Anti-leukemic Activity Of Novel Anti-CD47 Monoclonal Antibodies And Mechanism Of Regulating Expression Of CD47 In Acute Myeloid Leukemia

Objective:Acute myeloid leukemia(AML)is the most common type of acute leukemia,with an incidence that increases with advanced age.AML is difficult to treat due to a combination of biological heterogeneity and patient-related risk factors such as age or comorbidities,resulting in poor long-term overall survival(OS).It is presumed that successful treatment must effectively eradicate the leukemic stem cell and its subclones so that residual disease cannot act as the source of recurrence.Consequently,there is an urgent need for more specific and less toxic drugs that are rationally designed to target leukemia-specific abnormalities.CD47,also known as integrin-associated receptor protein(IAP),is a 50 kDa cell surface transmembrane immunoglobulin(Ig)superfamily member,which is expressed on a broad range of cell types.CD47 interacts with signal regulatory protein?(SIRP?),thrombospondin-1(TSP-1)and TSP-2 to mediate various cellular functions,including leukocyte adhesion and migration,T-cell activation,apoptosis and phagocytosis.In particular,CD47 signaling through SIRP?inhibits the phagocytosis of CD47-expressing target cells by SIRP?-expressing macrophages.Anti-CD47monoclonal antibodies inhibiting the interaction of CD47 and SIRP?promote the phagocytosis of tumor cells by macrophages.Blocking the CD47-SIRP?interaction with anti-CD47 monoclonal antibodies or anti-SIRP?antibodies have proven effective in inducing the phagocytosis of tumor cells in vitro and inhibiting the growth of the various hematological and solid tumors in vivo.Increased expression of CD47 has been reported to enable cancer cells to evade phagocytosis by macrophages,but the molecular mechanisms regulating CD47expression have not been determined.Here,we examined the expression of CD47 on the surface of AML cells,and the therapeutic potential of three new human anti-CD47monoclonal antibodies(made in China).At the same time,we first report the molecular mechanisms regulating CD47 expression in AML cells.Methods:We detected CD47 expression in 21 AML patients and 5 AML cell lines use flow cytometry.C57/BL mouse BM mononuclear cells were harvested and grown in IMDM containing 10%FBS supplemented with 50 ng/mL recombinant murine macrophage colony-stimulating factor(M-CSF)for 7 days to allow terminal differentiation of monocytes to macrophages.Human PB mononuclear cells were prepared from normal blood.Monocytes were isolated by adhering mononuclear cells to culture plates for 1 h at 37°C,after which non-adherent cells were removed by washing.Adherent cells were then incubated in IMDM plus 10%human serum for7–10 days to allow terminal differentiation of monocytes to macrophages.AML cells were marked with 2.5?M carboxyfluoresceinsuccinimidyl ester(CFSE).Macrophages were incubated in serum-free IMDM for 3h before adding 2×10~5CFSE-marked live AML cells.New antibodies(10?g/ml)or isotype control were added and incubated for 2h at 37°C.After coincubation,wells were washed thoroughly with PBS five times and subsequently imaged with fluorescence microscopy.Then,cells were harvested and stained with either mouse macrophage or human macrophage marker for 30 minutes followed by two washing steps with stain buffer.Subsequently,cells were analyzed by flow cytometry.Annexin V/PI method was used to detect the effect of CD47 monoclonal antibodies on AML cell apoptosis,and flow cytometry was used to detect the effect of CD47 monoclonal antibodies on AML cell proliferation.In vivo studies with human AML cell lines(U937)were conducted with NOG mice.We first preliminarily explored the mechanism of high expression of CD47 in AML cells use Western blot and PCR.Results:Flow cytometric analysis of patient bone marrow(BM)cells revealed that AML cells overexpress CD47 when compared with normal bone marrow cells.AML cells from 81%(17/21)of patients expressed at least 2-fold more CD47 than normal BM cells,while no patients had lower expression of CD47 on leukemia cells than on normal BM cells.The mean fluorescence of intensity(MFI)is 628.3(range 446-884)in normal group,however the MFI is 3430.6(range 952-9310)in AML patients group.Compared with IgG4 isotype control,the number of cell proliferation did not decrease at 0H,24H,48H and 72H after domestic CD47 monoclonal antibodies treated,and the rate of apoptosis did not increase.We found that all three domestic CD47 monoclonal antibodies can bind AML cells well.Further research found that these domestic CD47 monoclonal antibodies can competitively antagonize the binding sites of B6H12,providing a good theoretical basis for the promotion and application in the future.In vitro experiments found that domestic CD47 monoclonal antibodies can enhance phagocytosis and clearance of AML cell with high expression of CD47,but has no such effect for AML cell with low expression of CD47.Domestic CD47 monoclonal antibodies can promote phagocytosis and elimination of primary AML cells by macrophages.In vivo experiments found that domestic CD47monoclonal antibodies can significantly prolong the survival time of mice.CD47expression protects cells from phagocytosis by transmitting an inhibitory signal to macrophages.Here we show that blocking CD47 with new human anti-CD47monoclonal antibodies increased phagocytosis of AML cells in vitro and in vivo.Through Western blot and PCR,we have preliminarily explored the possible mechanism of overexpression of CD47 on the surface of AML cells.Happily,we found that hypoxia inducible factor 1 has obvious correlation with overexpression of CD47.Last but not least,we first report that hypoxia-inducible factor 1(HIF-1)can regulate the expression of CD47 in AML cells.Conclusions:These results show that domestic CD47 monoclonal antibodies can reactivate macrophage function by blocking CD47/SIRP?signaling pathway and enhance phagocytosis and clearance of AML cells by macrophages.In addition,this study preliminarily found that hypoxia inducible factor 1 can regulate the expression of CD47 in AML cells.