recombinant cd22 fc chimera protein  (R&D Systems)

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 1A: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 indicated Nalm6 leukemia on day -3, followed by 5e6 CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day 0, as well on days 5 and 11 post-CAR. 1B: Quantification of bioluminescence data in A. 1C: ELISA measuring Granzyme B in supernatant after 16 hour co-culture of CD22-CAR T cells with the indicated leukemia. 1D: Degranulation as measured by CD107a expression after 4 hour co-culture assay. 1E: Activation as measured by CD69 expression after 6 hour co-culture assay. 1F: Activation as measured by CD25 expression after 24 hour co-culture assay. All in vitro assays performed with n=3 technical replicates, 1 experiment. In vivo assay performed with n=5 mice per group, 1 experiment. Data represent mean +/-SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: For all CD22 CAR constructs, the CAR was detected by primary staining with a recombinant CD22-Fc chimera protein (R&D Systems) followed by secondary staining with PE-Conjugated Goat anti-Human IgG Fc secondary antibody (Thermo Fisher).

Techniques: In Vivo, Injection, Imaging, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Expressing, Co-culture Assay, Activation Assay, In Vitro

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 2A: Flow cytometry plots showing IL-2 by IFNg production after 6 hour coculture of the indicated CD22-CAR T cell with the indicated leukemia. 2B: Quantification of cytokine data in A. 2C: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 indicated Nalm6 leukemia on day -3, followed by 4e6 CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR injection. Mice were monitored for survival. 2D: Quantification of bioluminescence data against WT leukemia from C. 2E: Survival of mice bearing WT leukemia. 2D: Quantification of bioluminescence data against CD22 Lo leukemia from C. 2E: Survival of mice bearing CD22 Lo leukemia. All in vitro assays performed with n=3 technical replicates, and are representative of two experiments with two independent donors. In vivo assay performed with n=5 mice per group, 1 experiment. Data represent mean +/-SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: For all CD22 CAR constructs, the CAR was detected by primary staining with a recombinant CD22-Fc chimera protein (R&D Systems) followed by secondary staining with PE-Conjugated Goat anti-Human IgG Fc secondary antibody (Thermo Fisher).

Techniques: Flow Cytometry, In Vivo, Injection, Imaging, In Vitro

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: S1A: Cell-based direct antigen-binding affinity titration assay. Indicated CARs were stained with indicated concentrations of fluorophore-conjugated CD22 Protein Fc. MFI of CAR+ Populations were measured and normalized to peak protein binding for each individual CAR. Data represents one experiment with one replicate per concentration.

Article Snippet: For all CD22 CAR constructs, the CAR was detected by primary staining with a recombinant CD22-Fc chimera protein (R&D Systems) followed by secondary staining with PE-Conjugated Goat anti-Human IgG Fc secondary antibody (Thermo Fisher).

Techniques: Binding Assay, Titration, Staining, Protein Binding, Concentration Assay

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: Figures S2A to S2F quantify indicated metrics by flow cytometry after coculture of indicated CD22-CAR with indicated leukemia after 6 hour coculture. S2A: %+ and MFI for IFNg production against WT leukemia. S2B: %+ and MFI for IL2 production against WT leukemia. S2C: %+ of cells making IFNg and IL-2 against WT leukemia. S2D: %+ and MFI for IFNg production against CD22 Lo leukemia. S2E: %+ and MFI for IL2 production against CD22 Lo leukemia. S2F: %+ of cells making IFNg and IL-2 against CD22 Lo leukemia. Figures S2G to S2J quantify CAR and leukemia counts relative to a starting 5:1 ratio of leukemia and CAR to fluorescent counting beads. Aliquots were taken from each condition and analyzed by flow cytometry at each of the indicated time points. S2G: Quantification of CAR Count/Bead Count ratio against WT leukemia. S2H: Quantification of Leukemia Count/Bead Count ratio for WT leukemia. S2I: Quantification of CAR Count/Bead Count ratio against CD22 Lo leukemia. S2J: Quantification of Leukemia Count/Bead Count ratio for CD22 Lo leukemia. All in vitro assays performed with n=3 technical replicates. are representative of two experiments with two independent donors. are representative of one experiment. Data represent mean +/- SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: For all CD22 CAR constructs, the CAR was detected by primary staining with a recombinant CD22-Fc chimera protein (R&D Systems) followed by secondary staining with PE-Conjugated Goat anti-Human IgG Fc secondary antibody (Thermo Fisher).

Techniques: Flow Cytometry, In Vitro

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 3A: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 WT Nalm6 leukemia on day-3, followed by 2e6 of indicated CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR. 3B: Quantification of average bioluminescence data for each group in A. 3C: Quantification of individual bioluminescence data for each group in A. 3D: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 WT Nalm6 leukemia on day -3, followed by 4e6 of indicated CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR. 3E: Quantification of average bioluminescence data for each group in D. 3F: Survival of mice treated with 4e6 of the indicated CAR T cells. In vivo assay performed with n=5 mice per group, 1 experiment (3A to 3C) or 3 experiments with independent donors (3D to 3F). 3D to 3E are representative data from one experiment. 3F is pooled data, SA-SL (n=15), HA-SL (n=15), HA-LL (n=15). Data represent mean +/- SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: For all CD22 CAR constructs, the CAR was detected by primary staining with a recombinant CD22-Fc chimera protein (R&D Systems) followed by secondary staining with PE-Conjugated Goat anti-Human IgG Fc secondary antibody (Thermo Fisher).

Techniques: In Vivo, Injection, Imaging

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 4A: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 CD22 Lo Nalm6 leukemia on day -3, followed by 4e6 of indicated CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR. 4B: Quantification of average bioluminescence data for each group in A. 5C: Quantification of individual bioluminescence data for each group in A. For 4D to 4E, bone marrow was analyzed by flow cytometry at day 18 post-CAR for indicated cell population. 4D: % CAR+ of live marrow. 4E: % leukemia of live marrow. 4F: Survival of mice treated with 4e6 of indicated CAR T cells. In vivo assay performed with n=5 mice per group, 4 experiments with independent donors. Data in 4A to 4C is representative data from one experiment. Survival is pooled from 3 experiments with independent donors: Mock (n=10), SA-SL (n=15), HA-SL (n=10), HA-LL (n=15). Data represent mean +/- SD. * p<0.05, ** p<0.01, *** p<0.001, ****

Article Snippet: For all CD22 CAR constructs, the CAR was detected by primary staining with a recombinant CD22-Fc chimera protein (R&D Systems) followed by secondary staining with PE-Conjugated Goat anti-Human IgG Fc secondary antibody (Thermo Fisher).

Techniques: In Vivo, Injection, Imaging, Flow Cytometry

Journal: Cytotherapy

Article Title: Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in CAR T cell products

doi: 10.1016/j.jcyt.2022.09.004

Figure Lengend Snippet: Assay #2 was designed at the site between the two distinct CARs within the ORF of the vector, spanning the junction between the linker domains and the 5’-end of the FMC63 scFv of CD19. This primer-probe set was designed to be specific for the bicistronic CD19x22 CAR T cell product. A representative example of droplet readout is pictured here demonstrating that ddPCR confirms specificity of this assay to detect only the bicistronic CAR product as demonstrated with CAR positive droplets (FAM) in blue in the last column. Albumin control (VIC), depicted in green, appropriately demonstrates signal in all samples containing T cells. No CAR positive droplets (in blue) are detected in untransduced T cells or T cells transduced with either CD19 only CARs or CD22 only CARs. NTC = No template control, negative control.

Article Snippet: Following staining and subsequent wash, cells were stained with Recombinant Human CD19-Fc Chimera Atto 647N Protein (R&D Biotechne, ATM9269-020, 1ug/test), Recombinant Human Singlec2/CD22-Fc Chimera Atto 488 (R&D Biotechne, ATJ1968-050, 0.5ug/test), and lyophilized T cell cocktail 6-color Kit (Biolegend, San Diego, CA) containing CD45-PE/Dazzle 594 (clone HI30), CD3 BV510 (clone UCHT1), CD4 Alexa Fluor 700 (clone SK3), CD8 Pacific Blue (clone SK1), CD45RA PE (clone KI100), and CD196 (CCR7) PE/Cy7 (clone G043H7) for 15 minutes at room temperature.

Techniques: Plasmid Preparation, Transduction, Negative Control