Journal: Journal of Hematology & Oncology

Article Title: A bispecific CAR-T cell therapy targeting BCMA and CD38 in relapsed or refractory multiple myeloma

doi: 10.1186/s13045-021-01170-7

Figure Lengend Snippet: Preclinical evaluation of BM38 CAR-Ts. a Schematic structure of single-target BCMA CAR, CD38 CAR, bispecific 38BM CAR and BM38 CAR. All the CARs contained a granulocyte–macrophage colony-stimulating factor signal peptide (GM-CSF SP), a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain and a CD3ζ signaling domain. b CAR expression on lentivirus-transduced T cells and CD38 expression on CAR-Ts. Histograms are representative of 3 independent experiments. NT, nontransduced T. NT blank, NT without antibody staining. NT parallel, NT with antibody staining. c Phenotypic profiles of the final CAR-T products and NT cells. Differentiation phenotypes are depicted for naïve (CD45RA + CD62L + ), central memory (CM) (CD45RA − CD62L + ), effector memory (EM) (CD45RA − CD62L − ) and effector (CD45RA + CD62L − ) cells. n = 3, mean ± standard error of mean (SEM). Statistical analysis was performed using one-way ANOVA and subsequent multiple comparisons with BM38 CAR-Ts. *** P < 0.001; ns: not significant. d Expansion curves of the four CAR-Ts and NT cells after lentiviral transduction. n = 5, mean ± SEM. Repeated measures one-way ANOVA was used, ns: not significant. e Cytotoxicity of the four CAR-Ts and NT cells to target cells at an effector: target (E:T) ratio of 1:1, 5:1 and 10:1. n = 4, mean ± SEM. Dunnett’s multiple comparisons test was used at every E:T ratio with BM38 CAR-Ts as the control. * P < 0.05; ** P < 0.01; *** P < 0.001; ns: not significant. f Production of interferon γ (IFNγ) at an E:T ratio of 1:1. n = 3, mean ± SEM. Dunnett’s multiple comparisons test was used with BM38 CAR-Ts as control. ** P < 0.01; *** P < 0.001; ns: not significant. g Luciferase live imaging of MM.1s xenograft mice on day 0, 7, 14 and 23 after infusion of 3.0 × 10 6 NT cells, BCMA CAR-Ts, 38BM CAR-Ts or BM38 CAR-Ts. h Kaplan–Meier survival plot of MM xenograft mice. The log-rank test was used

Article Snippet: All the cell lines were authenticated for CD38 (anti-CD38-APC, 555335; BD Biosciences [BD], USA) and BCMA (anti-BCMA-PE, 130-119-147; Miltenyi Biotec, USA) expression by flow cytometry, and cultured in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (NQBB, China).

Techniques: Expressing, Staining, Transduction, Control, Luciferase, Imaging

Journal: Journal of Hematology & Oncology

Article Title: A bispecific CAR-T cell therapy targeting BCMA and CD38 in relapsed or refractory multiple myeloma

doi: 10.1186/s13045-021-01170-7

Figure Lengend Snippet: Clinical responses mediated of BM38 CAR-Ts. a Swimmer plot for the 23 patients treated in the study. Two patients were enrolled in the two low-dose groups for the consideration of risk–benefit trade-off. b Immunohistochemical staining of patient 13’s right parailiac mass before treatment, showing involvement of MM cells and positive expression of BCMA and CD38. c Computed tomography scans of patient 13 showing a large right parailiac plasmacytoma at enrollment that was partially eliminated in month 3 and completely disappeared in month 5. d Changes of serum M protein in patient 13 during BM38 CAR-T treatment. e BCMA and CD38 expression on MM cells in patient 15 and 20 at baseline. Representative staining and gating of MM cells are shown in Additional file : Fig. S6

Article Snippet: All the cell lines were authenticated for CD38 (anti-CD38-APC, 555335; BD Biosciences [BD], USA) and BCMA (anti-BCMA-PE, 130-119-147; Miltenyi Biotec, USA) expression by flow cytometry, and cultured in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (NQBB, China).

Techniques: Immunohistochemical staining, Staining, Expressing, Computed Tomography

Journal: Journal of Hematology & Oncology

Article Title: A bispecific CAR-T cell therapy targeting BCMA and CD38 in relapsed or refractory multiple myeloma

doi: 10.1186/s13045-021-01170-7

Figure Lengend Snippet: Predictive docking patterns of BM38 CAR to MM cells. a Hypothetical structure of the anti-BCMA scFv and anti-CD38 scFv joined with an (EAAAK) 3 linker (not shown). b Most favorable docking models of the anti-BCMA scFv with 51 amino acid residues of the extracellular domain of BCMA (2kn1.pdb); c Most favorable docking models of the anti-CD38 scFv with 257 amino acid residues of the extracellular domain of CD38 (1yh3.pdb); d Dual docking of BM38 CAR with BCMA and CD38. e Theoretical single combination pattern of the anti-CD38 scFv and CD38 on MM cells. f Theoretical single combination pattern of the anti-BCMA scFv and BCMA on MM cells. g Theoretical double-binding pattern of the bispecific BM38 CAR to BCMA and CD38 on MM cells. h–i The CD38 extracellular domain contains 257 amino acids and the BCMA extracellular chain consists of only 54 amino acids. The anti-BCMA scFv and anti-CD38 scFv contain 246 and 249 amino acids, respectively. Theoretically, CD38 binding might assist the anti-BCMA scFv in binding to BCMA on myeloma cells, and BCMA coalescence would reversely strengthen CD38 binding

Article Snippet: All the cell lines were authenticated for CD38 (anti-CD38-APC, 555335; BD Biosciences [BD], USA) and BCMA (anti-BCMA-PE, 130-119-147; Miltenyi Biotec, USA) expression by flow cytometry, and cultured in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (NQBB, China).

Techniques: Binding Assay

Journal: Frontiers in Oncology

Article Title: CD8+ T cell-associated genes MS4A1 and TNFRSF17 are prognostic markers and inhibit the progression of colon cancer

doi: 10.3389/fonc.2022.941208

Figure Lengend Snippet: Survival analysis and stemness scores. (A) Overall survival (OS) analysis of the high- and low-MS4A1-expression groups. (B) OS analysis of the high- and low-TNFRSF17-expression groups. (C) Landmark analysis results (D) Proportional risk assumption results (E) Progression-free survival (PFS) analysis of the high- and low-MS4A1-expression groups. (F) PFS survival analysis of the high- and low-TNFRSF17-expression groups. (G) Stemness scores of the high- and low-MS4A1-expression groups. (H) Stemness scores of the high- and low-TNFRSF17-expression groups. ****,<0.0001.

Article Snippet: After blocking (Albumin Bovine, BioFroxx 4240, China) both CC and peri-lesional tissues were incubated with MS4A1 (Kit-0001, MXB, China) and TNFRSF17 (A01014-1, 1:50, BOSTER, China) primary antibodies overnight at 4 ° C. The following day, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (IgG; PV-9000, ZSBIO, China) for 20 min at room temperature and washed thrice with phosphate-buffered solution.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: CD8+ T cell-associated genes MS4A1 and TNFRSF17 are prognostic markers and inhibit the progression of colon cancer

doi: 10.3389/fonc.2022.941208

Figure Lengend Snippet: Expression of MS4A1 and TNFRSF is lower in colon cancer (CC) and related to clinical features. (A) Expression of MS4A1 and TNFRSF17 at the transcription level was lower in colon cancer (CC) tissues than in healthy colon tissues. (B) qRT-PCR showed that the mRNA expression of MS4A1 and TNFRSF17 was higher in FHC cells than in SW480 and HCT116 cells. (C) Matched pairs of CC tissues and adjacent non-neoplastic colon tissues were stained with MS4A1- and TNFRSF17-specific antibodies for immunohistochemical analysis. Representative images of tissues and tumour-infiltrating cells are shown (scale bar = 20 μm). (D) The MOD of MS4A1 and TNFRSF17 is obtained by analysing the photo optical density with IPP software. (E, F) Western blotting revealed that the protein expression of MS4A1 and TNFRSF17 was higher in FHC cells than in SW480 and HCT116 cells. **,<0.01; ***,<0.001.

Article Snippet: After blocking (Albumin Bovine, BioFroxx 4240, China) both CC and peri-lesional tissues were incubated with MS4A1 (Kit-0001, MXB, China) and TNFRSF17 (A01014-1, 1:50, BOSTER, China) primary antibodies overnight at 4 ° C. The following day, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (IgG; PV-9000, ZSBIO, China) for 20 min at room temperature and washed thrice with phosphate-buffered solution.

Techniques: Expressing, Quantitative RT-PCR, Staining, Immunohistochemical staining, Software, Western Blot

Journal: Frontiers in Oncology

Article Title: CD8+ T cell-associated genes MS4A1 and TNFRSF17 are prognostic markers and inhibit the progression of colon cancer

doi: 10.3389/fonc.2022.941208

Figure Lengend Snippet: Analysis of correlation between expression of TNFRSF17 and clinical.

Article Snippet: After blocking (Albumin Bovine, BioFroxx 4240, China) both CC and peri-lesional tissues were incubated with MS4A1 (Kit-0001, MXB, China) and TNFRSF17 (A01014-1, 1:50, BOSTER, China) primary antibodies overnight at 4 ° C. The following day, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (IgG; PV-9000, ZSBIO, China) for 20 min at room temperature and washed thrice with phosphate-buffered solution.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: CD8+ T cell-associated genes MS4A1 and TNFRSF17 are prognostic markers and inhibit the progression of colon cancer

doi: 10.3389/fonc.2022.941208

Figure Lengend Snippet: Relationship between the target genes and immune infiltration. (A) Correlation between MS4A1 expression and immune scores. (B) Correlation between MS4A1 expression and stromal scores. (C) Correlation between TNFRSF17 expression and immune scores. (D) Correlation between TNFRSF17 expression and stromal scores. (E) Relationship between MS4A1 expression and immune cell infiltration in TME. (F) Correlation between TNFRSF17 expression and immune cell infiltration in TME. (G) Comparison of the immune status between the high- and low-MS4A1-expression groups. (H) Comparison of the immune status between the high- and low-TNFRSF17-expression groups. *,<0.05; **,<0.01; ***,<0.001; ns,>0.05.

Article Snippet: After blocking (Albumin Bovine, BioFroxx 4240, China) both CC and peri-lesional tissues were incubated with MS4A1 (Kit-0001, MXB, China) and TNFRSF17 (A01014-1, 1:50, BOSTER, China) primary antibodies overnight at 4 ° C. The following day, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (IgG; PV-9000, ZSBIO, China) for 20 min at room temperature and washed thrice with phosphate-buffered solution.

Techniques: Expressing, Comparison

Journal: Frontiers in Oncology

Article Title: CD8+ T cell-associated genes MS4A1 and TNFRSF17 are prognostic markers and inhibit the progression of colon cancer

doi: 10.3389/fonc.2022.941208

Figure Lengend Snippet: MS4A1 and TNFRSF17 are associated with CC drug sensitivity. (A) Differences in the expression of common immune checkpoints between the high- and low-MS4A1-expression groups. (B) Differences in the expression of common immune checkpoints between the high- and low-TNFRSF17-expression groups. (C) Differences in TIDE scores between the high- and low-MS4A1-expression groups. (D) Differences in response rates predicted based on TIDE scores between the high- and low-MS4A1-expression groups. (E) Differences in TIDE scores between the high- and low-TNFRSF17-expression groups. (F) Differences in response rates predicted based on TIDE scores between the high- and low-TNFRSF17-expression groups. (G) Correlation between drug sensitivity and gene expression. *, <0.05; ***, <0.001.

Article Snippet: After blocking (Albumin Bovine, BioFroxx 4240, China) both CC and peri-lesional tissues were incubated with MS4A1 (Kit-0001, MXB, China) and TNFRSF17 (A01014-1, 1:50, BOSTER, China) primary antibodies overnight at 4 ° C. The following day, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (IgG; PV-9000, ZSBIO, China) for 20 min at room temperature and washed thrice with phosphate-buffered solution.

Techniques: Expressing, Gene Expression

Journal: Frontiers in Oncology

Article Title: CD8+ T cell-associated genes MS4A1 and TNFRSF17 are prognostic markers and inhibit the progression of colon cancer

doi: 10.3389/fonc.2022.941208

Figure Lengend Snippet: Expression changes after transfection of MS4A1 and TNFRSF17. (A, B) Compared with the empty negative control group, both MS4A1 and TNFRSF17 gene overexpression plasmids transfected into SW480 and HCT116 cells, the RNA levels detected by QPCR were overexpressed (C, D) Compared with the empty negative control group, both MS4A1 and TNFRSF17 gene overexpression plasmids were transfected into SW480 and HCT116 cells, and the protein levels detected by WB had overexpression effects. *,<0.05; **,<0.01; ***,<0.001.

Article Snippet: After blocking (Albumin Bovine, BioFroxx 4240, China) both CC and peri-lesional tissues were incubated with MS4A1 (Kit-0001, MXB, China) and TNFRSF17 (A01014-1, 1:50, BOSTER, China) primary antibodies overnight at 4 ° C. The following day, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (IgG; PV-9000, ZSBIO, China) for 20 min at room temperature and washed thrice with phosphate-buffered solution.

Techniques: Expressing, Transfection, Negative Control, Over Expression

Journal: Frontiers in Oncology

Article Title: CD8+ T cell-associated genes MS4A1 and TNFRSF17 are prognostic markers and inhibit the progression of colon cancer

doi: 10.3389/fonc.2022.941208

Figure Lengend Snippet: CCK8 and Transwell experiments. (A) The proliferation ability of SW480 cells and HCT116 cells transfected with pCMV3-MS4A1 and pCMV3-TNFRSF17 was significantly reduced. (B) The invasive ability of SW480 cells and HCT116 cellstransfected with pCMV3-MS4A1 and pCMV3-TNFRSF17 was significantly reduced. *,<0.05; **,<0.01; ***,<0.001.

Article Snippet: After blocking (Albumin Bovine, BioFroxx 4240, China) both CC and peri-lesional tissues were incubated with MS4A1 (Kit-0001, MXB, China) and TNFRSF17 (A01014-1, 1:50, BOSTER, China) primary antibodies overnight at 4 ° C. The following day, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (IgG; PV-9000, ZSBIO, China) for 20 min at room temperature and washed thrice with phosphate-buffered solution.

Techniques: Transfection

Journal: Frontiers in Oncology

Article Title: CD8+ T cell-associated genes MS4A1 and TNFRSF17 are prognostic markers and inhibit the progression of colon cancer

doi: 10.3389/fonc.2022.941208

Figure Lengend Snippet: Cell scratch experiment. (A) The migration ability of SW480 cells transfected with pCMV3-MS4A1 and pCMV3-TNFRSF17 was significantly weakened. (B) The migration ability of HCT116 cells was significantly reduced after pCMV3-MS4A1 and pCMV3-TNFRSF17 were transfected. *,<0.05.

Article Snippet: After blocking (Albumin Bovine, BioFroxx 4240, China) both CC and peri-lesional tissues were incubated with MS4A1 (Kit-0001, MXB, China) and TNFRSF17 (A01014-1, 1:50, BOSTER, China) primary antibodies overnight at 4 ° C. The following day, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (IgG; PV-9000, ZSBIO, China) for 20 min at room temperature and washed thrice with phosphate-buffered solution.

Techniques: Migration, Transfection

Journal: bioRxiv

Article Title: Defining the cell surface proteomic landscape of multiple myeloma reveals immunotherapeutic strategies and biomarkers of drug resistance

doi: 10.1101/2021.01.17.427038

Figure Lengend Snippet: A. mRNA transcript data from the Human Blood Atlas (top) demonstrates that among hematopoietic cells CCR10 is most highly expressed in plasmablasts (we note that long-lived plasma cells are unfortunately not included in this dataset; plasmablasts used as closest proxy). Cancer Cell Line Encyclopedia (CCLE) (middle) data supports that on average myeloma cell lines express >20x more CCR10 mRNA than any other tumor cell type. GTex (bottom) data does suggest some low level CCR10 expression in non-hematopoietic tissues; B-lymphoblast expression can be compared to memory B-cell expression in the Human Blood Atlas. Plasmablast expression is therefore expected to be at least 20x higher than other non-hematopoietic tissues, supportive of a therapeutic index. B. Flow cytometry analysis confirms significantly higher CCR10 expression on myeloma plasma cells (OPM-2, MM.1S, ANBL-6, AMO-1) than B-cell malignancy cell lines (SEM, RS411 (B-ALL), HBL-1, OCI-LY10, TOLEDO (B-cell lymphoma)), with similar increase as seen for BCMA. Analysis of a primary patient plasma cell (CD19-/CD138+/CD38+) specimen confirms CCR10 expression (top right). C. Similar analysis as in A., illustrating highly enriched TXNDC11 expression on plasmablasts, highly increased expression in myeloma plasma cells versus any other cancer cell type in the CCLE, and moderate expression in other non-hematopoietic tissues. D. Data from the Cancer Dependency Map (depmap.org; Avana Public 20Q3) indicate that myeloma cell lines are among the most genetically dependent on CRISPR deletion of this gene, as noted by lowest CERES score when averaged across all included tumor cell lines.

Article Snippet: The following antibodies were used: CD138 (BD Biosciences, 562097, 552026), BCMA (BD Biosciences, 552026, BioLegend 357504), CD53 (BD Biosciences, 555508), CD10 (BD Biosciences, 561002), CD151 (BD Biosciences, 556057), CD50/ICAM3 (BD Biosciences, 555958), ITGB7 (BD Biosciences, 555945), GITR/CD357 (BioLegend 311604), MUC1/CD227 (BD Biosciences, 559774), CCR10 (BD Biosciences, clone 1B5).

Techniques: Expressing, Flow Cytometry, CRISPR