recombinant cd33 his tag protein  (R&D Systems)

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: 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