Journal: Cancers

Article Title: IOS-1002, a Stabilized HLA-B57 Open Format, Exerts Potent Anti-Tumor Activity

doi: 10.3390/cancers16162902

Figure Lengend Snippet: Structure, expression and receptor binding characteristics of IOS-1002. ( A ) Schematic representation of the IOS-1002 molecule constructed through the ligation of HLA-B57 (A46E/V97R) on N-terminus of human IgG4 Fc domain. ( B ) The topological structure of HLA-B57:01:01 including the B2m molecule. Mutation site residues A46 and V97 highlighted as spheres (PDB: 5VUF). ( C ) SEC-HPLC profile of purified IOS-1002. ( D ) Thermal unfolding profile of IOS-1002, determined by DSF. ( E ) Quantification of the binding affinities of IOS-1002 to LILRB1 ( n = 4), LILRB2 ( n = 5) and KIR3DL1 ( n = 1) surface receptors determined by SPR. Red line represents raw data and black line represents the fit of 1:1 binding. RU: response units; K D : binding constant represented as mean ± standard deviation. ( F ) The topological structure of the HLA-B57:01:01 interaction site generated by superimposing the HLA-B57 structure (PDB: 2HJK) onto LILRB1/HLA-G and LILRB2/HLA-G. The residues lining the binding interfaces between HLA-B57-B2m:LILRB1 and HLA-B57-B2m:LILRB2 are highlighted under the dashed circles and displayed as sticks. The crystal structure of HLA-B57:01 and KIR3DL1 allotype 015 (PDB: 5B39), which describes a separate epitope on the HLA-B57 α1-helix, incorporating residues 77–83, known as the Bw4 motif. Structural images generated using PyMOL. ( G ) Quantification of the binding affinity of IOS-1002 to FcγRI determined by SPR ( n = 1). The specified n indicates the number of independent experiments.

Article Snippet: H460, H1703, RPMI-8226 were cultured in RPMI1640 GlutaMax media (ThermoFisher, Waltham, MA, USA, 61870044) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher, Waltham, MA, USA, A3840102), 100 U/mL penicillin G and 100 µg/mL streptomycin (ThermoFisher, Waltham, MA, USA, 15070063), MIA PaCa-2 was cultured in DMEM GlutaMax media (ThermoFisher, Waltham, MA, USA, 10566016) supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, HCT116 was cultured in McCoy’s 5A media (ThermoFisher, Waltham, MA, USA, 16600082) supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin and 1mg/mL Geneticin (ThermoFisher, Waltham, MA, USA, 10131035), CHO expressing LILRB1 (Ham’s F-12 Nutrient Mix GlutaMAX media (ThermoFisher, Waltham, MA, USA, 31765068) supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 2 µg/mL Puromycin (Invivogen, Waltham, MA, USA, ant-pr-1)), CHO expressing LILRB2 (Ham’s F-12 Nutrient Mix GlutaMAX media supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 1 µg/mL Bleomycin (MedChemExpress, Monmouth Junction, NJ, US, HY-17565A)), CHO expressing KIR3DL1 (Ham’s F-12 Nutrient Mix GlutaMAX media supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 300 µg/mL Hygromycin (AG scientific, San Diego, CA, USA, HY-17565A)), CHO expressing LILRB1/LILRB2/CD64 (Ham’s F-12 Nutrient Mix GlutaMAX media supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 2 µg/mL Puromycin, 1 µg/mL Bleomycin and 400 µg/mL Hygromycin).

Techniques: Expressing, Binding Assay, Construct, Ligation, Mutagenesis, Purification, Standard Deviation, Generated

Journal: Cancers

Article Title: IOS-1002, a Stabilized HLA-B57 Open Format, Exerts Potent Anti-Tumor Activity

doi: 10.3390/cancers16162902

Figure Lengend Snippet: IOS-1002 binds to target receptors on human primary cells and inhibits the associated downstream signaling. ( A ) CHO cells were transduced with LILRB1, LILRB2 and CD64 FcγRI and interaction of AF488 labeled molecules was measured by flow cytometry ( n = 2). Mean ± standard deviation is presented. MFI: median of fluorescence intensity. ( B ) Dose-dependent binding of IOS-1002 on human primary monocytes and the monocyte-derived macrophages isolated from PBMCs ( n = 4). Mean ± standard deviation is presented. MFI: mean of fluorescence intensity. The non-linear regression curve and EC 50 (95% Confidential Interval) were calculated using the model agonist vs. response variable slope (four parameters) in A and B. ( C ) Competition between IOS-1002, anti-LILRB1, anti-LILRB2, dual anti-LILRB1/2 and anti-CD64 antibody for cell surface epitopes on monocytes. Fold change of background-subtracted MFI relative to the cells pre-treated with IgG1 null antibody is presented, ( n = 4). Mean ± standard deviation is presented. Statistical analysis of various conditions against IgG1 null control was performed using one-sample t -test (hypothetical mean = 1) and pre-treatment of combined dual anti-LILRB1/2 and anti-CD64 antibodies against anti-LILRB1/2 or anti-CD64 antibodies was analyzed by one-way ANOVA with Bonferroni multiple comparisons test. ( D ) Simple Western analysis showing expression and phosphorylation of ITIM-associated phosphatases, SHP-1 and SHP-2 in human primary monocytes-derived macrophages ( n = 5). Quantification of phosphorylation over total protein relative to isotype control is presented in the graph on the right. Mean ± standard deviation is presented. Stars indicate the statistical significance against IgG4 control (one-sample t -test, hypothetical mean = 1). * p < 0.05; ** p < 0.01; **** p < 0.0001. ns, non-significant. In ( A ) n indicates the number of independent experiments, in ( B – D ) n indicates the number of independent donors.

Article Snippet: H460, H1703, RPMI-8226 were cultured in RPMI1640 GlutaMax media (ThermoFisher, Waltham, MA, USA, 61870044) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher, Waltham, MA, USA, A3840102), 100 U/mL penicillin G and 100 µg/mL streptomycin (ThermoFisher, Waltham, MA, USA, 15070063), MIA PaCa-2 was cultured in DMEM GlutaMax media (ThermoFisher, Waltham, MA, USA, 10566016) supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, HCT116 was cultured in McCoy’s 5A media (ThermoFisher, Waltham, MA, USA, 16600082) supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin and 1mg/mL Geneticin (ThermoFisher, Waltham, MA, USA, 10131035), CHO expressing LILRB1 (Ham’s F-12 Nutrient Mix GlutaMAX media (ThermoFisher, Waltham, MA, USA, 31765068) supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 2 µg/mL Puromycin (Invivogen, Waltham, MA, USA, ant-pr-1)), CHO expressing LILRB2 (Ham’s F-12 Nutrient Mix GlutaMAX media supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 1 µg/mL Bleomycin (MedChemExpress, Monmouth Junction, NJ, US, HY-17565A)), CHO expressing KIR3DL1 (Ham’s F-12 Nutrient Mix GlutaMAX media supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 300 µg/mL Hygromycin (AG scientific, San Diego, CA, USA, HY-17565A)), CHO expressing LILRB1/LILRB2/CD64 (Ham’s F-12 Nutrient Mix GlutaMAX media supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 2 µg/mL Puromycin, 1 µg/mL Bleomycin and 400 µg/mL Hygromycin).

Techniques: Transduction, Labeling, Flow Cytometry, Standard Deviation, Fluorescence, Binding Assay, Derivative Assay, Isolation, Control, Simple Western, Expressing

Journal: Cancers

Article Title: IOS-1002, a Stabilized HLA-B57 Open Format, Exerts Potent Anti-Tumor Activity

doi: 10.3390/cancers16162902

Figure Lengend Snippet: IOS-1002 affects the differentiation of monocytes toward MDSCs and enhances phagocytosis of monocyte-derived macrophages. ( A ) Scheme of different monocyte-derived immune cell-based assays performed. ( B , C ) The effect of IOS-1002 on the differentiation potential of monocytes toward MDSCs ( n = 3) ( B ) and M2 macrophages ( n = 4) ( C ) is presented and compared with anti-LILRB2 antibody. Mean ± standard deviation is presented. In C, stars indicate the statistical significance toward IgG4 control. ( D ) Macrophage phagocytosis in the presence of different concentrations of IOS-1002 toward H460 (NSCLC cell line) ( n = 4). Mean ± standard deviation of 3 technical replicates is presented. A 4P-L curve was interpolated for quantification of the EC 50 . ( E ) Macrophage phagocytosis in the presence of IOS-1002 on different Fc backbones toward H460 cell line ( n = 2). Mean ± standard deviation is presented. Statistical analysis was performed using one-way ANOVA and Dunnett’s multiple comparisons test. Unless mentioned otherwise, all indicated compounds were used at a concentration of 20ug/mL. * p < 0.05, ** p < 0.01, *** p < 0.001. The specified n indicates the number of independent donors.

Article Snippet: H460, H1703, RPMI-8226 were cultured in RPMI1640 GlutaMax media (ThermoFisher, Waltham, MA, USA, 61870044) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher, Waltham, MA, USA, A3840102), 100 U/mL penicillin G and 100 µg/mL streptomycin (ThermoFisher, Waltham, MA, USA, 15070063), MIA PaCa-2 was cultured in DMEM GlutaMax media (ThermoFisher, Waltham, MA, USA, 10566016) supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, HCT116 was cultured in McCoy’s 5A media (ThermoFisher, Waltham, MA, USA, 16600082) supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin and 1mg/mL Geneticin (ThermoFisher, Waltham, MA, USA, 10131035), CHO expressing LILRB1 (Ham’s F-12 Nutrient Mix GlutaMAX media (ThermoFisher, Waltham, MA, USA, 31765068) supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 2 µg/mL Puromycin (Invivogen, Waltham, MA, USA, ant-pr-1)), CHO expressing LILRB2 (Ham’s F-12 Nutrient Mix GlutaMAX media supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 1 µg/mL Bleomycin (MedChemExpress, Monmouth Junction, NJ, US, HY-17565A)), CHO expressing KIR3DL1 (Ham’s F-12 Nutrient Mix GlutaMAX media supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 300 µg/mL Hygromycin (AG scientific, San Diego, CA, USA, HY-17565A)), CHO expressing LILRB1/LILRB2/CD64 (Ham’s F-12 Nutrient Mix GlutaMAX media supplemented with 10% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, 2 µg/mL Puromycin, 1 µg/mL Bleomycin and 400 µg/mL Hygromycin).

Techniques: Derivative Assay, Standard Deviation, Control, Concentration Assay