Mesenchymal stem cells (MSC) are adult multipotent cells found in bone marrow, adipose tissue, and other adult tissues. MSC have been shown to improve regeneration of injured tissues in vivo, but the mechanisms remain unclear. Typically, MSC are cultured under ambient, or normoxic, conditions (21% oxygen). However, the physiological niches for MSC in the bone marrow and other sites have much lower oxygen tension. When used as a therapeutic tool to repair tissue injuries, MSC cultured in standard conditions must adapt from 21% oxygen in culture to less than 1% oxygen in the ischemic tissue. We therefore examined the effects of preculturing human bone marrow-derived MSC in hypoxic conditions (1%-3% oxygen) to elucidate the best conditions that enhance their tissue regenerative potential. We demonstrated that MSC cultured in hypoxia activate the Akt signaling pathway while maintaining their viability and cell cycle rates. We also showed that MSC cultured in hypoxia induced expression of cMet, the major receptor for hepatocyte growth factor (HGF), and enhanced cMet signaling. MSC cultured in hypoxic conditions increased their migration rates. Since migration and HGF responsiveness are thought to be key mediators of MSC recruitment and/or activation in vivo, we next examined the tissue regenerative potential of MSC cultured under hypoxic conditions, using a murine hind limb ischemia model. We showed that local expression of HGF is increased in ischemic muscle in this model. Intra-arterial injection of MSC cultured in either normoxic or hypoxic conditions 24 hours after surgical induction of hind limb ischemia enhanced revascularization compared with saline controls. However, restoration of blood flow was observed significantly earlier in mice that had been injected with hypoxic preconditioned MSC. Collectively, these data suggest that preculturing MSC under hypoxic conditions prior to transplantation improves their tissue regenerative potential. STEM CELLS
Upregulation of tumor CD47, the "don't eat me" signal, to evade immune surveillance is a common escape mechanism that evolves during cancer development, progression, and relapse. Previous studies have shown multiple myeloma (MM) cells leverage this mechanism through broad upregulation of CD47 compared to non-malignant plasma cells, making CD47 an attractive therapeutic target for this disease. 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, a lack of binding to red blood cells, non-ADCC direct tumor killing and elicits immunogenic cell death with DAMP induction, all in addition to single-agent phagocytosis. In this study, AO-176's anti-tumor activity in MM was evaluated. Immunohistochemical analyses of MM patient tumors with upregulated CD47 expression showed infiltration of innate immune cells such as macrophages and dendritic cells, both previously shown to be involved in anti-CD47 antibody mechanisms of action. AO-176 binding was confirmed on human cell lines frequently used in MM xenograft models. AO-176 exerted substantial single agent in vivo anti-tumor activity in multiple MM xenograft models when dosed at 25 mg/kg, including significant tumor growth inhibition of RPMI-8226 xenografted mice, and complete responses (CRs) in (10/10) NCI-H929 xenografted mice. These CRs were durable, with treated mice tumor-free up to 120 days post antibody dosing. Immunohistochemical analysis of AO-176 treated tumors from both models showed increased numbers of macrophages and dendritic cells compared to controls. An AO-176 dose response study resulted in CRs and increases in overall survival down to 10 mg/kg, with CRs observed as low as 3 mg/kg during dosing. In addition, we found that large NCI-H929 tumors (up to 1600mm3) showed pronounced regression after AO-176 treatment. The anti-tumor activity of AO-176 was also evaluated in combination with several standard of care MM therapies. When combined with the proteasome inhibitor bortezomib, AO-176 treatment at both 10 mg/kg and 25 mg/kg resulted in profound RPMI-8226 xenograft growth inhibition, near-total CRs (19/20 mice), and extended survival at both doses. Combining AO-176 and the anti-CD38 antibody daratumumab or immunomodulatory drugs (lenalidomide/pomalidomide) both produced significant enhancement of anti-tumor activity in xenograft models. The combined regimen of AO-176 with daratumumab led to significant MM.1S tumor growth inhibition compared to AO-176 or daratumumab alone. Both lenalidomide and pomalidomide combined with AO-176 resulted in significantly increased MM.1S tumor growth inhibition and extended survival compared to AO-176 alone, with an increased number of CRs observed in the combination groups compared to monotherapy groups. In summary, the pre-clinical potent single agent activity and enhanced activity when combined with standard of care anti-MM agents, warrants further development of AO-176 in MM treatment. AO-176 is being evaluated in phase 1 clinical trials for the treatment of patients with solid tumors (NCT03834948) and with MM (NCT04445701). Disclosures Wilson: Arch Oncology: Current Employment, Current equity holder in private company. Richards:Arch Oncology: Current Employment, Current equity holder in private company. Puro: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. Donio:Arch Oncology: Current Employment, Current equity holder in private company. Hiebsch:Arch Oncology: Current Employment, Current equity holder in private company. Chakraborty:Arch Oncology: Current Employment, Current equity holder in private company. Sung:Arch Oncology: Current Employment, Current equity holder in private company. Pereira:Arch Oncology: Current Employment, Current equity holder in private company.
Hematopoietic stem cell transplantation has traditionally been used to reconstitute blood cell lineages that had formed abnormally because of genetic mutations, or that had been eradicated to treat a disease such as leukemia. However, in recent years, much attention has been paid to the new concept of "stem cell plasticity," and the hope that stem cells could be used to repair damaged tissues generated immense excitement. The field is now in a more realistic and critical period of intense investigation and the concept of cell fusion to explain some of the observed effects has been shown after specific types of damage in liver and muscle, both organs that contain a high number of multinucleate cells. The field is still an extremely exciting one, and many questions remain to be answered before stem cell therapy for tissue repair can be used effectively in the clinic. Immune deficient mouse models of tissue damage provide a system in which human stem cell migration to sites of damage and subsequent contribution to repair can be carefully evaluated. This chapter gives detailed instructions for methods to study human stem cell contribution to damaged liver and to promote repair of damaged vasculature in immune deficient mouse models.
AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.
Purpose of study: To determine whether AO-176, a highly differentiated, humanized antibody targeting CD47, shows efficacy alone or in combination with a variety of approved anti-cancer drugs in solid tumors. Methods: We investigated AO-176 as a single agent and in combination with chemotherapies and targeted antibodies, utilizing standard in vitro phagocytosis assays, tumor cell killing assays, and in vivo xenograft models. Results: AO-176 is a highly-differentiated anti-CD47 antibody that not only blocks the CD47/SIRPα interaction to stimulate phagocytosis of tumor cells, but also exerts direct killing activity on tumor cells (non-ADCC), induces immunogenic cell death, and exhibits preferential binding to tumor cells compared to normal cells. In addition, tumor-specific CD47 binding by AO-176 increases in the acidic tumor microenvironment. As a single agent, AO-176 induced cell killing (14-52% Annexin V positivity, EC50 = 1-30 μg/ml) and phagocytosis (10-34%, EC50 = 0.8-3.3 μg/ml) in ovarian, gastric, non-small cell lung, head and neck, colorectal, thyroid, pancreatic and endometrial solid tumor cell lines. When combined with tumor-targeted antibodies (i.e. cetuximab against head and neck and colorectal cancer cell lines, or the checkpoint inhibitor avelumab against ovarian cancer cell lines), AO-176 significantly enhanced phagocytosis of the tumor cells in vitro. In combination with the chemotherapeutics paclitaxel and cisplatin, AO-176 also potentiated direct tumor killing of gastric cancer cells in vitro. In vivo, AO-176 showed potent single-agent anti-tumor activity against ovarian and gastric tumor xenografts. These data add to the previous pre-clinical anti-tumor activity of AO-176 reported in breast cancer, multiple myeloma, and non-Hodgkin’s lymphoma xenografts. When cisplatin or paclitaxel was added to the AO-176 treatment regimen against xenografted ovarian tumors in vivo, significant combination anti-tumor activity was observed. We then sought to extend our promising in-vitro findings from combining AO-176 with a checkpoint inhibitor to in-vivo models. As AO-176 is human CD47-specific, we utilized an in-house murine reactive anti-CD47 blocking antibody and combined it with a murine-reactive anti-PDL1 antibody to treat MC38 murine tumors established in syngeneic mice. Combination of the two antibodies significantly improved anti-tumor efficacy compared to either single agent. Conclusions: AO-176 has demonstrated broad in vitro phagocytosis/killing as well as in vivo efficacy that supports its development, both as a single agent and in combination with other anti-cancer drugs. With a highly differentiated mechanism of action and binding profile, AO-176 may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Citation Format: Casey Wilson, Myriam Bouchlaka, Robyn Puro, Ben Capoccia, Ronald Hiebsch, Prabir Chakraborty, Michael Donio, Vicki Sung, Daniel Pereira. AO-176, a highly differentiated humanized anti-CD47 antibody, exhibits single-agent and combination antitumor efficacy with chemotherapy and targeted antibodies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B100. doi:10.1158/1535-7163.TARG-19-B100
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