Journal: Scientific reports

Article Title: Preclinical pharmacology characterization of HX009, a novel PD1 x CD47 Bi-specific antibody.

doi: 10.1038/s41598-024-79865-3

Figure Lengend Snippet: Fig. 1. HX009 structure and in vitro characterization. (A) Molecular structure of HX009: 2 × 2 symmetric BsAb molecule; (B) HX009 binding to the recombinant CD47 proteins of different species including human, cynomolgus monkey (upper) and mouse (lower), binding EC50 (half maximal effective concentration) were determined per four-parameter equation fitting curves; (C) competitive binding to CD47 single-positive (upper panel) or CD47-PD1 double positive (lower panel) cells by HX009. Labeled B6H12 (anti-CD47) was competed out by SIRPα-Fc, but not by HX009 (weakened binding) in single-positive cell assay, while it was competed out in double-positive cell assays by both SIPRα-Fc and HX009. (D) T-cell activation luciferase report assays: upper panel: HX009 had enhanced T-cell activation (cis-binding) over HX008 (4X), whereas the enhancement is diminished by an anti-SIRPα neutralizing antibody but not by CD47-Fc (soluble CD47). Lower panel: there was no enhancement of T-cell activation by HX008 and SIRPα-Fc combo treatment due to trans-binding.

Article Snippet: By coupling the 7.5 μg/mL of HX009 antibody on the chip surface, the recombinant human (Atagenix), cynomolgus monkey (Acrobiosystems), mouse (Sinobiological) and rat (Sinobiological) CD47 proteins, at serial loading concentrations from 2 to 128 nM (two-fold dilution), or the recombinant human (Atagenix), cynomolgus monkey (Sinobiological), mouse (Sinobiological) and rat (Sinobiological) PD1 proteins at serial loading concentrations from 1 to 64 nM (two-fold dilution), flowed through the chip surface, a binding of 200-s to reach the target level above 770 RU, and a 20-min dissociation time with 10 mM of Glycine pH 1.7.

Techniques: In Vitro, Binding Assay, Recombinant, Concentration Assay, Labeling, Activation Assay, Luciferase

Journal: Scientific reports

Article Title: Preclinical pharmacology characterization of HX009, a novel PD1 x CD47 Bi-specific antibody.

doi: 10.1038/s41598-024-79865-3

Figure Lengend Snippet: Fig. 3. HX009 anti-tumor pharmacology studies in preclinical cancer models. (A) Pharmacology evaluation of HX009 in a humanized MC38-huCD47 mouse colon cancer model in huPD1-HuGEMM mice; (B) Pharmacology evaluation of HX009 in a humanized MC38-huCD47 mouse colon cancer model in huPD1 × huPD-L1 × huCD47 × huSIRPα-HuGEMM model; (C) Pharmacology evaluation of HX009 in three AML-PDX models as shown in the figures. Columns from Left to right: Leukemic burden was measured as percentage of human CD45+ cells in peripheral blood; survival was displayed as in Caplan-Meier plot; Leukemic loads in different organs at the termination, as % of human CD45+. SP spleen, BM bone marrow, PB peripheral blood. Bottom: The differential expressions of CD47 per IHC and RNAseq in three AML-PDX models are shown in the bottom table. Graphs in A-D showed mean tumor volume ± standard error of the mean (SEM). Significance was calculated using one-way ANOVA with post-hoc comparisons or Welch’s t- test between treatment groups and vehicle group. ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001. (D) Correlation of OX40 mRNA levels with HX009 anti-lymphoma activity represented by TGI (tumor growth inhibition) in DLBCL-PDX trial.

Article Snippet: By coupling the 7.5 μg/mL of HX009 antibody on the chip surface, the recombinant human (Atagenix), cynomolgus monkey (Acrobiosystems), mouse (Sinobiological) and rat (Sinobiological) CD47 proteins, at serial loading concentrations from 2 to 128 nM (two-fold dilution), or the recombinant human (Atagenix), cynomolgus monkey (Sinobiological), mouse (Sinobiological) and rat (Sinobiological) PD1 proteins at serial loading concentrations from 1 to 64 nM (two-fold dilution), flowed through the chip surface, a binding of 200-s to reach the target level above 770 RU, and a 20-min dissociation time with 10 mM of Glycine pH 1.7.

Techniques: Activity Assay, Inhibition

Journal: Blood

Article Title: Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages.

doi: 10.1182/blood-2009-07-231449

Figure Lengend Snippet: Figure 1. Galectin-5 is present on the surface of rat red cells. (A) Freshly isolated reticulocytes or erythrocytes were adsorbed on glass coverslips and processed for immunofluorescence as described in “Fluorescence-activated cell-sorting analysis of exosomes and red cells, fluorescence microscopy of red cells.” Transmission images of red cells (left) and corresponding fluorescence imaging (right) were recorded on cells by the use of purified rabbit anti–galectin-5 antibody followed by incubation withAlexa 488 anti–rabbit antibody. (B) Young reticulocytes and erythrocytes were analyzed by flow cytometry by the use of antibodies raised against Gal-2 (dashed line), Gal-4 (dotted line), and Gal-5 (solid line), already tested for their specificity (top), or the produced anti–galectin-5 serum (solid line) and the preimmune serum (bottom, dashed line). Tinted patterns indicate cell labeling obtained in the absence of primary antibodies. (C) Lymphocytes isolated from rat blood, as described in “Cells,” were analyzed by flow cytometry for Gal-5 (left, solid line), CD47 (middle, solid line) and Syto 16 green (right, solid line). Tinted patterns indicate cell labeling in the absence of primary antibodies. (D) Ghost and raft extracts isolated from reticulocytes or mature erythrocytes, as described in “Red cell subcellular fractionation,” were processed by SDS-PAGE and analyzed by Western blot for the indicated proteins. The molecular mass (kDa) standards are indicated on the left.

Article Snippet: Mouse anti–rat CD47 was from Serotec Limited.

Techniques: Isolation, Fluorescence, FACS, Microscopy, Transmission Assay, Imaging, Incubation, Cytometry, Produced, Labeling, Fractionation, SDS Page, Western Blot

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) ATAC-Seq analysis for JUN and the hedgehog genes Gli1 and Ptch1 in scleroderma fibroblasts (SSCL), scleroderma fibroblasts after JUN knockout (JUN-KO) or under vismodegib, and normal skin fibroblasts. The promoter regions are highlighted with red boxes. n = 2. ( B ) ATAC-Seq analysis for the immune checkpoints CD47 and PD-L1 and the interleukin IL-6 in scleroderma fibroblasts, scleroderma fibroblasts after JUN knockout or under vismodegib, and normal skin fibroblasts. The promoter regions are highlighted with red boxes. n = 2. ( C ) Heatmap of differential open chromatin regulatory elements characterized from ATAC-Seq. The color bar shows the relative ATAC-Seq signal ( Z score of normalized read counts) as indicated. Samples 1 and 2 in both groups are individual samples. n = 2. ( D ) Fibrosis-linked genes with a 5-fold decline in promoter accessibility after JUN knockout. n = 2. ( E ) p-JUN expression in pulmonary fibroblasts and scleroderma fibroblasts on a 70 kPa hydrogel or a regular polystyrene plastic dish. Two-sided t test. ** P < 0.01; *** P < 0.001. n = 4. Tukey’s multiple comparison test. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Knock-Out, Expressing, Comparison

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) Immunofluorescence stains against CD47 and FSP1 with and without JUN induction. Scale bar: 25 μm. n = 5. ( B ) Histogram of CD47 expression in fibroblasts with and without JUN induction. n = 5. ( C ) Percentage of CD47 positivity in different fibroblast populations with and without JUN induction. Fisher’s multiple comparison test. ** P < 0.01; *** P < 0.01. n = 5. Bars represent means with standard deviations. ( D ) Representative optical images of ectopically transplanted JUN-inducible mouse dermal fibroblasts ± CD47 inhibition. n = 4. ( E ) Corresponding quantification of photon emissions. Values are normalized to day 0. Fisher’s multiple comparison test. ** P < 0.01. n = 4. Bars represent means with standard deviations. ( F ) Fluorescent graft visualization under the dissection microscope after 7 days of CD47 inhibition. Scale bar: 5 mm. n = 2. ( G ) FACS plot for PE/RFP + CD11b + macrophages ± JUN induction ± CD47 inhibition in an in vitro phagocytosis assay. n = 3. ( H ) Corresponding quantification of RFP + macrophages. Tukey’s multiple comparison test. * P < 0.05; *** P < 0.01. n = 3. Bars represent means with standard deviations. ( I ) Schema of a macrophage depletion trial with subsequent skin fibrosis induction. n = 5. ( J ) Corresponding trichrome stains. Scale bar: 500 μm. n = 5. ( K ) Corresponding hydroxyproline assay. Two-sided t test. *** P < 0.001. n = 5. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Immunofluorescence, Expressing, Comparison, Inhibition, Dissection, Microscopy, In Vitro, Phagocytosis Assay, Hydroxyproline Assay

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) Schematic outline of the therapeutic trial. ( B ) Representative H&E and trichrome stains of the different groups. Scale bar: 500 μm. n = 4. ( C ) Hydroxyproline content of the skin. Tukey’s multiple comparison test. * P < 0.05; ** P < 0.01. n = 6. Graph bars represent means with standard deviations. ( D ) Amount of fat tissue. Values indicate μm 2 /μm skin width. Tukey’s multiple comparison test. * P < 0.05. n = 8. Graph bars represent means with standard deviations. ( E ) Representative optical images of ectopically transplanted GFP/luciferase-labeled human scleroderma fibroblasts ± CD47/IL-6 inhibition. ( F ) Optical imaging of explanted kidneys on day 5. ( G ) Quantification of photon emissions of explanted kidney grafts normalized to the values of the untreated mice. Two-sided t test. * P < 0.05. n = 3–4. Bars represent means with standard deviations. ( H ) Corresponding caspase-3 staining of kidney grafts. Scale bar: 25 μm. ( I ) Corresponding percentage of caspase-3 + GFP + fibroblasts. Two-sided t test. ** P < 0.01. n = 4–5. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Comparison, Luciferase, Labeling, Inhibition, Optical Imaging, Staining

Journal: Journal of Neuroinflammation

Article Title: Deletion of SIRPα (signal regulatory protein-α) promotes phagocytic clearance of myelin debris in Wallerian degeneration, axon regeneration, and recovery from nerve injury

doi: 10.1186/s12974-019-1679-x

Figure Lengend Snippet: Macrophages present in Wallerian degeneration express SIRPα in wild-type mice. a – f Cryostat sections in which CD47 ( a , c , e ) and SIRPα ( b , d , f ) were visualized by immunofluorescence microscopy using anti-CD47 and anti-SIRPα mAbs. Sections were taken from intact sciatic nerve ( a , b ), sciatic nerve undergoing in vivo Wallerian degeneration for 5 days ( c , d ), and cultured sciatic nerve explant undergoing in vitro Wallerian degeneration for 5 days ( e , f ). All three tissues displayed immunoreactivity to CD47 whereas only sciatic nerve undergoing in vivo Wallerian degeneration displayed immunoreactivity to SIRPα. Bar in f : 100 μm, for a through f . g – o Three macrophages ( g – i ), ( j – l ), and ( m – o ) from 7 days in vivo Wallerian degenerating nerve segments were visualized by immunofluorescence microscopy using anti-SIRPα ( g , j , m ; red) and anti-αM/CD11b subunit of CR3 ( h , k , n ; green) Abs. SIRPα and CR3 combined ( i , l , o ). Hoechst staining visualized nuclei (blue). The three cells displayed immunoreactivity to both SIRPα and CR3. Bar in o : 5 μm, for g through o .

Article Snippet: To visualize CD47 and SIRPα, sections were incubated in rat anti-mouse CD47 and SIRPα monoclonal antibody (mAb) (provided by Dr. Oldenborg, Umea, Sweden).

Techniques: Immunofluorescence, Microscopy, In Vivo, Cell Culture, In Vitro, Staining

Journal: Journal of Neuroinflammation

Article Title: Deletion of SIRPα (signal regulatory protein-α) promotes phagocytic clearance of myelin debris in Wallerian degeneration, axon regeneration, and recovery from nerve injury

doi: 10.1186/s12974-019-1679-x

Figure Lengend Snippet: SIRPα inhibits the phagocytosis of myelin debris in cultured BMDM (bone marrow-derived macrophages). a BMDM from wild-type (WT; +/+) but not SIRPα−/− (−/−) mice expressed SIRPα protein. Immunoblot of BMDM lysates using anti-SIRPα Ab determined the presence/absence of SIRPα protein (130KD). Immunoblot of same lysates using anti-α-tubulin Ab (55KD) verified equal protein load. b BMDM from SIRPα−/− mice (SIRPα−/−) phagocytosed more myelin debris than BMDM from wild-type mice (WT). BMDM plated at low cell density were exposed to myelin debris for 40 min and levels of phagocytosed myelin debris quantified by ELISA. Phagocytosis by SIRPα−/− BMDM is presented as percentage of phagocytosis by wild-type BMDM normalized to 100%. Box and whisker plot of eight experiments is given. The line represents the median, the box outlines the 25 to 75% range, and the whiskers extend to the highest and lowest observations. Double tailed value of significance, **** p < 0.0001, by unpaired t test

Article Snippet: To visualize CD47 and SIRPα, sections were incubated in rat anti-mouse CD47 and SIRPα monoclonal antibody (mAb) (provided by Dr. Oldenborg, Umea, Sweden).

Techniques: Cell Culture, Derivative Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Whisker Assay

Journal: Journal of Neuroinflammation

Article Title: Deletion of SIRPα (signal regulatory protein-α) promotes phagocytic clearance of myelin debris in Wallerian degeneration, axon regeneration, and recovery from nerve injury

doi: 10.1186/s12974-019-1679-x

Figure Lengend Snippet: The in vivo removal of myelin debris is faster in SIRPα−/− mice than in wild-type mice. Sciatic nerve segments undergoing Wallerian degeneration were removed from wild-type (WT) and SIRPα−/− mice at the indicated days after surgery, immediately lysed and protein content in lysates quantified. Levels of myelin-specific MBP (Myelin/MBP) in lysate samples of equal protein content were quantified using ELISA. Levels of Myelin/MBP in Wallerian degenerating nerve segments are presented as percentage of levels in intact nerves (time 0) normalized to 100%. Box and whisker plot of Myelin/MBP levels in five to ten different nerves is given. The line represents the median, the box outlines the 25 to 75% range, and whiskers extend to the highest and lowest observations. Significance of difference of WT mice from SIRPα−/− mice at the indicated days after surgery, ^ p < 0.05 and ^^^ p < 0.001, by two-way ANOVA and the Bonferroni multiple comparisons posttest. Significance of difference between levels of Myelin/MBP in intact nerves (day 0) and those at the indicated days after surgery, ** p < 0.001 and *** p < 0.0001, by one-way ANOVA and the Dunnett posttest calculated for each mice strain separately

Article Snippet: To visualize CD47 and SIRPα, sections were incubated in rat anti-mouse CD47 and SIRPα monoclonal antibody (mAb) (provided by Dr. Oldenborg, Umea, Sweden).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Whisker Assay

Journal: Journal of Neuroinflammation

Article Title: Deletion of SIRPα (signal regulatory protein-α) promotes phagocytic clearance of myelin debris in Wallerian degeneration, axon regeneration, and recovery from nerve injury

doi: 10.1186/s12974-019-1679-x

Figure Lengend Snippet: Sensory and motor functions recover faster in SIRPα−/− mice than in wild-type mice. The a flexor-withdrawal reflex and b toe-spreading reflex recovery curves display the cumulative percentage of mice that regained ( a ) sensory and ( b ) motor functions at each of the indicated days after surgery. a Sensory function was tested in 14 wild-type (WT) and 11 SIRPα−/− mice. Significance of difference of WT mice from SIRPα−/− mice, p < 0.001, by the log-rank Mantel-Cox test. b Motor function was tested in 12 wild-type (WT) and 11 SIRPα−/− mice using the toe-spreading reflex. Significance of difference of wild-type mice from SIRPα−/− mice, p < 0.01, by the log-rank Mantel-Cox test.

Article Snippet: To visualize CD47 and SIRPα, sections were incubated in rat anti-mouse CD47 and SIRPα monoclonal antibody (mAb) (provided by Dr. Oldenborg, Umea, Sweden).

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Deletion of SIRPα (signal regulatory protein-α) promotes phagocytic clearance of myelin debris in Wallerian degeneration, axon regeneration, and recovery from nerve injury

doi: 10.1186/s12974-019-1679-x

Figure Lengend Snippet: Severed axons regenerate faster in SIRPα−/− mice than in wild-type mice. Axons and macrophages were visualized in intact and Wallerian degenerating ( a ) saphenous and ( b ) sciatic nerves by immunofluorescence microscopy using Abs against neurofilaments (NF; red) for labeling axons and mAbs against the αM/CD11b subunit of CR3 (green) for labeling macrophages. The overlay of NF/red over CR3/green is yellow. Hoechst staining visualized nuclei (blue). a Intact (0d) and freeze-crushed saphenous nerves were sampled at distances ranging from 8 to 10 mm distal to lesion sites 2, 4, and 7 days (2d, 4d, and 7d) after surgery. b Intact (0d) and freeze-crushed sciatic nerves were sampled at distances ranging from 8 to 10 mm distal to lesion sites 3, 7, and 9 days (3d, 7d, and 9d) after surgery. Initially, on days 2 and 3 after surgery, NF immunoreactivity decreased in wild-type (WT) and SIRPα−/− mice. Then, at the indicated days thereafter, NF immunoreactivity increased more in SIRPα−/− mice than in wild-type mice. CR3 immunoreactivity that was hardly detected in intact nerves (0d) increased as of days 2 and day 3 after surgery onwards in saphenous and sciatic nerves of both WT and SIRPα−/− mice; arrows mark some of the CR3 expressing cells. Shown are representative images from four nerves that were sampled at each time point. Bars: 20 μm in a and 100 μm in b

Article Snippet: To visualize CD47 and SIRPα, sections were incubated in rat anti-mouse CD47 and SIRPα monoclonal antibody (mAb) (provided by Dr. Oldenborg, Umea, Sweden).

Techniques: Immunofluorescence, Microscopy, Labeling, Staining, Expressing

Journal: Journal of Neuroinflammation

Article Title: Deletion of SIRPα (signal regulatory protein-α) promotes phagocytic clearance of myelin debris in Wallerian degeneration, axon regeneration, and recovery from nerve injury

doi: 10.1186/s12974-019-1679-x

Figure Lengend Snippet: Regenerating axons appear faster in SIRPα−/− than in wild-type mice but the number of CR3 expressing cells present in Wallerian degeneration are comparable in the two mice strains. a , b Neurofilaments (NF) were counted in cryostat sections from wild-type (WT) and SIRPα−/− mice (Fig. ) in 100 μm 2 areas of a saphenous nerves 4 days after surgery and b sciatic nerves 7 days after surgery. The number of NF images that label axons was about twofold higher in SIRPα−/− than in wild-type mice in the two nerves. Significance of difference of the number of NF images between SIRPα−/− and wild-type mice, * p < 0.05, by Mann-Whitney test. c , d Cells that express the phagocytic receptor CR3 were counted in cryostat sections from wild-type (WT) and SIRPα−/− mice (Fig. ) at the indicated days after surgery in c 100 μm 2 area of saphenous nerves and d 200 μm 2 area of sciatic nerves. Green images (CR3-labeled cells) and yellow images (green, CR3-labeled cells overlaying red, NF-labeled axon) were identified as CR3-expressing cells. The number of CR3 expressing cells increased significantly with time after surgery in both sciatic and saphenous nerves but to same levels in the two mice strains. Significance of difference of the number of CR3 expressing cells between days 2 and 4 in saphenous nerves and days 3 and 7 in sciatic nerves from SIRPα−/− and wild-type (WT) mice, * p < 0.05, by Mann-Whitney test. Box and whisker plots of the number of NF and CR3 expressing cells from four different nerves at each indicated day after surgery are given. The line represents the median, the box outlines the 25% to 75% range, and whiskers extend to the highest and lowest observations

Article Snippet: To visualize CD47 and SIRPα, sections were incubated in rat anti-mouse CD47 and SIRPα monoclonal antibody (mAb) (provided by Dr. Oldenborg, Umea, Sweden).

Techniques: Expressing, MANN-WHITNEY, Labeling, Whisker Assay