biotinylated cd33 protein  (ACROBiosystems)

Journal: bioRxiv

Article Title: Chimeric Antigen Receptors Transmit Piconewton Forces that are Coupled with T Cell Function

doi: 10.1101/2025.10.23.684052

Figure Lengend Snippet: A. Proposed mechanism of action for dasatinib in CAR T cells which leads to Lck inhibition. B . Representative RICM and tension images of CAR T cells treated with escalating doses of dasatinib. C and D. Plots quantifying CAR T cell forces and cell spread area as a function of dasatinib treatment. N ≥ 162 from 5 independent transductions. ***= 0.0001, ****<0.0001. E . Plot showing cytotoxicity as a function of dose-dependent dasatinib CAR T cell treatment. Data is averaged from two technical replicates using the same batch of CAR T cells. F . Relationship between CAR T cell tension and cytotoxicity showing correlation. G . Schematic showing three additional engineered CARs with 4-1BB costimulatory domain, ITAM mutants lacking key tyrosine residues, and finally CAR lacking a cytoplasmic domain along with representative RICM and 8 pN locked tension (Cy3B) images. H . Plot of single cell tension levels for each CAR T cell construct tested. N ≥ 162 from 5 independent transductions. One-way ANOVA. ***= 0.0001 , ****<0.0001. I . Representative RICM and 8 pN tension images showing the tension and spread area of cells on CD19 and CD33 antigens. J. Quantification of cellular tension levels from n≥90 cells/condition from 3 separate transductions. Each color indicates a biological replicate from a unique transduction using the same human donor. The larger data points indicate the mean of each replicate while the small data points represent the single cell measurements. N ≥ 106 cells/condition from 3 independent transductions. Student’s Two-Tailed Paired T Test, ****<0.0001.

Article Snippet: Biotinylated CD19 protein (Cat#CD9-H82E9-25ug), biotinylated CD33 protein (Cat#CD3-H82E7-25ug), and AlexaFluor 647 Anti-FMC63 Ab (Cat#FM3-AM534-25tests) were purchased from AcroBioSystems (Newark, DE).

Techniques: Inhibition, Construct, Transduction, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: Tmod can be adapted for blood cancer. (A) Diagram for Tmod system showing the two receptors that comprise the NOT gate targeting HLA loss of heterozygosity (LOH) in solid tumors. (B) Diagram for Tmod utilizing tandem receptors for blood cancer. (C) CD33 and CD16b mRNA expression in primary AML and healthy blood cells including T cells, neutrophils, monocytes, and hematopoietic stem cells (HSC) (data from sources shown; see Supplementary Table 1 ). (D) mRNA expression of targets in AML cell lines (n=43; DepMap).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: CD33 | CD16b Tmod functions robustly in Jurkat and primary T cells. (A) Diagram of functional screen in Jurkat reporter cell line cocultured with K562 target cells transfected with different amounts of CD16b mRNA. Tmod transgene expression in Jurkat cells was detected by staining with recombinant human (rh) CD16b and CD33. (B) Diagram of functional parameters estimated from the Jurkat cell assay data. (C) Functional readout from 8-point mRNA titration curves. Three CARs combined with 4 blockers, that were selected for further analysis, are shown in color. Data are shown as mean ± standard deviation of technical replicates (n=2), normalized to each sample’s maximum activation. (D) Left, diagram of non-viral construct-screening in primary T cells using PiggyBac transposase and single vectors (BA vectors). Right, metrics used to quantify the potency and selectivity of the Tmod pair. (E) Flow cytometry analysis of stable integrants via staining with labeled recombinant human CD33 (see Methods). (E, F) T cell cytotoxicity curves generated from GFP signal at 48 hour time point with each well normalized to the zero time point. Tumor (CD33(+)CD16(-)) target-cell curves are shown with dashed lines and “normal” (CD33(+)CD16(+)) target-cell curves with solid lines. Black lines are CAR constructs and colored lines are Tmod constructs. Tumor cells are K562 cells engineered with CD33 and normal cells are K562 cells engineered to overexpress CD33 and CD16b. Data are shown as mean ± standard deviation of technical replicates (n=3). (G) Potency calculated as ET50 of Tmod cells cocultured with tumor cells. (H) Selectivity ratios are calculated as ET50 on normal cells divided by ET50 on tumor cells. (I) Kinetic cytotoxicity analysis of the most selective and potent construct compared to the CAR-T. GFP(+) area was used as proxy for target cell viability. Data are shown as mean ± standard deviation of technical replicates (n=3).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: Functional Assay, Transfection, Expressing, Staining, Recombinant, Titration, Standard Deviation, Activation Assay, Construct, Flow Cytometry, Labeling, Generated

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: CD33 | CD16b Tmod cells selectively kill tumor but not “normal” cells in vivo . (A) Schema for in vivo experiment. 2 million MV-4-11 AML cells or MV-4-11 cells that overexpress CD16b were injected into NSG-SGM3 mice and 6 days later 7.5 million T cells were injected. (B) Selectivity in vitro using MV-4-11 cells. Surrogate normal cells were generated by overexpression of CD16b in the AML cells. E:T cytotoxicity curves were generated from firefly luciferase bioluminescence at 48 hours. Data are shown as mean ± standard deviation of technical replicates (n=3). Inset: ET50 values of depicted curves. Data shown are interpolated values with 95% CI. (C) Flow cytometry analysis of construct expression by staining with labeled recombinant human CD16b and CD33. (D) Bioluminescence imaging (BLI) at 20 days post target-cell injection. (E) Flow cytometry analysis of MV-4-11 cells in the bone marrow 27 days post target-cell injection. (F) Quantification of data shown in panel. (E) Statistics were calculated using a non-parametric Kruskal-Wallis H test; *: 0.01 < adjusted p < 0.05; **: adjusted p value < 0.01; ns: not significant (adjusted p > 0.05).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: In Vivo, Injection, In Vitro, Generated, Over Expression, Luciferase, Standard Deviation, Flow Cytometry, Construct, Expressing, Staining, Labeling, Recombinant, Imaging

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: Tandem Tmod constructs for blood cancer. (A) Diagram of a Tmod cell with bispecific activator to target AML (CD33) and other blood cancers (SPN) and bispecific blocker to protect HSCs (CLEC9A) and neutrophils (CD16b). (B) Target expression in primary blood cancers and healthy blood cells (data from sources shown; see Supplementary Table 1 ). (C) Target expression in blood cancer cell lines (DepMap). (D) Jurkat cell (SPN KO) functional readout of SPN | CD16b Tmod with blocker titration curves. (E) Jurkat cell (SPN KO) functional readout of SPN-CD33 tandem CAR activation and blocking by CD16b blocker in the presence of SPN and CD33 antigens. (F) Jurkat functional readout of binders cloned as CARs with titration of primary HSCs. (G) Jurkat functional readout of CD33 CAR4 blocked by tandem CLEC9A-CD16b blocker. (H) Jurkat functional readout of CD33 and/or SPN monospecific or bispecific activators paired with CD16b and/or CLEC9A monospecific or bispecific blockers. Data are shown as mean ± standard deviation of technical replicates (n=2).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: Construct, Expressing, Functional Assay, Titration, Activation Assay, Blocking Assay, Clone Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: ( A ) Schematic representation of an Nb in the phagemid vector pMECS. Downstream of the PelB secretion sequence, the Nb-sequence is followed by a triple alanine linker, a hemagglutinin (HA), and hexa-histidine (His) tags. ( B ) Amino acid sequences of the anti-CD33 Nbs (numbering according to IMGT) . The CDR1, CDR2, and CDR3 regions are highlighted in cyan, green, and pink, respectively. The amino acid sequence of the CDR3 region is displayed in alphabetical order. Position 10 in the framework region-1 (FR1-IMGT) and position 73 in the FR3-IMGT are gaps introduced to align to other V-GENE groups or subgroups. For Nb_7, Nb_21, and Nb_22, an amino acid deletion relative to other sequences occurred at position 85 (represented by a dash).

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: Plasmid Preparation, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Purity of the anti-CD33 Nb preparations. ( A ) SEC profile of the Nb_12, showing a single peak of protein. (All other Nbs gave comparable chromatograms). ( B , C ) SDS-PAGE under reducing conditions, where proteins are revealed after staining with Coomassie blue ( B ) or by western blot, using a mouse anti-HA tag monoclonal antibody and a goat anti-mouse IgG horse radish peroxidase (HRP)-conjugated antibody ( C ). For both staining conditions and for each Nb preparation, only one single band was revealed.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: SDS Page, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Six anti-CD33 Nbs bind native CD33 protein expressed on THP-1 cells, without affecting the cells’ in vitro proliferative capacity. ( A ) Individual histogram plots of flow cytometry analysis from the selected Nbs (clear peak) versus a non-targeting Nb (tinted peak). An anti-CD33 monoclonal antibody was used as positive control (clear peak), with an isotype-matched antibody as negative control (tinted peak). ( B ) Graphical representation of the ΔMFI values for the generated Nbs. The ΔMFI is defined as the MFI signal from THP-1 cells treated with Nb and HA-labeled monoclonal antibody subtracted with the MFI signal from cells and labeled monoclonal, but without Nb. An Nb was selected as a binder if its ΔMFI signal was at least three times higher than the one obtained with non-targeting binder (αBabA Nb_19), which defined the threshold (dashed line). ( C ) The impact of ant-CD33 Nbs on the proliferation of THP-1 cells was determined by the alamarBlue assay, in which the proliferative status measured by absorbance is translated into a bar plot. THP-1 cells were incubated for 48 h, with 5 µg of the selected anti-CD33 Nbs (gray bars) or a non-targeting Nb (αBabA Nb_19; light grey bar), or they were left untreated (patterned bar). Medium alone was also included as extra control condition (black bar).

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: In Vitro, Flow Cytometry, Positive Control, Negative Control, Generated, Labeling, Alamar Blue Assay, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Kinetic association (k a ), dissociation (k d ), and equilibrium (K D ) constants of the six anti-CD33 Nbs for CD33 ectodomain measured by SPR. The Tm value representing the thermal stability of the six Nbs was measured with the ThermoFluor ® method.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: SPR sensorgram of Nb_12 on the CD33 ectodomain and epitope binning by SPR: ( A ) sensorgram of different concentrations (as indicated in the graph) of anti-CD33 Nb_12 binding to biotinylated recombinant human CD33 ectodomain. Kinetics were measured with a two-fold dilution series of Nbs (250–1.95 nM). The fitting of the binding curves using the 1:1 binding mathematical model calculated a K D of 3.9 nM. ( B , C ) Examples of a noncompetitive (independent) and a competitive (overlapping) epitope binding of two Nbs for the same antigen, respectively.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: Binding Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Overview of the epitope binning experiments. Each pair of anti-CD33 Nbs is classified as noncompetitive if it recognizes a non-overlapping epitope, or as competitive if it binds to an overlapping epitope.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: An example of Tm measurement of anti-CD33 Nb_12, as determined by ThermoFluor ® assay. The samples were heated from 10 to 95 °C, with stepwise increments of 0.5 °C per 30 s. AU stands for arbitrary units. The Tm for this Nb (58.00 ± 0.23 °C) was determined by using Boltzmann’s Equation.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Biodistribution of 99m Tc-labeled anti-CD33 Nbs, and the non-targeting 99m Tc-ctrl_Nb in mice bearing THP-1 tumors. Micro-SPECT/CT images were obtained 1 h after intravenous injection of 99m Tc-labeled Nbs. Arrows indicate THP-1 tumors. K: kidney; B: bladder; and L: liver.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: Labeling, Micro-SPECT, Injection

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Graphical presentation of the biodistribution of the 99m Tc-labeled anti-CD33 Nbs and non-targeting control Nb (ctrl_Nb) in mice bearing subcutaneous THP-1 tumors ( n = 3). ( A ) Ex vivo biodistribution results obtained 1.5 h after injection. Results are presented as mean %IA/g ± standard deviation. ( B ) Mice injected with 99m Tc-labeled Nb_12, Nb_87, Nb_21, and Nb_7 showed significant higher tumor uptake compared to the group of mice injected with non-targeting 99m Tc labeled ctrl_Nb.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: Labeling, Control, Ex Vivo, Injection, Standard Deviation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies

doi: 10.1073/pnas.1819992116

Figure Lengend Snippet: Deletion of CD33 does not impair engraftment, hematopoietic repopulation, and function in NSGS mice. ( A ) Schematic of experimental design. ( B and C ) BM-derived CD34 + cells engraftment and repopulation: ( B ) Peripheral blood (7 wk) and ( C ) whole BM (21 wk) posttransplant analyzed for cells of various lineages, as indicated. CD34 + CD33 Del cells show the same engraftment (CD45 + ) as control cells as well as comparable percentage of mature myeloid and lymphoid cells. BM CD34 + CD33 Del cells show comparable percentage of myeloid (progenitor CD123 + , mature CD14 + ), and lymphoid (progenitor CD10 + , mature CD19 + ) T cells (CD3 + ) and stem cells CD34 + 38 − . ( D and E ) CB-derived CD34 + cell engraftment and repopulation: ( D ) Peripheral blood (9 wk) and ( E ) BM (21 wk) posttransplant analyzed for cells of various lineages, as indicated. CD34 + CD33 Del cells show same engraftment (CD45 + ) as control cells, as well as comparable percentage of mature myeloid and lymphoid cells. BM CD34 + CD33 Del cells show comparable percentage of myeloid (progenitor CD123 + , mature CD14 + ) and lymphoid (progenitor CD10 + , mature CD19 + ), T cells (CD3 + ), and stem cells CD34 + 38 − . Data were analyzed using unpaired t test and no significant differences were found in all of the groups examined ( P > 0.05). All data are represented as mean ± SEM (two independent experiments, two donors). ( F – I ) In vitro and in vivo functional assays. ( F ) CD34 + CD33 Del show comparable development of myeloid lineage than CD34 + CD33 WT in NSGS mice. Frequencies of neutrophils, monocytes, cDC, pDC, mast cells, and basophils in the BM aspirates of NSGS mice injected with CB CD34 + CD33 WT or CD34 + CD33 Del cells. (Control n = 12, CD34 + CD33 Del n = 13). ( G ) In vitro E. coli bioparticles phagocytosis assay of in vitro CD33 WT or CD33 Del differentiated monocytes. CD33 Del monocytes show similar phagocytosis capacity (two independent experiments, two donors). ( H ) Response to LPS-induced Toll-like receptor activation is similar in NSGS mice injected with CD34 + CD33 WT or CD34 + CD33 Del cells. Analysis of plasma cytokines level at 0 and 4h30 after intraperitoneal injection of 15 μg LPS (Control n = 12, CD34 + CD33 Del n = 13). ( I ) Peritoneal cavity analysis 2 h after intravenous injection of E. coli bioparticles (Control n = 3 CD34 + CD33 Del n = 5), untreated mice (♦). Mouse and syringe images designed by Freepik and Kiranshastry from Flaticon .

Article Snippet: CAR expression and their ability to recognize and bind CD33 was assessed by incubating CAR-T cells with biotinylated human CD33 protein (ACRO biosystem) for 20 min at 4 °C and then stained with fluorochrome-conjugated streptavidin.

Techniques: Derivative Assay, Control, In Vitro, In Vivo, Functional Assay, Injection, Phagocytosis Assay, Activation Assay, Clinical Proteomics