scholarly journals The therapeutic effect of a novel anti-TNF-α/IL-6R triple-specific fusion protein under experimental septic condition

2021 ◽  
Author(s):  
Xiaole Chen ◽  
Shuangyu Tan ◽  
Mengru Yan ◽  
Kaimei Nie ◽  
Qingmei Zheng ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Expression System ◽  
Cecal Ligation ◽  
Mouse Fibroblast ◽  
Potential Candidate ◽  
Tnf Α ◽  
Prokaryotic Expression System

Abstract A novel anti-TNF-α/IL-6R triple-specific fusion protein, by linking 3 single domain chains, was designed and constructed in our lab. The high purity fusion proteins were obtained by our developed prokaryotic expression system process with high binding affinity with TNF-α (94.75 pM), Human Serum Albumin (1.83 nM) and IL-6R (2.29 nM). In this study, the anti-TNF-α/IL-6R triple-specific fusion protein protected the mouse fibroblast fibrosarcoma cell line (L929) from the apoptosis effects induced by TNF-α, establishing that the expressed fusion proteins can selectively combine with TNF-α in vitro. In vivo, the survival rate of cecal ligation and puncture (CLP) was notably increased in the group with anti-TNF-α/IL-6R triple-specific fusion protein treatment, and meaningfully higher compared with the single-targeted IL-6R and TNF-α fusion protein at the same dose. After the treatment with anti-TNF-α/IL-6R triple-specific fusion protein, the level of serum TNF-α, IL-1β and IL-6 were significantly decreased, and sepsis-induced pathological injuries in the kidney were remarkably attenuated. The anti-TNF-α/IL-6R triple-specific fusion protein can be the potential candidate for the development of new drug design against sepsis.

α2-Adrenoceptor agonist dexmedetomidine protects septic acute kidney injury through increasing BMP-7 and inhibiting HDAC2 and HDAC5

2012 ◽  
Vol 303 (10) ◽  
pp. F1443-F1453 ◽  
Author(s):  
Chung-Hsi Hsing ◽  
Chiou-Feng Lin ◽  
Edmund So ◽  
Ding-Ping Sun ◽  
Tai-Chi Chen ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Histone Deacetylases ◽  
Trichostatin A ◽  
Histone H3 ◽  
Cecal Ligation ◽  
Kidney Injury ◽  
Protective Effects ◽  
Tnf Α

Bone morphogenetic protein (BMP)-7 protects sepsis-induced acute kidney injury (AKI). Dexmedetomidine (DEX), an α2-adrenoceptor (α2-AR) agonist, has anti-inflammatory effects. We investigated the protective effects of DEX on sepsis-induced AKI and the expression of BMP-7 and histone deacetylases (HDACs). In vitro , the effects of DEX or trichostatin A (TSA, an HDAC inhibitor) on TNF-α, monocyte chemotactic protein (MCP-1), BMP-7, and HDAC mRNA expression in LPS-stimulated rat renal tubular epithelial NRK52E cells, was determined using real-time PCR. In vivo, mice were intraperitoneally injected with DEX (25 μg/kg) or saline immediately and 12 h after cecal ligation and puncture (CLP) surgery. Twenty-four hours after CLP, we examined kidney injury and renal TNF-α, MCP-1, BMP-7, and HDAC expression. Survival was monitored for 120 h. LPS increased HDAC2, HDAC5, TNF-α, and MCP-1 expression, but decreased BMP-7 expression in NRK52E cells. DEX treatment decreased the HDAC2, HDAC5, TNF-α, and MCP-1 expression, but increased BMP-7 and acetyl histone H3 expression, whose effects were blocked by yohimbine, an α2-AR antagonist. With DEX treatment, the LPS-induced TNF-α expression and cell death were attenuated in scRNAi-NRK52E but not BMP-7 RNAi-NRK52E cells. In CLP mice, DEX treatment increased survival and attenuated AKI. The expression of HDAC2, HDAC5, TNF-α, and MCP-1 mRNA in the kidneys of CLP mice was increased, but BMP-7 was decreased. However, DEX treatment reduced those changes. DEX reduces sepsis-induced AKI by decreasing TNF-α and MCP-1 and increasing BMP-7, which is associated with decreasing HDAC2 and HDAC5, as well as increasing acetyl histone H3.

scholarly journals In Vivo and In Vitro Evaluation of the Protective Effects of Hesperidin in Lipopolysaccharide-Induced Inflammation and Cytotoxicity of Cell

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 478 ◽  
Author(s):  
Rasha Al-Rikabi ◽  
Hanady Al-Shmgani ◽  
Yaser Hassan Dewir ◽  
Salah El-Hendawy
Keyword(s):  
Breast Cancer ◽  
Chromatin Condensation ◽  
Control Group ◽  
Potential Candidate ◽  
Dependent Manner ◽  
Protective Effects ◽  
Mcf7 Cells ◽  
Tnf Α

(1) Background: Plant flavonoids are efficient in preventing and treating various diseases. This study aimed to evaluate the ability of hesperidin, a flavonoid found in citrus fruits, in inhibiting lipopolysaccharide (LPS) induced inflammation, which induced lethal toxicity in vivo, and to evaluate its importance as an antitumor agent in breast cancer. The in vivo experiments revealed the protective effects of hesperidin against the negative LPS effects on the liver and spleen of male mice. (2) Methods: In the liver, the antioxidant activity was measured by estimating the concentration of glutathione (GSH) and catalase (CAT), whereas in spleen, the concentration of cytokines including IL-33 and TNF-α was measured. The in vitro experiments including MTT assay, clonogenity test, and sulforhodamine 101 stain with DAPI (4′, 6-diamidino-2-phenylindole) were used to assess the morphological apoptosis in breast cancer cells. (3) Results: The results of this study revealed a significant increase in the IL-33 and TNF-α cytokine levels in LPS challenged mice along with a considerable elevation in glutathione (GSH); moreover, the catalase (CAT) level was higher compared to that of the control group. Cytotoxicity of the MCF-7 cell line revealed significant differences among the groups treated with different concentrations when compared to the control groups, in a concentration-dependent manner. Hesperidin significantly inhibited the colony formation of MCF7 cells when compared to that of control. Clear changes were observed in the cell shape, including cell shrinkage and chromatin condensation, which were associated with a later apoptotic stage. (4) Conclusion: The results indicate that hesperidin might be a potential candidate in preventing diseases.

scholarly journals Adenosine Receptors as Neuroinflammation Modulators: Role of A1 Agonists and A2A Antagonists

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1739
Author(s):  
Aleix Martí Navia ◽  
Diego Dal Ben ◽  
Catia Lambertucci ◽  
Andrea Spinaci ◽  
Rosaria Volpini ◽  
...  
Keyword(s):  
Partial Agonist ◽  
Pathological Condition ◽  
Neuroprotective Effect ◽  
Antioxidant Properties ◽  
Potential Candidate ◽  
In Vivo Models ◽  
Tnf Α

The pathological condition of neuroinflammation is caused by the activation of the neuroimmune cells astrocytes and microglia. The autacoid adenosine seems to be an important neuromodulator in this condition. Its main receptors involved in the neuroinflammation modulation are A1AR and A2AAR. Evidence suggests that A1AR activation produces a neuroprotective effect and A2AARs block prevents neuroinflammation. The aim of this work is to elucidate the effects of these receptors in neuroinflammation using the partial agonist 2′-dCCPA (2-chloro-N6-cyclopentyl-2′-deoxyadenosine) (C1 KiA1AR = 550 nM, KiA2AAR = 24,800 nM, and KiA3AR = 5560 nM, α = 0.70, EC50A1AR = 832 nM) and the newly synthesized in house compound 8-chloro-9-ethyl-2-phenethoxyadenine (C2 KiA2AAR = 0.75 nM; KiA1AR = 17 nM and KiA3AR = 227 nM, IC50A2AAR = 251 nM unpublished results). The experiments were performed in in vitro and in in vivo models of neuroinflammation. Results showed that C1 was able to prevent the inflammatory effect induced by cytokine cocktail (TNF-α, IL-1β, and IFN-γ) while C2 possess both anti-inflammatory and antioxidant properties, counteracting both neuroinflammation in mixed glial cells and in an animal model of neuroinflammation. In conclusion, C2 is a potential candidate for neuroinflammation therapy.

Expression of Recombinant Human Cytomegalovirus Fusion Proteins pp150-pp65 Fragments and its Application

2013 ◽  
Vol 634-638 ◽  
pp. 1313-1318
Author(s):  
Yu Fen Jin ◽  
Yan Lei Li ◽  
Yan Hua ◽  
Xiao Gang Zhang ◽  
Ting Yu
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Human Cytomegalovirus ◽  
Colloidal Gold ◽  
Fusion Proteins ◽  
Prokaryotic Expression ◽  
Expression System ◽  
Serum Samples ◽  
Igg Antibody ◽  
Prokaryotic Expression System

Objective To evaluate the effectiveness of prokaryotic expression of fusion proteins pp150-pp65 of human cytomegalovirus (hCMV) for its application as antigen, the fusion protein of pp150-pp65 were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column for preparing the colloidal gold kit. Methods Using DNA from HCMV strain as template, the genes encoding pp150 and pp65 protein fragment were amplified by PCR technique, respectively. After confirmed by DNA sequence analysis, the recombinant plasmid pET28a-pp150-pp65 was transformed into E.Coil.BL21(DE3) and induced to express with IPTG. The expressed fusion protein was characterized by SDS-PAGE and western blot after purified, then we used the purified fusion protein to develop combined detection kit of IgM/IgG antibody against HCMV (colloidal gold method) with Beijing Innovita Bio-tech Co., Ltd for detecting the samples, compared with the imported kits (ELISA). Results The gene of fusion fragment pp150-pp65 was correctly amplified and the recombinant vector was successfully constructed. The purified protein with the molecular weight of 45KD had good antigenicity by western blot. The protein was subjected to assay with an ELI SA capture kit in its specific and sensitive assay based on colloidal gold nanoparticles, testing of 600 serum samples indicated that this kit had a sensitivity of 92.7%;, specificity of 83.1%, crude consistency o f 90.2%, compared to the imported HCMV-IgG kit; the sensitivity of the kit was 88.1%, specificity was 89.2%, coarse consistency was 88.5%, compared to the imported HCMV-IgM kit; Conclusion In this experiment, the HCMV antigen with high purity and specificity (pp150-pp65 recombinant protein) was prepared effectively through genetic engineering technology. Compared to imported reagents, the colloidal gold kit consisting of fusion protein in a capture assay had high sensitivity and specificity. Preliminary clinical use warrants further development and use of this kit. Furthermore, it provides a technological basis for detection of HCMV in different stages of clinical infection.

scholarly journals Exosomes from adipose-derived mesenchymal stem cells alleviate sepsis-induced lung injury in mice by inhibiting the secretion of IL-27 in macrophages

2022 ◽  
Vol 8 (1) ◽  
Author(s):  
Xiaoyan Wang ◽  
Danyong Liu ◽  
XiHe Zhang ◽  
LiuMing Yang ◽  
Zhengyuan Xia ◽  
...  
Keyword(s):  
Lung Injury ◽  
Pulmonary Edema ◽  
Tissue Injury ◽  
Cecal Ligation ◽  
Wild Type Mouse ◽  
Vascular Leakage ◽  
Inflammatory Disorder ◽  
Tnf Α

AbstractAcute lung injury (ALI) represents a frequent sepsis-induced inflammatory disorder. Mesenchymal stromal cells (MSCs) elicit anti-inflammatory effects in sepsis. This study investigated the mechanism of exosomes from adipose-derived MSCs (ADMSCs) in sepsis-induced ALI. The IL-27r−/− (WSX-1 knockout) or wild-type mouse model of sepsis was established by cecal ligation and puncture (CLP). The model mice and lipopolysaccharide (LPS)-induced macrophages were treated with ADMSC-exosomes. The content of Dil-labeled exosomes in pulmonary macrophages, macrophages CD68+ F4/80+ in whole lung tissues, and IL-27 content in macrophages were detected. The mRNA expression and protein level of IL27 subunits P28 and EBI3 in lung tissue and the levels of IL-6, TNF-α, and IL-1β were measured. The pulmonary edema, tissue injury, and pulmonary vascular leakage were measured. In vitro, macrophages internalized ADMSC-exosomes, and ADMSC-exosomes inhibited IL-27 secretion in LPS-induced macrophages. In vivo, IL-27 knockout attenuated CLP-induced ALI. ADMSC-exosomes suppressed macrophage aggregation in lung tissues and inhibited IL-27 secretion. ADMSC-exosomes decreased the contents of IL-6, TNF-α, and IL-1β, reduced pulmonary edema and pulmonary vascular leakage, and improved the survival rate of mice. Injection of recombinant IL-27 reversed the protective effect of ADMSC-exosomes on sepsis mice. Collectively, ADMSC-exosomes inhibited IL-27 secretion in macrophages and alleviated sepsis-induced ALI in mice.

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase

1993 ◽  
Vol 13 (3) ◽  
pp. 1737-1745
Author(s):  
J A Cooper ◽  
A Kashishian
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Expression System ◽  
Sh2 Domain ◽  
Phosphorylation Sites ◽  
Phosphatidylinositol 3 Kinase ◽  
Binding Properties ◽  
Sh2 Domains ◽  
Phosphatidylinositol 3

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of Recombinant HIV-1 p24-Nef Protein in Two Forms as Potential Candidate Vaccines in Three Vehicles

2020 ◽  
Vol 17 (5) ◽  
pp. 387-395
Author(s):  
Mona Sadat Larijani ◽  
Mohammad Hassan Pouriayevali ◽  
Seyed Mehdi Sadat ◽  
Amitis Ramezani
Keyword(s):  
Fusion Protein ◽  
Western Blotting ◽  
Highly Active Antiretroviral Therapy ◽  
Fusion Proteins ◽  
Therapeutic Vaccine ◽  
Potential Candidate ◽  
Conserved Regions ◽  
Bioinformatics Tools ◽  
Hiv 1

Background: Different approaches have been investigated to develop a preventive or therapeutic vaccine, although none of them has been fully practical. Therapeutic vaccines against HIV-1 have been studied with the aim of eliminating the virus from reservoir cells with or without HAART (Highly Active Antiretroviral Therapy). Fusion proteins with the most immunogenic features among conserved regions can facilitate this achievement in such a variable virus. To achieve the most immunogenic and also conserved regions, bioinformatics tools are widely used to predict antigens’ features before applying them. Objective: This study aimed at the in vitro evaluation of p24 -Nef fusion protein based on the previous in silico design to achieve a potential therapeutic subunit vaccine against HIV-1. Methods: The truncated form of p24-Nef using AAY flexible linker and the full protein were expressed and evaluated in the prokaryotic system and confirmed by western blotting. We also used pcDNA3.1 to transfect Lenti-X 293T cells. Moreover, lentiviral vectors were applied to produce recombinant virions harboring the genes of interest and cell transduction. Results: Both fusion proteins in a truncated and a full form were expressed and confirmed by Anti Nef polyclonal antibody in western blotting. Recombinant virions were generated and transduced Lenti-X 293T cells confirming by immunofluorescence microscope and p24 ELISA assay kit. Transduced cells were analyzed by SDS-PAGE and western blotting, which resulted in approved protein expression. Conclusion: Fusion protein of p24 and Nef is well expressed in eukaryotic cell lines according to its pre-evaluated features by bioinformatics tools.

In Vivo and In Vitro Anti-inflammatory Activities of Neoandrographolide

2007 ◽  
Vol 35 (02) ◽  
pp. 317-328 ◽  
Author(s):  
Jun Liu ◽  
Zheng-Tao Wang ◽  
Li-Li Ji
Keyword(s):  
Oral Administration ◽  
Potential Candidate ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Factor Alpha ◽  
Anti Inflammatory ◽  
Necrosis Factor ◽  
Dimethyl Benzene

Neoandrographolide, one of the principal diterpene lactones, isolated from a medicinal herb Andrographis paniculata Nees, was tested in vivo and in vitro for its anti-inflammatory activities and mechanism. Oral administration of neoandrographolide (150 mg/kg) significantly suppressed ear edema induced by dimethyl benzene in mice. Oral administration of neoandrographolide (100–150 mg/kg) also reduced the increase in vascular permeability induced by acetic acid in mice. In vitro studies were performed using the macrophage cell line RAW264.7 to study the effect of neoandrographolide on suppressing phorbol-12-myristate-13-acetate (PMA)-stimulated respiratory bursts and lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α). Respiratory bursts were quantified by chemiluminescence (CL) measurements.Results showed that neoandrographolide suppressed PMA-stimulated respiratory bursts dose-dependently from 30 μM to 150 μM. Neoandrographolide also inhibited NO and TNF-α production in LPS-induced macrophages, contributing to the anti-inflammatory activity of A. paniculata. These results indicate that neoandrographolide possesses significant anti-inflammatory effects, which implies that it would be one of the major contributing components to participate in the anti-inflammatory effect of A. paniculata. and a potential candidate for further clinical trial.

Preclinical Research on Idiotype Vaccine Against B Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4744-4744
Author(s):  
Fuxu Wang ◽  
Bing Zhao ◽  
Ling Pan ◽  
Xuejun Zhang ◽  
Jianmin Luo ◽  
...  
Keyword(s):  
Fusion Protein ◽  
B Cell ◽  
Fusion Proteins ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Host Cells ◽  
Fusion Genes ◽  
Preclinical Research

Abstract The idiotype (Id) of immunoglobulin expressed by B cell lymphoma can serve as the only widely accepted tumor associated antigen. But the Id vaccines have failed to elicit anti-tumor immunity for its weak immunogenic. Monocyte chemoattractant protein-3 (MCP3) can recruit various subsets of immune cells, such as DCs, which would uptake and properly process and present Id, activating both arms of the immune system, humoral and cellular. So the Id-MCP3 fusion proteins are potential vaccines for immunotherapy of B cell lymphoma. In this study, two vaccine candidates were constructed by fusing allogeneous MCP3 to the amino-(MCP3-scFv) or carboxyl-(scFv-MCP3) terminus of the A20 (BABL/c murine B-lymphocyte) Id scFv with a flexible polypeptide spacer encoding NDAQAPKS to prevent dissociation and keep their respective natural construction and function. And VH and VL domains were linked with a current linker encoding (Gly4Ser)3. Firstly, the cDNAs of Ig VH and Ig VL were amplified by RT-PCR from A20 mRNA, and then assembled into scFv by recombinant PCR method. Secondly the fusion genes of scFv/MCP3 were formed using the same method. After sequencing, MCP3/scFv fusion genes were cloned into pET-39b vector. Lastly MCP3/scFv fusion proteins were expressed in E.coli BL21. And the fusion protein is about 62 kD. We found that, under the same condition, MCP3-scFv fusion protein was expressed successfully and accounted for 40% of the total protein of the bacteria but not scFv-MCP3. Our result indicated that fusing MCP3 to carboxyl-terminus of scFv protein may have cytotoxicity to the host cells or maybe not stable inside the host cells. Next we will determine the activity of the fusion protein MCP3-scFv with cell-chemotatic-experiment in vitro and bearing-tumor mice experiment in vivo. Once the results would suggest that there may be an anti-tumor effect, we can make individual vaccines to lead to a better survival.

Blockade Of CD47 Using SIRPαFc: Role Of The Fc Region In Anti-Leukemic Activity and Tolerability

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3935-3935 ◽  
Author(s):  
Robert A. Uger ◽  
Xinli Pang ◽  
Mark Wong ◽  
Violetta House ◽  
Karen Dodge ◽  
...  
Keyword(s):  
Stem Cell ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Research Funding ◽  
Cell Therapeutics ◽  
Human Igg1 ◽  
Effector Activity ◽  
Stem Cell Therapeutics

Abstract Introduction CD47 binds to SIRPα on the surface of macrophages and delivers a “do not eat” signal that suppresses phagocytosis. There is increasing evidence that acute myeloid leukemia (AML) stem cells exploit the CD47-SIRPα pathway to escape macrophage-mediated destruction. Blockade of CD47 using a soluble SIRPα-Fc fusion protein (SIRPαFc) has emerged as a promising strategy to neutralize the suppressive effects of CD47 and promote the eradication of AML cells. However, little information is available regarding the optimal structure of SIRPαFc. In particular, the influence of the Fc region, which can mediate antibody-dependent cellular cytotoxicity and complement activation, on anti-leukemic activity and toxicity has not been explored. Results We have generated three unique human SIRPαFc fusion proteins that vary in their Fc regions: SIRPα-G1, which contains the Fc region from human IgG1 with full effector activity; SIRPα-G4, bearing the Fc region from human IgG4, which has low effector activity; and SIRPα-G4m, which possesses a mutated human IgG4 Fc region that is devoid of any effector activity. These three fusion proteins were tested for their ability to promote macrophage-mediated phagocytosis of patient-derived AML cells in vitro. Although all three proteins were able to stimulate tumor cell destruction, SIRPα-G4m was clearly the least potent, while SIRPα-G1 and SIRPα-G4 showed similar activity. Next, the anti-leukemic activity of the fusion proteins was assessed in an AML xenograft model in NOD.SCID mice. SIRPα-G1 induced a profound anti-leukemic effect and was superior to both SIRPα-G4 and SIRPα-G4m, particularly with respect to eradicating tumor cells within the transplanted femur. Thus, while only a low level of Fc activity was required for maximal pro-phagocytic activity in vitro, full effector activity (human IgG1) provided superior anti-leukemic activity in vivo. The strong anti-tumor activity of this fusion protein presumably results from the simultaneous delivery of a positive macrophage activating signal (through Fc receptors) and blockade of the negative “do not eat” signal from CD47. Increased Fc effector activity could also carry the risk of increased toxicity. Since human SIRPα has no measurable binding to mouse CD47, to assess tolerability in mice we generated a surrogate fusion protein consisting of NOD mouse SIRPα linked to a mouse IgG2a Fc region with full effector function (mSIRPα-G2a). Repeat administration of high dose mSIRPα-G2a to mice (50 mg/kg IP twice per week for 8 weeks) produced no adverse clinical effects. No abnormalities were observed in hematological parameters, (including erythrocyte, platelet and leukocyte counts) or bone marrow CD150+CD48- LSK hematopoietic stem cells, nor were gross or microscopic changes noted in any tissue. Furthermore, taking advantage of a fortuitous cross-reactivity between NOD SIRPα and human CD47, we conducted a xenograft study with patient-derived AML cells using the mSIRPα-G2a fusion protein. Compared to control Fc, mSIRPα-G2a profoundly reduced leukemic burden in both the injected femur and non-injected bone marrow at doses significantly below the 50 mg/kg used in the tolerability studies. Thus, a mouse surrogate fusion that can bind both human CD47 on xenograft AML cells and endogenous CD47 on host tissue is both safe and effective. A pilot repeat-dose toxicity study using various human SIRPαFc proteins is currently underway in non-human primates. Conclusions These results demonstrate that SIRPαFc fusion proteins that combine Fc activity with CD47 blockade lead to effective AML destruction in vitro and in vivo, and are well tolerated in mice. Thus the therapeutic window in a homologous model system appears to be sufficiently wide to proceed with formal IND-enabling studies. On the basis of these findings we are moving forward with the development of a SIRPαFc therapeutic for the treatment of AML. Disclosures: Uger: Trillium Therapeutics/Stem Cell Therapeutics: Employment. Pang:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Wong:Trillium Therapeutics/Stem Cell Therapeutics: Employment. House:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Dodge:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Viau:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Vigo:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Tam:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Truong:Trillium Therapeutics/Stem Cell Therapeutics: Employment. Jin:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Malko:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Ho:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Prasolava:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Danska:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Wang:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding. Petrova:Trillium Therapeutics/Stem Cell Therapeutics: Employment.

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