Journal: Scientific reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression.

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Figure 1. Evidence for domain-specific and concentration dependent associations between C1q and CD33 using purified proteins in slot blot assays. Unlabeled proteins were immobilized on membranes; biotin labeled proteins were in solution. (A–C) Dose related binding of immobilized whole C1q and gC1q, but not CLR, to biotin-CD33M (5 ug/ml). (D) Biotin-whole C1q (10 ug/ml) binds to immobilized CD33m (lacking the extracellular V-like domain and expressing the C2-like domain) in a dose specific manner. (HSA = human serum albumin control showing no reactivity. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). Pixel density was determined by densitometry. (C,D) Bar graphs represent the mean ± SE of pooled data; one-way ANOVA followed by Tukey’s pairwise multiple comparison was used to determine significance. N ≥ 3.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described12, 52.

Techniques: Concentration Assay, Purification, Dot Blot, Labeling, Binding Assay, Expressing, Control, Comparison

Journal: Scientific reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression.

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Figure 2. C1q binds to CD33M and CD33m isoforms on the cell surface. (A) A representative flow cytometry plot showing binding of biotin C1q (20 ug/ml) to HEK293T cells transfected with plasmids encoding CD33m and CD33M and control mock plasmid. Cells were incubated with C1q as outlined in Materials and Methods. Numbers in graphs = % positive/MFI. Debris/dying cells was excluded on the basis of forward and side scatter. (B) Pooled flow cytometry data illustrating binding of C1q (at comparable levels) on the surface of CD33m/M transfected HEK293T cells and a lack of C1q binding on mock transfected cells. For A and B, N = 3. (C) Representative slot blot showing that C1q binds to cell-associated CD33m, CD33M or LAIR-1 in a dose dependent manner. Protein prepared from whole cell lysates (HEK293T cells transfected with plasmid encoding CD33m, CD33M or LAIR-1) was immobilized on membranes at increasing doses; biotin-whole C1q was in solution at 10 ug/ml. HSA = human serum albumin control. Protein concentration was calculated by the BCA method. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). (D) Pooled data analyses illustrating dose related interactions between biotin-C1q and cell-associated CD33m or CD33M represented in (C). (B,D), the mean ± SE of pooled data is shown; for all proteins and doses, N = 3–5. *P = < 0.05, **P = <0.01, ***P < 0.001. One-way ANOVA followed by Tukey’s pairwise multiple comparison was used.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described12, 52.

Techniques: Flow Cytometry, Binding Assay, Transfection, Control, Plasmid Preparation, Incubation, Dot Blot, Protein Concentration, Comparison

Journal: Scientific reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression.

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Figure 3. C1q triggers CD33 ITIM phosphorylation and CD33-LAIR-1 physical associations in human monocytes. Monocytes were either untreated (utx), treated with whole C1q, gC1q, C1q CLR or pervanadate (PV) as detailed in Materials and Methods. (A,B) Immunoblot analyses showing increased phosphorylated (p) CD33 after treatment with C1q and gC1q, but not C1q CLR. CD33 was immunoprecipitated with anti-CD33M antibody; tyrosine phosphorylation of CD33 (pCD33M) was detected with anti-phosphotyrosine (4G10) antibody. The membrane was stripped and re-probed with anti-CD33 WM53 antibody (CD33M). Arrows denote molecular weight of 67kD. Bar graphs denote pooled data expressed as fold change relative to total CD33 and corresponding statistical analyses. One-way ANOVA with post-hoc Tukey multiple comparisons was used to determine significance. N = 4 for A; N = 3 for B. (C) The addition of whole C1q (20 ug/ml) prompts concurrent increases in pLAIR-1 and pCD33 in a phospho-immunoreceptor array. (D) C1q CLR “tails” (20 ug/ml) do not elicit increases in pCD33M in the phospho-immunoreceptor array. Bar graphs represent fold change (plus/minus C1q), calculated from the mean pixel density of duplicates, as determined by densitometry analysis. Corresponding C1q minus/plus array data is located below each bar graph in C and D. Data from a typical array are shown. N = 3 for C and D. (E) Proximity ligation assay, performed on freshly isolated human monocytes as detailed in materials and methods, showing that whole C1q is required for CD33-LAIR-1 crosslinking. Red fluorescent dots represent molecular associations between CD33 and LAIR-1; blue represents nuclear staining with DAPI. CLR and gC1q represent the C1q collagen tail and globular heads of C1q, respectively. Original magnification = 60X. One of three representative experiments is shown.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described12, 52.

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Membrane, Molecular Weight, Proximity Ligation Assay, Isolation, Staining

Journal: bioRxiv

Article Title: Rational design of oxidation-resistant antibodies through local electrostatic modulation

doi: 10.1101/2025.06.29.662139

Figure Lengend Snippet: a) Cryo-EM structure of 15G15.3 Fab (gray) bound to CD33 (violet), showing Trp96 forming key interactions with Lys52 (CD33) and Asp101 (Fab). Trp96 oxidizes at 97% under AAPH stress; W96F mutation abolishes binding ( > 1000-fold loss). b) Electrostatic potential of the lead candidate, with 12 mutated residues shown as pink spheres. c) Scatter plot of Trp oxidation vs. Epot for the lead and 13 variants; point color reflects relative KD. d) Summary of 13 engineered variants. S11 and S13 show improved oxidation resistance with preserved binding.

Article Snippet: Briefly, each antibody variant was captured by Protein A sensor chip (Series S) on the different flow cell to achieve approximately 150 response units (RU), followed by the injection of fivefold serial dilutions of human CD33 protein (R&D Systems; 0.16 nM to 100 nM) in HBS-EP buffer.

Techniques: Cryo-EM Sample Prep, Mutagenesis, Binding Assay

Journal: Blood Cancer Journal

Article Title: Primary CD33-targeting CAR-NK cells for the treatment of acute myeloid leukemia

doi: 10.1038/s41408-022-00660-2

Figure Lengend Snippet: A Schematic representation and surface expression of the CD33-directed second-generation CAR used in this study. Expression was analyzed by flow cytometry 12 days after transgene transfer into primary NK cells. B Time-lapsed expansion of CAR-transduced (CD33-CAR) and untransduced (UTD)-NK cells in the presence of IL-2 (500 IU/mL) and IL-15 (140 IU/mL) ( n = 5). C Expanded NK cells show high cytotoxic activity against various AML cell lines except OCI-AML2. On day 14 of expansion, NK cells were co-incubated with various AML target cells at indicated E:T-ratios. After 24 h, the fraction of viable target cells was quantified by flow cytometry. Data shown are representative of results from two independent experiments. D The AML cell line OCI-AML2 displays high CD33 surface expression. E , F NK cells equipped with a CD33-CAR become highly cytotoxic against OCI-AML2 and CD33-positive primary AML cells. Cells were co-cultivated for 4 h and the viability of target cells was quantitated by flow cytometry. Two representative experiments are shown. G Dynamic monitoring of CAR-NK cell-mediated cytotoxicity. On day 12 after transduction, CAR-NK cells were co-cultured with (GFP + ) OCI-AML2 cells and fluorescence emission was measured in the IncuCyte S3 imaging platform over 4 days. Shown is one representative from three separate experiments with a total of 5 donors. H Repetitive tumor-challenge assay revealed superior serial killing capacity of CD33-CAR-NK cells compared to UTD-NK cells. Expanded NK cells at day 12 post transduction were co-cultured with OCI-AML2 cells at an E:T-ratio of 1:1 and re-challenged with AML cells every other day. Shown is one representative experiment with a total of two donors. All graphs show mean of replicated ± SD.

Article Snippet: An anti-CD56-BV786 antibody (Clone NCAM16.2, BD Biosciences) and a CD33-CAR Detection Reagent (containing a recombinantly expressed fusion protein consisting of the human CD33 extracellular domains and a specifically mutated human IgG1 Fc region, Miltenyi Biotec) followed by an anti-biotin-PE antibody (clone REA746, Miltenyi Biotec) were used to determine CD56 and CD33-CAR expression, respectively.

Techniques: Expressing, Flow Cytometry, Activity Assay, Incubation, Transduction, Cell Culture, Fluorescence, Imaging

Journal: Blood Cancer Journal

Article Title: Primary CD33-targeting CAR-NK cells for the treatment of acute myeloid leukemia

doi: 10.1038/s41408-022-00660-2

Figure Lengend Snippet: A Scheme of the in vivo evaluation of a single treatment with CD33-CAR-NK cells (1 × 10 7 intravenously) followed by subcutaneous treatment with IL-2 in OCI-AML2 (Luc + ) xenograft NSG-SGM3 mice. B Total flux analysis as well as representative BLI images of differently treated OCI-AML2 (Luc + ) engrafted NSG-SGM3 mice over time (d7 n = 7; d14 n = 6; d21 n = 5 per group). Mice received a single dose of 1 × 10 7 NK cells day 3 post AML cell injection. At day 21, 4 out of 5 mice (80%) that were treated with CD33-CAR-NK cells show severely reduced leukemic burden compared to untreated mice (UT) or mice which received untransduced (UTD)-NK cells. C Serum analysis of blood day 3 before AML injection and day 1 post first NK cell application shows significantly increased levels of GM-CSF as well as INF-γ for mice that had received CD33-CAR-NK cells ( n = 3). Mean ± SD. D Total flux analysis of femurs/tibiae and spleens, as well as flow cytometry analysis of isolated cells from BMs or spleens at day 7, 14, and 21 post tumor cell injection, revealed the absence of GFP-positive tumor cells in CD33-CAR-NK-treated mice as well as increased NK cell infiltration (day 7/14 n = 1; day 21 n = 2 per group). Values of zero were set to 1 for total flux analysis. Median ± range. Flow cytometry-based CAR expression analysis of BM- ( E ) or spleen- ( F ) infiltrating NK cells at day 14 and 21 revealed the presence of mainly CAR-positive cells (day 14 n = 1; day 21 n = 2 per group). Mean ± SD. G Confocal microscopy imaging shows GFP-positive leukemia cells in BM of UTD-NK treated NSG-SGM3 mice at day 21 while absent in mice that received CD33-CAR-NK cells. Images from one representative animal are shown. Statistical analysis was performed by Student’s t test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: An anti-CD56-BV786 antibody (Clone NCAM16.2, BD Biosciences) and a CD33-CAR Detection Reagent (containing a recombinantly expressed fusion protein consisting of the human CD33 extracellular domains and a specifically mutated human IgG1 Fc region, Miltenyi Biotec) followed by an anti-biotin-PE antibody (clone REA746, Miltenyi Biotec) were used to determine CD56 and CD33-CAR expression, respectively.

Techniques: In Vivo, Injection, Flow Cytometry, Isolation, Expressing, Confocal Microscopy, Imaging

Journal: Blood Cancer Journal

Article Title: Primary CD33-targeting CAR-NK cells for the treatment of acute myeloid leukemia

doi: 10.1038/s41408-022-00660-2

Figure Lengend Snippet: A Scheme of the in vivo evaluation of a repetitive treatment with CD33-CAR-NK cells (1 × 10 7 intravenously) combined with subcutaneous IL-2 treatment in OCI-AML2 (Luc + ) xenograft NSG-SGM3 mice. B Total flux analysis, as well as representative BLI images of differently treated OCI-AML2 (Luc+), engrafted NSG-SGM3 mice over time ( n = 7 per group). Mice received a total of three weekly doses of 1 × 10 7 NK cells. Mice that were treated with CD33-CAR-NK cells show severely reduced leukemic burden compared to untreated mice (UT) or mice which received untransduced (UTD)-NK cells. C Total flux analysis of femurs/tibiae and spleens, as well as flow cytometry analysis of isolated cells from BMs or spleens at day 22 post AML-injection, revealed the absence of GFP-positive leukemic cells in CD33-CAR-NK treated mice as well as increased NK cell infiltration ( n = 6–7 per group). Values of zero were set to 1 for total flux analysis. Median ± range. D Chimerism analysis d22 post AML-injection revealed high amounts of DNA from human NK cells without detectable DNA of AML in blood of mice that were treated with CD33-CAR-NK cells ( n = 6–7 per group). Mean ± SD. E Serum analysis of blood day 3 before AML injection and day 1 post first NK cell application showed significantly increased pro-inflammatory human cytokines for mice that received CD33-CAR-NK cells ( n = 6–7 per group). F Flow cytometry-based CAR-expression analysis of BM- or spleen-infiltrating NK cells in CD33-CAR-NK treated mice revealed the presence of mainly CAR-positive cells ( n = 6–7 per group). Mean ± SD. G Confocal microscopy imaging demonstrated GFP-positive leukemia cells in BM of UTD-NK treated NSG-SGM3 mice while absent in mice that received CD33-CAR-NK cells. Additionally, CAR-NK cells could be detected in the BM of CD33-CAR-NK treated mice. One representative image from a total of four are shown. Statistical analysis was performed by Mann–Whitney-test (for total flux analysis) or Student’s t test (for the rest) (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: An anti-CD56-BV786 antibody (Clone NCAM16.2, BD Biosciences) and a CD33-CAR Detection Reagent (containing a recombinantly expressed fusion protein consisting of the human CD33 extracellular domains and a specifically mutated human IgG1 Fc region, Miltenyi Biotec) followed by an anti-biotin-PE antibody (clone REA746, Miltenyi Biotec) were used to determine CD56 and CD33-CAR expression, respectively.

Techniques: In Vivo, Flow Cytometry, Isolation, Injection, Expressing, Confocal Microscopy, Imaging, MANN-WHITNEY

Journal: Molecules

Article Title: In Vitro and In Vivo Characterization of 89 Zirconium-Labeled Lintuzumab Molecule

doi: 10.3390/molecules27196589

Figure Lengend Snippet: Radiolabeling of lintuzumab with 89 Zr and its in vitro binding to CD33. ( A ) Binding of Lintuzumab−DFO conjugate to recombinant human CD33 protein is demonstrated via ELISA. ( B ) Binding of Lintuzumab−DFO conjugate to human cancer cell lines that express CD33 is demonstrated via immunofluorescence staining using a flow cytometer. ( C ) HPLC trace chromatograms ran on purified antibody conjugate (upper panel) and radiolabeled conjugate at a wavelength of 280 nm (middle panel UV trace, lower panel radioactivity trace).

Article Snippet: ELISA: Nunc MaxiSorp flat-bottomed 96-well plates were coated with 100 ng/well of human recombinant CD33 His-Tag protein (R&D Systems) in PBS and incubated overnight at 4 °C.

Techniques: Radioactivity, In Vitro, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Flow Cytometry, Purification

Journal: Molecules

Article Title: In Vitro and In Vivo Characterization of 89 Zirconium-Labeled Lintuzumab Molecule

doi: 10.3390/molecules27196589

Figure Lengend Snippet: 89 Zr-lintuzumab Selectively Accumulates in CD33 Expressing Tumors. ( A ) PET/CT imaging of mice administered with 5.55 MBq 89 Zr-DFO-Lintuzumab only ( n = 4). Images were taken on Days 1, 2, 3, and 7 post administration. The radioconjugate was cleared from blood and other organs except for the tumor after Day 1 and accumulated in CD33-positive OCI-AML-3 tumors. ( B ) Cohort of mice ( n = 4) pre-blocked with cold Lintuzumab 24hr prior to administration of radioconjugate. PET/CT images were taken on Days 3 and 7 post radioconjugate administration. The clearance of radioconjugate from blood and other organs in mice pre-blocked with Lintuzumab was not as effective as in non-blocked mice: radioactivity was detected in various organs by PET/CT imaging even on Day 7 post administration. ( C ) Standardized uptake values (SUV) analysis of PET/CT images taken on Days 3 and 7. Tumor volumes of interest (VOI) were drawn, and SUV were calculated. 89 Zr-DFO-Lintuzumab showed a significantly higher uptake in the tumors of non-blocked mice than in the tumors of pre-blocked mice. ( D ) Time–activity curves (TAC) show that the radiolabeled antibody remains mostly constant over the 7-day period after initial uptake for both non-blocked and pre-blocked tumors, with the pre-blocked has significantly less uptake. All images are displayed as maximum intensity projections (MIP). ** and *** mean p = 0.001 and p < 0.0001, respectively.

Article Snippet: ELISA: Nunc MaxiSorp flat-bottomed 96-well plates were coated with 100 ng/well of human recombinant CD33 His-Tag protein (R&D Systems) in PBS and incubated overnight at 4 °C.

Techniques: Expressing, Positron Emission Tomography-Computed Tomography, Imaging, Radioactivity, Activity Assay