cd2 selection kit  (Miltenyi Biotec)

Journal: PLoS ONE

Article Title: Cross-linking of T cell to B cell lymphoma by the T cell bispecific antibody CD20-TCB induces IFNγ/CXCL10-dependent peripheral T cell recruitment in humanized murine model

doi: 10.1371/journal.pone.0241091

Figure Lengend Snippet: a) Workflow schematics: skinfold chambers were installed on NSG mice (day -2). 48 hours later (day 0): WSU DLCL2 (blue), unstained CT26 cells, and CD2 + T cells (pink) freshly purified from human PBMCs or from HSC-NSG mice were injected intra-dermally in the skinfold chamber together with labeled CD20-TCB (0.5 mg/kg) or with suitable vehicle. Cells were imaged 2 hours post treatment by MP-IVM. Adapted from https://smart.servier.com/ . b) 3D representative rendering of MP-IVM imaging on skinfold chamber of HSC-NSG-NSG mice showing localization of therapy (white) at the contact site between WSU DLCL2 cells (blue) and T cells (pink), 2 hours post treatment. c) MP-IVM analysis of T cells tracks in the skin fold chamber of PBMC-NSG (top) vs HSC-NSG-NSG mice (bottom), +/- CD20-TCB. T cell tracks are plotted according to their displacement in the X and Y axes. Total number of tracks for each plot is: Top left: Vehicle n = 330. Top right: CD20-TCB n = 759. Bottom left: Vehicle n = 741. Bottom right: CD20-TCB n = 185. d-e) Quantification of (d) Track Speed (μm/min) and (e) Track displacement (μm) of T cells in PBMC-NSG or HSC-NSG-NSG mice, +/- CD20-TCB. Shown in yellow are mean values +/- s.d. Unpaired t-test; ****p<0.0001; n.s.: not significant.

Article Snippet: Conventional pan T cells from the spleen of HSC-NSG mice were isolated using a CD2 + selection kit (Miltenyi Biotech, 130-091-114) and directly injected.

Techniques: Purification, Injection, Labeling, Imaging

Journal: PLoS ONE

Article Title: Cross-linking of T cell to B cell lymphoma by the T cell bispecific antibody CD20-TCB induces IFNγ/CXCL10-dependent peripheral T cell recruitment in humanized murine model

doi: 10.1371/journal.pone.0241091

Figure Lengend Snippet: a-c) Top: Representative histological staining of WSU DLCL2 tumors 24h post second treatment (0.5 mg/kg CD20-TCB or suitable vehicle i.v.). Bottom: Quantification of total number of cells/mm 2 from histological images of vehicle vs CD20-TCB treatment. Whole slide scans quantification of 4 μm FFPE sections with the software (a) Definiens; (b-c) Halo. Statistical analysis: Unpaired 2-tailed t-test with Welch’s correction. *p<0.05, **p<0.005 (a) Red: CD3 staining, brown: CD31 staining. Quantification: Number of CD3 + cells b) red: Ki67, yellow: CD3, blue: DAPI. Quantification: Number of CD3 + Ki67 + cells c) Red: CXCR3, yellow: CD3, Blue: DAPI. Quantification of CD3 + CXCR3 + T cells. d) Percentage of proliferating CD8 + T cells, as assessed by CFSE dilution, freshly purified from PBMCs. Proliferation has been evaluated at 24h, 48h and 72h post CD20-TCB treatment, at the indicated doses, in the presence of WSU DLCL2 cells as target. n = 3 per group, mean and s.d. are shown. One-way Anova, *p<0.05, **p<0.005, ****p<0.0001. e) Workflow schematics: Skinfold chamber were installed on NSG mice. 48h later, WSU DLCL2 (Blue), unstained CT26 cells, and CD2 + T cells freshly purified from HSC-NSG spleens (pink) were injected intra-dermally in the skinfold chamber, together with 0.25 mg/kg of CD20-TCB or with suitable vehicle. Concomitantly, freshly purified CD2 + T cells from HSC-NSG spleens (orange) were injected i.v. to allow visualization of peripheral blood T cells. Cells were imaged 72h post treatment by MP-IVM. f) Representative MP-IVM imaging of the tumors. Blue: WSU DLCL2 cells; Pink: Resident T cells; Orange: Recruited T cells. Images were acquired 72h post intradermal treatment with 0.25 mg/kg CD20-TCB or suitable vehicle. Adapted from https://smart.servier.com/ g) Quantification of peripheral T cells (number/mm 2 ) 72h post treatment. Mean +/- s.d. are shown. Unpaired 2-tailed t-test with Welch’s correction. **p<0.005. h) In the context of the skinfold chamber model, increasing number of T cells (Resident) were co-injected with the tumor and 0.25 mg/kg of CD20-TCB intradermally, while 2.5*10 6 T cells were injected intravenously (Peripheral). 72h post treatment, peripheral blood T cells were counted for each tumor from 5 representative fields. 4 tumors per group were analyzed. Shown is the count of peripheral T cells/mm 2 , Mean +/- s.d. per group. Statistical analysis: One-way Anova. **** p<0.0001. i) 3 hours in vitro chemotaxis assay of T cells toward preconditioned medium derived from WSU DLCL2 co-culture with CD3/CD28 pre-activated T cells. Pre-activated CD8 T cells have been plated with WSU DLCL2 cells at decreasing T cells: Tumor cells ratios, in the presence of 200 ng/ml of CD20-TCB. 24h later the supernatant has been collected and transferred to the bottom chamber of a 24-Transwell plate. In the top chamber 100.000 pre activated T cells, labeled with CFSE, have been seeded and let to migrate for 3 hours. Migration has been evaluated by counting total amount of CFSE positive migrated cells in the bottom chamber, by flow cytometry at constant volume and acquisition speed. Mean fold change and +/- s.d. are shown. n = 5, from two independent experiments 2-way Anova; **p<0.005.

Article Snippet: Conventional pan T cells from the spleen of HSC-NSG mice were isolated using a CD2 + selection kit (Miltenyi Biotech, 130-091-114) and directly injected.

Techniques: Staining, Software, Purification, Injection, Imaging, In Vitro, Chemotaxis Assay, Derivative Assay, Co-Culture Assay, Labeling, Migration, Flow Cytometry

Journal: PLoS ONE

Article Title: Cross-linking of T cell to B cell lymphoma by the T cell bispecific antibody CD20-TCB induces IFNγ/CXCL10-dependent peripheral T cell recruitment in humanized murine model

doi: 10.1371/journal.pone.0241091

Figure Lengend Snippet: a) IFNγ protein quantification by multiplex analysis of supernatant derived from co-culture of WSU DLCL2 cells with CD8 + T cells freshly purified from PBMCs and stimulated with CD20-TCB (200 ng/ml) at the indicated time points. n = 3 per group. Two-way Anova. *p< 0.05, **p< 0.05, ***p < 0.001, ****p < 0.0001, n.s.: not significant. b) Flow cytometry analysis of CD8 + INFγ + T cells, at 24, 48 and 72 hours post in vitro CD20-TCB (200 ng/ml) treatment. c) Quantification of released cytokines (pg/mL) upon IFNγ stimulation of WSU DLCL2 tumor cells for 48 hours. Shown is Mean +/- s.d. of 3 replicates. d) Quantification of released cytokines (pg/mL) upon CD20-TCB treatment (200 ng/ml) of WSU DLCL2 co-cultured with CD3/CD28 pre-activated CD8 + T cells. Shown is Mean +/- s.d. n = 4 per group. Two-way Anova *p< 0.05, n.s.: not significant. e) CXCL10 protein quantification by multiplex analysis of supernant derived from co-culture of WSU DLCL2 cells with CD8 + T cells freshly purified from PBMCs and stimulated with CD20-TCB (200 ng/ml) at the indicated time points. n = 3 per group. Two-way Anova. ***p < 0.001, ****p < 0.0001, n.s. not significant. f) Representative images from MP-IVM imaging in the skinfold chamber of HSC-NSG-NSG mice. WSU DLCL2 cells (blue) pre-treated or not with IFNγ were injected intra-dermally together with CD2 + T cells derived from the spleen of HSC-NSG (pink) and 0.25 mg/kg CD20-TCB or suitable vehicle. CD2 + T cells derived from the spleen of HSC-NSG (orange) where concomitantly injected intravenously. Top row: Tumor cells (blue) and peripheral T cells (orange). Bottom row: Tumor cells (blue), resident T cells (pink) and peripheral T cells (orange). Where indicated, antibodies against CXCL10 or IFNγ were injected intravenously. g) Quantification of peripheral blood T cells (count/mm 2 ) 72 hours post treatment. Shown are individual counts/mm 2 and mean +/- s.d. Statistical analysis: One-way Anova. ***p < 0.001, ****p < 0.0001.

Article Snippet: Conventional pan T cells from the spleen of HSC-NSG mice were isolated using a CD2 + selection kit (Miltenyi Biotech, 130-091-114) and directly injected.

Techniques: Multiplex Assay, Derivative Assay, Co-Culture Assay, Purification, Flow Cytometry, In Vitro, Cell Culture, Imaging, Injection

Journal: iScience

Article Title: Second generation CD2-targeting LFA-3 fusion protein SBT115301 to restore immune homeostasis in autoimmune disease

doi: 10.1016/j.isci.2025.112447

Figure Lengend Snippet: Sequence alignment of the engineered CD2 binding domain of SBT115301 with human WT LFA-3 Yellow boxes denote amino acids identified as being involved in binding to CD2, with white, gray, and black dots showing the effects of single amino acid mutagenesis on binding of human LFA-3 to human CD2. Red arrows and boxes identify residues that were altered in SBT115301.

Article Snippet: EasySep Human CD2 Positive Selection Kit II , Stemcell Technologies , Cat#17883.

Techniques: Sequencing, Binding Assay, Mutagenesis

Journal: iScience

Article Title: Second generation CD2-targeting LFA-3 fusion protein SBT115301 to restore immune homeostasis in autoimmune disease

doi: 10.1016/j.isci.2025.112447

Figure Lengend Snippet: SBT115301 preferentially depletes CD2 hi -expressing T cells in vitro (A) Median fluorescence intensity (MFI) of CD2 expression on NK cells (CD3 − CD56 + ) and CD3 + CD4 + T reg (CD25 hi FOXP3 + T n (CD45RA + CCR7 + ), T cm (CD45RA − CCR7 + ), and T em (CD45RA − CCR7 - ) cell subsets ( n = 30). Boxes represent the interquartile range (25th to 75th percentile). Individual data points are shown. (B) Activation of the high affinity (V158; each condition in triplicate, 5 experiments) and low affinity (F158; each condition in triplicate, 4 experiments) CD16a variants by SBT115301 as measured by mean ± SD relative light units (RLU) in a Jurkat ADCC reporter assay. (C) Representative histogram demonstrating the difference in CD2 expression of a PBMC sample incubated for 20 h with 100 nM SBT115301 or control (left). PBMC were incubated with increasing concentrations of SBT115301 or control in triplicate and mean ± SD cell numbers were quantified at the end of the assay (right). (D) Mean ± SD cell counts normalized to no test or control added of naive (CD45RA + ) and proliferating (Prolif) and non-proliferating (Non-prolif) memory (CD45RO + ) CD4 + T cells following incubation of CMV-reactive PBMC with CMV antigen and SBT115301 for 6 days. Each condition run in triplicate. Wilcoxon rank-sum ∗∗∗∗ p ≤ 0.001 by Wilcoxon rank-sum test (A).

Article Snippet: EasySep Human CD2 Positive Selection Kit II , Stemcell Technologies , Cat#17883.

Techniques: Expressing, In Vitro, Fluorescence, Activation Assay, Reporter Assay, Incubation, Control

Journal: iScience

Article Title: Second generation CD2-targeting LFA-3 fusion protein SBT115301 to restore immune homeostasis in autoimmune disease

doi: 10.1016/j.isci.2025.112447

Figure Lengend Snippet: SBT115301 depletes T cell subsets with higher CD2 expression in an NHP model (A) Comparison of ADCC activity of SBT115301 in human versus non-human primate (NHP) in a primary PBMC in vitro assay. Boxes represent the interquartile range (25th to 75th percentile). Individual data points are shown. (B and C) Mean ± SD numbers of (B) circulating total (CD3 + ) T cells, CD4 + T cells, and CD8 + T cells, (C) CD4 + T em (CD45RA − CCR7 - ), T cm (CD45RA − CCR7 + ), T n (CD45RA + CCR7 + ), and T reg (CD4 + CD25 hi FOXP3 + ) expressed as % of baseline (pre-dose) cell counts after a single dose of SBT115301. (D) Mean ± SD numbers of circulating (CD3 + ) T cells in combined male and female NHP in a multi-dose (weekly) study. Data were collected pre-treatment (pre-Tx; Day 1) and up to 168 h post-Dose 1 ( n = 5 animals/sex/group), pre-Dose 5 (Day 29) and up to 168 h post-Dose 5 ( n = 5 animals/sex/group), and at the end of the study (Day 92; n = 2 animals/sex/group). (E) Mean ± SD anti-drug antibody (ADA) titers in NHP receiving multiple doses of SBT115301.

Article Snippet: EasySep Human CD2 Positive Selection Kit II , Stemcell Technologies , Cat#17883.

Techniques: Expressing, Comparison, Activity Assay, In Vitro

Journal: iScience

Article Title: Second generation CD2-targeting LFA-3 fusion protein SBT115301 to restore immune homeostasis in autoimmune disease

doi: 10.1016/j.isci.2025.112447

Figure Lengend Snippet: Safety, PK, and ADA profiles of a single dose of SBT115301 in healthy participants (A) Mean ± SD numbers of circulating CD4 + T cells over time in participants that received SBT115301 or placebo expressed as absolute counts (left) and as % of baseline (pre-dose; right). (B) PK of SBT115301 as demonstrated by the mean ± SD serum concentration of SBT115301 in participants over time. (C) Mean ± SD ADA titers of ADA + participants over time. (D) The effects of ADA on PK as measured by dose normalized AUC inf (left) and on PD as measured by the decrease of T em from baseline at days 36 and 72 post-dose (right). Boxes represent the interquartile range (25th to 75th percentile). Individual data points are shown. (E) The effect of ADA + serum on SBT115301-mediated ADCC activity as measured by a Jurkat ADCC-reporter assay, comparing pre-dose and Day 72 serum samples (left), and correlated with ADA titer (right). Boxes represent the interquartile range (25th to 75th percentile); individual data points are shown (left). Blue lines and gray shades (right) are loess smooth lines with the associated 95% confidence bands. (F) The effect of ADA + serum in a Jurkat activation reporter assay examining activation of Jurkat cells following binding of recombinant LFA-3 protein to Jurkat-expressed CD2. Boxes represent the interquartile range (25th to 75th percentile). Individual data points are shown. (G) The differential binding of ADA + serum to CD2-binding domains of SBT115301 and WT LFA-3. Boxes represent the interquartile range (25th to 75th percentile); individual data points are shown. Lines connect paired measurements from the same donor at pre-treatment and D72 timepoints.

Article Snippet: EasySep Human CD2 Positive Selection Kit II , Stemcell Technologies , Cat#17883.

Techniques: Concentration Assay, Activity Assay, Reporter Assay, Activation Assay, Binding Assay, Recombinant

Journal: iScience

Article Title: Second generation CD2-targeting LFA-3 fusion protein SBT115301 to restore immune homeostasis in autoimmune disease

doi: 10.1016/j.isci.2025.112447

Figure Lengend Snippet: Pharmacodynamics of a single dose of SBT115301 in healthy participants (A) Mean ± SD numbers of circulating CD4 + T em (CD45RA − CCR7 - ), T cm (CD45RA − CCR7 + ), and T n (CD45RA + CCR7 + ) subsets reported as % change of pre-treatment values over time. (B and C) Maximum reduction of (B) CD4 + T em and (C) T reg (CD4 + CD127 lo CD25 hi FOXP3 + ) in each cohort reported in % change from baseline as measured by the net area under the effect curve from Day 3 to Day 8 using % change from baseline based on cell counts divided by duration of time (E avg(3-8) ). Boxes represent the interquartile range (25th to 75th percentile). One-way analysis of variance and Dunnett’s test were used to compare between treated groups and placebo groups. Statistical significance (compared to placebo) was set at ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01. (D) Mean ± SD ratio of T reg to T em cells reported as a % change of pre-treatment values following dosing in the 10 and 30 mg IM cohorts compared to participants who received placebo. (E) Durability of the CD4 + T em PD response at timepoints after 32 days compared to the initial PD response measured by E avg(3-8) . (F) Correlation of baseline CD2 median fluorescence intensity on all cell subsets tested (CD4 + and CD8 + T cell subsets) with the PD responses of each subset as measured by E avg(3-36) . Linear regressions were used to depict relations between E avg(3-36) and baseline CD2 median fluorescence intensity within each dose. Fitted regression lines (solid lines) with 95% confidence bands (dotted lines) are displayed. Colored dots represent estimated E avg(3-36) for each cell subset at the average CD2 level and vertical edges represent 95% confidence intervals. ∗ p < 0.05, ∗∗ p < 0.01, ns (not significant) by one-way analysis of variance and Dunnett’s test (B and C).

Article Snippet: EasySep Human CD2 Positive Selection Kit II , Stemcell Technologies , Cat#17883.

Techniques: Drug discovery, Fluorescence