile as a result of its preferential binding to tumor versus normal cells, lack of RBC binding, and enhanced binding to tumor cells at acidic pH. CD47 has previously been shown to be upregulated on acute myeloid leukemia (AML) leukemic stem cells (LSC), enabling their expansion through evasion from phagocytic clearance. As a result, patients with increased CD47 on AML LSCs have worse overall survival. In this study, AO-176 efficacy was evaluated in AML cell lines as a single agent and in combination with azacitidine and venetoclax which are approved therapies for AML. Azacitidine is a cytosine analogue which acts to inhibit DNA methylation, and venetoclax is a potent Bcl-2 inhibitor. Previous studies have shown that azacitidine induces apoptosis of tumor cells and increases cell surface exposure of calreticulin, a DAMP (Damage Associate Molecular Pattern) which provides a strong pro-phagocytic signal. From these findings, it was hypothesized that azacitidine would enable increased tumor cell phagocytosis when combined with AO-176. The potential for a similar enhancement with a combination of AO-176 and venetoclax was also explored. The ability of AO-176, with or without azacitidine or venetoclax, to induce DAMPs on the surface of AML cells was assessed. Cell surface expression of DAMPs, calreticulin and PDIA3, were measured by flow cytometry. AO-176, azacitidine, and venetoclax as single agents potently increased both calreticulin and PDIA3 in a dose-dependent manner on AML cell lines such as HL60 This is the first time, to our knowledge, that venetoclax has been shown to induce DAMPs. To better understand the functional implications of these findings, in vitro phagocytosis assays were performed. Azacitidine and venetoclax significantly enhanced AO-176-mediated phagocytosis of AML cells compared to any of the agents alone. Moreover, when AO-176 was combined with azacitidine in direct tumor cell killing assays, enhanced activity was observed in a subset of AML cell lines. In conclusion, AO-176 combined with either azacitidine or venetoclax, resulted in significant enhancement of phagocytic AML cell clearance in vitro which also correlated with the ability of these agents to induce DAMPs. In vivo treatment with AO-176 in combination with these agents is in progress. AO-176 is being evaluated in phase 1 clinical trials for the treatment of patients with solid tumors (NCT03834948) and multiple myeloma (NCT04445701). Disclosures Donio: Arch Oncology: Current Employment, Current equity holder in private company. Wilson:Arch Oncology: Current Employment, Current equity holder in private company. Darwech:Arch Oncology: Current Employment, Current equity holder in private company. Andrejeva:Arch Oncology: Current Employment, Current equity holder in private company. Capoccia:Arch Oncology: Current Employment, Current equity holder in private company. Puro:Arch Oncology: Current Employment, Current equity holder in private company. Kashyap:Arch Oncology: Current Employment, Current equity holder in private company. Pereira:Arch Oncology: Current Employment, Current equity holder in private company.

Background Overexpression of CD47 by tumor cells exploits an immune checkpoint preventing tumor recognition and destruction by innate immune cells. Binding of tumor CD47 to SIRPα on macrophages and dendritic cells triggers a ‘don’t eat me’ signal that inhibits phagocytosis and allows escape from innate immune surveillance. Blockade of the CD47/SIRPα axis, however, enables immune recognition and phagocytic clearance of tumor cells. We have developed a clinical stage CD47 targeting antibody AO-176 that is highly differentiated among agents in this class. AO-176 not only blocks the CD47/SIRPα interaction and induces phagocytosis of tumor cells, but it also has a direct killing mechanism (via PCDIII) and induction of immunogenic cell death, leveraged by preferential binding to tumor versus normal cell CD47. Methods CD47 and β1 integrin expression and localization were evaluated using a combination of flow cytometry, western blotting, confocal microscopy and immunohistochemistry. Results Previously, we described that the preferential binding of AO-176 to tumor versus normal cells was due to its interaction with CD47 molecules that were pre-complexed to β1 integrin. This finding was particularly important and suggestive of why AO-176 does not bind red blood cells since they do not express β1 integrin. We have extended these findings to show that β1 integrin as well as CD47 are also expressed at lower levels in normal versus tumor cells, and that solid and hematologic tumor cells overexpress both CD47 and β1 integrin which correlate with poor prognosis in cancer. In addition, we show that AO-176 is able to bind and occupy CD47/β1 integrin complexes to a greater extent at acidic versus physiologic pH such as would be found in tumor microenvironments, an observation that also contributes to the enhanced targeting of AO-176 to tumor cells. Taken together, these findings add further insight into the preferential binding of AO-176 to tumor versus normal cells. Conclusions The context dependent binding of AO-176 to CD47, when complexed to β1 integrin, is unique among CD47 axis targeting agents and together with its direct killing mechanism of action offers a potentially better safety profile and opportunity for a therapeutic advantage. AO-176 is currently being evaluated in Phase 1 clinical trials for the treatment of patients with select solid tumors (NCT03834948) and multiple myeloma (NCT04445701). Trial Registration NCT03834948, NCT04445701.

Abstract Recent success in cancer immunotherapy has targeted immune checkpoints such as PD-1, PDL-1, and CTLA-4 to enhance the cytotoxic activity of the adaptive T-cell immune response. While the clinical response to these therapies has been dramatic for some, many others have shown partial or even no response highlighting the need for alternative or synergistic approaches that activate innate immunity. Disruption of the interaction between SIRP alpha and CD47, an innate checkpoint inhibitor, using anti-CD47 antibodies, for example, is known to enhance innate immunity by increasing the phagocytosis of tumor cells by macrophages and dendritic cells (DCs) leading to processing and presentation of tumor antigens. Recently, we described AO-176, a next generation anti-CD47 antibody that blocks the CD47/SIRP alpha interaction, induces phagocytosis and causes a direct tumor cell-autonomous death while negligibly binding RBCs. Herein, we characterize the ability of our CD47 antibodies such as AO-176 to induce immunogenic cell death (ICD) and damage-associated molecular patterns (DAMPs) in tumor cells and to potentiate chemotherapy-induced ICD/DAMPs. ICD is a process whereby an agent induces cell surface exposure and release of DAMPs from dying cells which stimulates DCs and adaptive immune responses. Tumor cells were treated in vitro with our CD47 antibodies either alone or in combination with chemotherapeutics followed by assessment of ICD/DAMPs using flow cytometry and biochemical assays. RNAseq was also performed on cells undergoing CD47 antibody mediated ICD/DAMP induction to better understand how CD47 inhibition may regulate ICD. AO-176 and other CD47 antibodies, developed by Arch Oncology, caused mitochondrial stress and loss of outer-membrane integrity, typically observed prior to cells undergoing apoptosis. In addition, CD47 antibody treatment induced a significant ER stress response at the genetic level resulting in the surface exposure of ER chaperone proteins calreticulin, Hsp90, and PDIA3. Concomitantly, our CD47 antibodies increased autophagy and JAK/STAT signaling, which resulted in both ATP and HMGB1 release, respectively. Finally, we demonstrated that in combination, our antibodies potentiated the effects of ICD/DAMP-inducing chemotherapy (e.g., doxorubicin). Here, we describe the unique ability of a specific subset of next generation CD47 antibodies, such as AO-176 to induce ICD/DAMPs. RNAseq analysis of treated cells also revealed alteration of several pathways, including those where DAMPs play a role. In summary, next-generation CD47 antibodies such as AO-176 may provide a novel approach to enhancing the current landscape of checkpoint immunotherapy by enhancing both the innate and adaptive immune responses against tumors. Citation Format: Daniel S. Pereira, Benjamin J. Capoccia, Ronald R. Hiebsch, Michael J. Donio, Alun J. Carter, Robyn J. Puro, W. Casey Wilson, Pamela T. Manning, Robert W. Carr. AO-176, a next-generation anti-CD47 antibody, induces immunogenic cell death [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 A147.

AO-176 is a next generation humanized anti-CD47 IgG2 that binds human and cynomolgus monkey CD47 equivalently. AO-176, like other CD47 antibodies, blocks the interaction of CD47 with SIRPα, inducing phagocytosis of tumor cells by activated macrophages. Unique to AO-176 is its ability to directly kill tumor cells via programmed cell death type III and immunogenic cell death in a cell autonomous (non-ADCC) manner. AO-176 also exhibits preferential binding to tumor vs. normal cells, a notable characteristic, as CD47 is expressed on many normal cells including red blood cells (RBC), platelets, T cells and endothelial cells. While other CD47 antibodies induce hematological toxicities such as anemia and thrombocytopenia in both primate models and in patients, AO-176 negligibly binds RBCs and platelets and even at high doses, minimally impacts hematology in cynomolgus monkey toxicology studies. AO-176 is currently being evaluated in phase 1 clinical trials for the treatment of solid tumors.In recent head to head experiments conducted with other CD47 antibodies, AO-176 bound all normal cells tested to a significantly lower degree. For example, unlike anti-CD47 antibodies such as Hu-5F9-G4, we observed negligible and minimal ex vivo binding to healthy human RBCs and platelets respectively and significantly lower binding of AO-176 to other hematologic and non-hematologic cells such as T cells and endothelial cells. Although AO-176 binds cynomolgus monkey RBCs to a slightly greater degree than human RBCs ex vivo, AO-176 versus comparative published Hu5F9-G4 findings has demonstrated dramatically reduced receptor occupancy when evaluated in monkey toxicology studies. We have also developed a clinic-ready receptor occupancy assay to measure AO-176 binding to circulating PBMCs in patients treated with AO-176 and to demonstrate minimal binding to CD47 on normal human cells.We have begun to evaluate mechanisms that may underlie the normal cell sparing effects of AO-176. Published studies have demonstrated that in addition to CD47 protein expression level, clustering and mobility of CD47 at the cell surface may impact binding to ligands, downstream signaling and subsequent cellular responses such as apoptosis. For example, avidity for SIRPα and clearance by macrophages is modulated by CD47 clustering that can be modulated by protein glycosylation, association with lipid rafts, cytoskeleton, integrins or other cis-acting factors. Here, we compare the distribution of CD47 on tumor and RBCs to evaluate receptor association with other cellular membrane components that may impact distribution on the cell surface.In summary, AO-176 is a next generation anti-CD47 antibody that, aside from induction of phagocytosis, possesses additional attributes that include direct tumor cell killing and preferential binding to tumor versus normal cells. These attributes of AO-176 differentiate it from other CD47 axis targeting agents currently in clinical evaluation.Citation Format: Prabir Chakraborty, Myriam N. Bouchlaka, Benjamin J. Capoccia, Ronald R. Hiebsch, Michael J. Donio, Robyn J. Puro, Vicki Sung, Daniel S. Pereira. AO-176, a normal cell sparing humanized anti-CD47 antibody [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 540.

Targeting immune checkpoints of adaptive immunity has shown great therapeutic efficacy in oncology, but in a limited fraction of patients. Innate immune cells represent the most abundant immune cell types in many solid tumors and are often linked to a poor prognosis.SIRPα is expressed by innate immune cells and its interaction with CD47, expressed by most tumor cells, is an important immune checkpoint of the innate response, involved in the regulation of phagocytosis by macrophages, dendritic cells and neutrophils.Recently, first generation agents targeting CD47 (CD47 antibodies and SIRPα-Fc fusion proteins) have shown promise in clinical trials, but they have also experienced hematological toxicities such as anemia or thrombocytopenia. Consequently, we have previously reported on the development of AO-176, a next generation anti-CD47 antibody that not only blocks the CD47/SIRPα interaction and induces phagocytosis, but also preferentially binds tumor versus normal cells (including RBCs where it binds negligibly) and directly kills tumor cells via a programmed cell death type III and an immunogenic cell death process.Here we report the discovery of novel anti-SIRP antibodies that recognize either SIRPα selectively or SIRPα/γ. These antibodies are being evaluated for their ability to induce phagocytosis of tumor cells - we have identified antibodies that induce phagocytosis of tumor cells alone and in combination with Rituxan. The ability of our anti-SIRP antibodies to induce immunomodulatory activities in a variety of ex vivo cultured immune cells expressing either SIRPα or SIRPα/γ is also under investigation and will be presented.Citation Format: Ronald R. Hiebsch, Myriam N. Bouchlaka, Benjamin J. Capoccia, Michael J. Donio, Prabir Chakraborty, W. Casey Wilson, Robyn J. Puro, Daniel S. Pereira. Evaluation of novel SIRP antibodies as potential cancer therapeutics [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 548.

Abstract Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and overexpressed on many tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggers a “don't eat me” signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models. We have developed a next-generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPα interaction to induce tumor cell phagocytosis, but also induces tumor cytotoxicity in hematologic and solid human tumor cell lines, but not normal noncancerous cells, by a cell autonomous mechanism (not ADCC). AO-176 also binds preferentially to tumor versus many normal cell types. In particular, AO-176 binds negligibly to RBCs in contrast to tumor cells, even at high concentrations up to 200 μg/mL and does not agglutinate RBCs up to 1 mg/mL in vitro. These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. When tested in cynomolgus monkeys, AO-176 was well tolerated with no adverse effects. Finally, we show that AO-176 demonstrates dose-dependent antitumor activity in tumor xenograft models. Taken together, the unique properties and antitumor activity of our next-generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPα axis targeting agents in clinical development.

CD47 is a cell surface glycoprotein that interacts with signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggering a “don9t eat me” signal that inhibits phagocytosis. Many tumors evade immune surveillance by overexpressing CD47, thereby preventing their recognition by phagocytes. Blocking the interaction of SIRPα/CD47 promotes phagocytosis and tumor cell destruction leading to a reduction in tumor burden. We have developed a humanized anti-CD47 antibody, AO-176, that blocks the interaction between CD47 and SIRPα and exhibits several additional novel functional characteristics. These characteristics include the induction of cell death in multiple human tumor cell lines in a cell autonomous manner (not ADCC), assessed by an increase in phosphatidylserine/7AAD positive staining. A second novel characteristic is enhanced binding to tumor cells at acidic pH. AO-176 binds to human tumor cell lines in the high pM to low nM range at physiologic pH, however, binding is enhanced up to 20-fold at an acidic pH of 6.5. The acidic pH of the tumor microenvironment which ranges from 6.4-7.2 is characteristic of solid tumors and correlates with tumor progression and metastasis. As a result of this enhanced binding at acidic pH, AO-176 has the potential added advantage of tumor-specific targeting. A third novel characteristic exhibited by AO-176 is its selective binding to tumor cells while exhibiting reduced binding to normal cells including red blood cells (cynomolgus monkey and human), endothelial, epithelial and skeletal muscle cells. In addition to these novel characteristics, AO-176 also exhibits dose-dependent efficacy in multiple mouse tumor models. Taken together, the unique combination of functional characteristics of AO-176, including induction of cell-autonomous killing, enhanced binding to tumor cells at acidic pH, significantly reduced binding to normal cells and potent in vivo efficacy provides the preclinical rationale for further development. Citation Format: Robyn Puro, Katherine Liu, Benjamin Capoccia, Michael Donio, Ronald Hiebsch, Myriam Bouchlaka, Alun Carter, Pamela Manning, Kathleen Crowley, Robert Karr. A humanized anti-CD47 monoclonal antibody that directly kills human tumor cells and has additional unique functional characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1765.