Journal: Nature communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: Fig. 2 SPPL3KO suppresses CD19 CAR T cell activation. a Schematic of T cell activation studies. b–d, Expression of b CD69, c PD1, and d Tim3 on T cells after overnight co-culture with WT or SPPL3KO Nalm6 cells or alone (control). e expression of CD107a on T cells after a 4-h co-culture with WT or SPPL3KO

Article Snippet: Normalized lysates were cleared by incubation with protein A beads (Invitrogen) on a rotator (1 h, 4 °C) and then incubated with 2 uL anti-CD19 antibody (clone FMC63, Novus Biologicals) on a rotator (overnight, 4 °C).

Techniques: Activation Assay, Expressing, Co-Culture Assay, Control

Journal: Nature communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: Fig. 3 Hyperglycosylation impairs detection of CD19. a Western blot of lysates from WT or SPPL3KO Nalm6 and OCI-Ly10 cells probed for CD19. Protein electrophoresis was performed on a 6% polyacrylamide gel. Representative of n = 4 individual experiments. b Western blot of lysates from WT or SPPL3KO

Article Snippet: Normalized lysates were cleared by incubation with protein A beads (Invitrogen) on a rotator (1 h, 4 °C) and then incubated with 2 uL anti-CD19 antibody (clone FMC63, Novus Biologicals) on a rotator (overnight, 4 °C).

Techniques: Western Blot, Protein Electrophoresis

Journal: Nature communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: Fig. 5 Overexpression of SPPL3 results in loss of CD19 surface expression. a Sequential flow cytometric evaluation of FMC63-APC binding on SPPL3KO + Nalm6 after overexpression of SPPL3. b Western blot of fractionated cellular lysates from WT, SPPL3KO or SPPL3KO with overexpression of SPPL3 (SPPL3KO+) Nalm6 cells probed for CD19 performed 20 days after engineering. Representative of n = 2 individual experiments. c Survival of WT, SPPL3KO, or SPPL3KO + Nalm6 over time when established on 20 days after re-introduction of SPPL3 (E:T 0.25:1). Representative data from n = 2 experiments with independent T cell donors. Error bars reflect mean ± standard error of the mean (s.e.m.). ****P < 0.0001 by two-way ANOVA with Bonferroni correction for multiple comparisons. Statistical annotations reflect differences between WT and SPPL3KO (upper) or WT and SPPL3KO + Nalm6 (lower). Source Data are provided as a Source Data file.

Article Snippet: Normalized lysates were cleared by incubation with protein A beads (Invitrogen) on a rotator (1 h, 4 °C) and then incubated with 2 uL anti-CD19 antibody (clone FMC63, Novus Biologicals) on a rotator (overnight, 4 °C).

Techniques: Over Expression, Expressing, Binding Assay, Western Blot

Journal: Nature communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: Fig. 6 Proposed model for the mechanism of glycosylation-mediated antigen escape. (left) Normal SPPL3 function results in standard glycosylation of CD19. (Middle) Loss of SPPL3 results in increased and altered glycosylation, disrupting the CAR binding epitope. (Right) Increased SPPL3 results initially in decreased glycosylation followed by degradation of CD19, preventing surface presenation.

Article Snippet: Normalized lysates were cleared by incubation with protein A beads (Invitrogen) on a rotator (1 h, 4 °C) and then incubated with 2 uL anti-CD19 antibody (clone FMC63, Novus Biologicals) on a rotator (overnight, 4 °C).

Techniques: Glycoproteomics, Binding Assay

Journal: Nature Communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: a Schematic of T cell activation studies. b – d , Expression of b CD69, c PD1, and d Tim3 on T cells after overnight co-culture with WT or SPPL3 KO Nalm6 cells or alone (control). e expression of CD107a on T cells after a 4-h co-culture with WT or SPPL3 KO Nalm6 cells. f , g Expression of f GFP and g eCFP, reflecting binding of NFAT and NFκB to their respective promoter sites in CD19 CAR-expressing Jurkat cells. h Schematic of long-term co-culture and re-challenge study. i Survival of WT Nalm6 over time after the combination of CART19 cells previously cultured with either WT or SPPL3 KO Nalm6 cells. b – e , i Representative of n = 2 individual experiments with distinct donor T cells. f , g representative of n = 4 individual experiments. Error bars reflect mean ± standard error of the mean (s.e.m.). **** P < 0.0001 by two-way ANOVA with Bonferroni correction for multiple comparisons. Source Data are provided as a Source Data file.

Article Snippet: Cells were resuspended in FACS staining buffer (PBS + 3% fetal bovine serum) using the following antibodies: CD3 (clone OKT3, BD Biosciences 555342, diluted 1:50), PD1 (clone EH12.2H7, BioLegend, 329928, diluted 1:80), Tim3 (clone 7D3, BD Biosciences 565566, diluted 1:100), CD22 (clone HIB22, BD Biosciences 562860, diluted 1:100), CD19 (clone FMC63, Novus Biologicals 52716, diluted 1:80-1:32000; clone HIB19, BD Biosciences 555413, diluted 1:50), CD107a-PECy7 (clone H4A3, Biolegend 328607, diluted 1:100), CD34 APC (BD 555824, diluted 1:50).

Techniques: Activation Assay, Expressing, Co-Culture Assay, Control, Binding Assay, Cell Culture

Journal: Nature Communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: a Western blot of lysates from WT or SPPL3 KO Nalm6 and OCI-Ly10 cells probed for CD19. Protein electrophoresis was performed on a 6% polyacrylamide gel. Representative of n = 4 individual experiments. b Western blot of lysates from WT or SPPL3 KO Nalm6 cells that were either untreated (UT), treated with PNGase F (PNG), or treated with Endoglycosidase H (Endo H) and then probed for CD19. Representative data of n = 3 individual experiments. Electrophoresis performed on a 6% polyacrylamide gel. c Median Fluorescence Intensity of CD19 as detected by FMC63-APC on the surface of WT or SPPL3 KO Nalm6 cells. d Western blot analysis of lysates from cells treated with kifunensine (16 ng/mL) for 10 days and then probed for CD19. Representative of n = 2 individual experiments. e Survival of WT, untreated SPPL3 KO , and kifunensine-treated SPPL3 KO Nalm6 over time in co-culture with CART19 cells (E:T ratio 0.25:1) statistical annotations reflect differences between SPPL3 KO + kifu and WT and SPPL3 KO + kfiu and SPPL3 KO (lower). f Western blot of lysates from WT or SPPL3 KO Nalm6 cells probed for CD22. Representative of n = 4 individual experiments. g Survival of WT or SPPL3 KO Nalm6 cells over time after combination with CART22 (E:T ratio 0.25:1). e , g Representative data from n = 3 individual experiments with distinct donor T cells. Error bars reflect mean ± standard error of the mean (s.e.m.). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by two-way ANOVA with Bonferroni correction for multiple comparisons. Source Data are provided as a Source Data file.

Article Snippet: Cells were resuspended in FACS staining buffer (PBS + 3% fetal bovine serum) using the following antibodies: CD3 (clone OKT3, BD Biosciences 555342, diluted 1:50), PD1 (clone EH12.2H7, BioLegend, 329928, diluted 1:80), Tim3 (clone 7D3, BD Biosciences 565566, diluted 1:100), CD22 (clone HIB22, BD Biosciences 562860, diluted 1:100), CD19 (clone FMC63, Novus Biologicals 52716, diluted 1:80-1:32000; clone HIB19, BD Biosciences 555413, diluted 1:50), CD107a-PECy7 (clone H4A3, Biolegend 328607, diluted 1:100), CD34 APC (BD 555824, diluted 1:50).

Techniques: Western Blot, Protein Electrophoresis, Electrophoresis, Fluorescence, Co-Culture Assay

Journal: Nature Communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: a Structural modeling of the FMC63 binding epitope on CD19. Residues in blue (R144, W140, and P203) are essential for antibody binding and proximal asparagine residues are in red (N125 and N114). b Median Fluorescence Intensity of CD19 as detected by FMC63-APC on the surface of WT, SPPL3 KO or SPPL3 KO CD19 KO (dual KO) Nalm6 cells engineered to express mutated CD19 molecules. Representative data from n = 2 flow cytometry experiments. c Survival of Nalm6 cells over time after combination with CART19 (E:T ratio 0.25:1). Representative data from n = 2 experiments with independent T cell donors. Error bars reflect mean ± standard error of the mean (s.e.m.). **** P < 0.0001 by two-way ANOVA with Bonferroni correction for multiple comparisons. Source Data are provided as a Source Data file.

Article Snippet: Cells were resuspended in FACS staining buffer (PBS + 3% fetal bovine serum) using the following antibodies: CD3 (clone OKT3, BD Biosciences 555342, diluted 1:50), PD1 (clone EH12.2H7, BioLegend, 329928, diluted 1:80), Tim3 (clone 7D3, BD Biosciences 565566, diluted 1:100), CD22 (clone HIB22, BD Biosciences 562860, diluted 1:100), CD19 (clone FMC63, Novus Biologicals 52716, diluted 1:80-1:32000; clone HIB19, BD Biosciences 555413, diluted 1:50), CD107a-PECy7 (clone H4A3, Biolegend 328607, diluted 1:100), CD34 APC (BD 555824, diluted 1:50).

Techniques: Binding Assay, Fluorescence, Flow Cytometry

Journal: Nature Communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: a Sequential flow cytometric evaluation of FMC63-APC binding on SPPL3 KO + Nalm6 after overexpression of SPPL3. b Western blot of fractionated cellular lysates from WT, SPPL3 KO or SPPL3 KO with overexpression of SPPL3 ( SPPL3 KO +) Nalm6 cells probed for CD19 performed 20 days after engineering. Representative of n = 2 individual experiments. c Survival of WT, SPPL3 KO , or SPPL3 KO + Nalm6 over time when established on 20 days after re-introduction of SPPL3 (E:T 0.25:1). Representative data from n = 2 experiments with independent T cell donors. Error bars reflect mean ± standard error of the mean (s.e.m.). **** P < 0.0001 by two-way ANOVA with Bonferroni correction for multiple comparisons. Statistical annotations reflect differences between WT and SPPL3 KO (upper) or WT and SPPL3 KO + Nalm6 (lower). Source Data are provided as a Source Data file.

Article Snippet: Cells were resuspended in FACS staining buffer (PBS + 3% fetal bovine serum) using the following antibodies: CD3 (clone OKT3, BD Biosciences 555342, diluted 1:50), PD1 (clone EH12.2H7, BioLegend, 329928, diluted 1:80), Tim3 (clone 7D3, BD Biosciences 565566, diluted 1:100), CD22 (clone HIB22, BD Biosciences 562860, diluted 1:100), CD19 (clone FMC63, Novus Biologicals 52716, diluted 1:80-1:32000; clone HIB19, BD Biosciences 555413, diluted 1:50), CD107a-PECy7 (clone H4A3, Biolegend 328607, diluted 1:100), CD34 APC (BD 555824, diluted 1:50).

Techniques: Binding Assay, Over Expression, Western Blot

Journal: Nature Communications

Article Title: Antigen glycosylation regulates efficacy of CAR T cells targeting CD19

doi: 10.1038/s41467-022-31035-7

Figure Lengend Snippet: (left) Normal SPPL3 function results in standard glycosylation of CD19. (Middle) Loss of SPPL3 results in increased and altered glycosylation, disrupting the CAR binding epitope. (Right) Increased SPPL3 results initially in decreased glycosylation followed by degradation of CD19, preventing surface presenation.

Article Snippet: Cells were resuspended in FACS staining buffer (PBS + 3% fetal bovine serum) using the following antibodies: CD3 (clone OKT3, BD Biosciences 555342, diluted 1:50), PD1 (clone EH12.2H7, BioLegend, 329928, diluted 1:80), Tim3 (clone 7D3, BD Biosciences 565566, diluted 1:100), CD22 (clone HIB22, BD Biosciences 562860, diluted 1:100), CD19 (clone FMC63, Novus Biologicals 52716, diluted 1:80-1:32000; clone HIB19, BD Biosciences 555413, diluted 1:50), CD107a-PECy7 (clone H4A3, Biolegend 328607, diluted 1:100), CD34 APC (BD 555824, diluted 1:50).

Techniques: Glycoproteomics, Binding Assay

Journal: Nature Medicine

Article Title: BCMA CAR T cells in a patient with relapsing idiopathic inflammatory myositis after initial and repeat therapy with CD19 CAR T cells

doi: 10.1038/s41591-025-03718-3

Figure Lengend Snippet: a , Serum CK levels after CD19 CAR T cell therapy and at relapse. The horizontal dotted line indicates the upper limit of normal range (<170 U per l). b , Muscle strength as indicated by MMT8 score. c , CD19 CAR + CD3 + T cell counts in blood. d , CD19 + B cell counts in blood. e – h , Counts of leukocytes ( e ); neutrophils ( f ); CD4 + T cells ( g ) and CD8 + T cells ( h ) in blood after lymphodepletion (in cells per µl). i , ELISA quantification of the humoral response to CAR (FMC63). j , Quantification of the anti-CAR T cell response in control donors and the index patient. k , Sequence of events demonstrating the onset of the anti-CAR T cell response and humoral response. Unstim, no T cell stimulation; +, stimulated T cells.

Article Snippet: Anti-FMC63 antibodies were detected using the ACROBIO Anti-CD19 (FMC63) CAR Immunogenicity ELISA Kit following kit instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Sequencing, Cell Stimulation