cd70 monoclonal antibody  (Proteintech)

Journal: Frontiers in Immunology

Article Title: Single-cell and bulk transcriptomics reveal a CD8 + T-cell gene signature predicting prognosis in diffuse large B-cell lymphoma

doi: 10.3389/fimmu.2025.1685541

Figure Lengend Snippet: Validation cohort for predicting the risk signature of DLBCL survival based on the discovery cohort. (A) Expression of CD69 on infiltrating CD8 + T cells in DLBCL (400X). CD69 (red), CD8 (green), DAPI (blue). (B) Kaplan–Meier curves of OS in DLBCL patients with CD69 + /CD8 + and CD69 + /CD8 + . Cases were classified as CD69 + /CD8 + when ≥10% of infiltrating CD8 + T cells expressed CD69. (C) Expression of CD70 on infiltrating CD8 + T cells in DLBCL (400X). CD70 (green), CD8 (red), DAPI (blue). (D) Kaplan–Meier curves of OS in DLBCL patients with CD70 + /CD8 + and CD70 + /CD8 + . Cases were classified as CD70 + /CD8 + when ≥10% of infiltrating CD8 + T cells expressed CD70. (E) The Kaplan-Meier OS curve of the validation cohort (this work. (n=66)) patients between low risk group (n=34) and high risk group (n=32). This work samples were stratified by risk score. (F) Univariate Cox regression analysis of Risk Score: high risk, IPI: low-mid, IPI: mid-high and IPI: high in this work. (G) The Kaplan-Meier OS curve of the validation cohort ( GSE181063 (n=773)) patients between low risk group (n=387) and high risk group (n=386). GSE181063 samples were stratified by risk score. (H) Univariate Cox regression analysis of Risk Score: high risk, IPI: low-mid, IPI: mid-high and IPI: high in GSE181063 . (I) The Kaplan-Meier OS curve of the validation cohort ( GSE117556 (n=469)) patients between low risk group (n=235) and high risk group (n=234). GSE117556 samples were stratified by risk score. (J) Univariate Cox regression analysis of Risk Score: high risk, IPI: low-mid, IPI: mid-high and IPI: high in GSE117556 . Log-rank tests were used to derive p-values for comparisons between two groups.

Article Snippet: The primary antibodies are as follows: CD69 Polyclonal antibody (Proteintech, 10803-1-AP), CD70 Monoclonal antibody (Proteintech, 67749-1-Ig), CD8a Monoclonal antibody (Proteintech, 66868-1-Ig), Anti-CD8 alpha antibody (Abcam, ab93278).

Techniques: Biomarker Discovery, Expressing

Journal: Clinical and Translational Medicine

Article Title: The dual role of CD70 in B‐cell lymphomagenesis

doi: 10.1002/ctm2.1118

Figure Lengend Snippet: The workflow of the diffuse large B‐cell lymphoma (DLBCL) cohorts. The workflow of the Chinese (A,B) and Swedish (C,D) DLBCL cohorts. In CD70 mutation analysis, some samples were examined by more than one sequencing method. Detailed information of each patient is shown in Tables and respectively.

Article Snippet: The cells were lysed 48 h after transfection, and CD70 expression was detected by Western blotting using a rabbit anti‐human CD70 monoclonal antibody (orb213695, Biorbyt, recognising amino acids 76–115, Cambridge, UK). β‐Actin was served as a loading control (#4967; Cell Signaling, Danvers, MA).

Techniques: Mutagenesis, Sequencing

Journal: Clinical and Translational Medicine

Article Title: The dual role of CD70 in B‐cell lymphomagenesis

doi: 10.1002/ctm2.1118

Figure Lengend Snippet: The CD70 gene is frequently targeted by mutations and deletions in diffuse large B‐cell lymphoma (DLBCL). (A) Schematic diagram representing the distribution of mutations identified in the CD70 gene in tumour samples from Chinese (CHN, red symbols) and Swedish DLBCL (SWE, purple symbols) patients. Mis, missense mutation; Mut, mutation; Trunc, truncation mutation. (B) Distribution of CD70 homozygous (copy number = 0) and heterozygous (copy number = 1) deletions in CHN and SWE DLBCL tumour samples. χ 2 ‐test, P > 0.6585. (C) Comparison of the frequency of CD70 mutations and copy number variations in CHN and SWE DLBCL tumour samples. Mut and copy number loss: mutation and copy number loss; χ 2 ‐test, ** P = 0.0095.

Article Snippet: The cells were lysed 48 h after transfection, and CD70 expression was detected by Western blotting using a rabbit anti‐human CD70 monoclonal antibody (orb213695, Biorbyt, recognising amino acids 76–115, Cambridge, UK). β‐Actin was served as a loading control (#4967; Cell Signaling, Danvers, MA).

Techniques: Mutagenesis, Comparison

Journal: Clinical and Translational Medicine

Article Title: The dual role of CD70 in B‐cell lymphomagenesis

doi: 10.1002/ctm2.1118

Figure Lengend Snippet: CD70 genetic alterations resulted in reduced/loss of protein expression or CD27 binding. (A) Immunoblotting analysis of exogenous CD70 expression in HEK293T cells transfected with equimolar amounts of vectors expressing HA‐His‐tagged wild‐type (WT) or mutant (identified from our clinical cohorts) CD7 0 alleles. Representative images of three independent experiments are shown. (B) CD70 expression on the cytomembrane was analysed by flow cytometry using a mAb against CD70‐transfected HEK293T cells. Three independent experiments were performed. (C) The binding of overexpressed WT (red) or mutant CD70 (p.Y15N) (blue) to recombinant human CD27 was measured by flow cytometry. The shaded area represents nontransfected HEK293T cells. The binding of anti‐CD5 mAb to cells transfected with WT (yellow) is shown. Three independent experiments were performed. (D) Representative images of CD70 staining by immunohistochemical (IHC) (left panel) and quantification of CD70 expression on the cytomembrane (right panel) in Chinese diffuse large B‐cell lymphoma (DLBCL) samples and reactive lymph nodes. Scale bar, 50 μm. Mann–Whitney U ‐test, * P = 0.0163, *** P = 0.0005. (E) Quantification of CD70 expression on the cytomembrane by IHC in Swedish DLBCL samples. The same reactive lymph node samples were used. Mann–Whitney U ‐test, * P = 0.0285, ** P = 0.0006.

Article Snippet: The cells were lysed 48 h after transfection, and CD70 expression was detected by Western blotting using a rabbit anti‐human CD70 monoclonal antibody (orb213695, Biorbyt, recognising amino acids 76–115, Cambridge, UK). β‐Actin was served as a loading control (#4967; Cell Signaling, Danvers, MA).

Techniques: Expressing, Binding Assay, Western Blot, Transfection, Mutagenesis, Flow Cytometry, Recombinant, Staining, Immunohistochemical staining, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: The dual role of CD70 in B‐cell lymphomagenesis

doi: 10.1002/ctm2.1118

Figure Lengend Snippet: Hepatitis B virus (HBV) but not Epstein‒Barr virus (EBV) infection contributes to the increased CD70 genetic alterations in Chinese diffuse large B‐cell lymphoma (DLBCL) patients. (A) CD70 genetic aberration rates in tumour samples were similar in EBV‐positive and EBV‐negative DLBCL patients. (B) Compared with those from HBsAg‐negative DLBCL patients, tumour samples from HBsAg + DLBCL patients were more frequently targeted by CD70 genetic aberrations. χ 2 ‐test, P = 0.0236.

Article Snippet: The cells were lysed 48 h after transfection, and CD70 expression was detected by Western blotting using a rabbit anti‐human CD70 monoclonal antibody (orb213695, Biorbyt, recognising amino acids 76–115, Cambridge, UK). β‐Actin was served as a loading control (#4967; Cell Signaling, Danvers, MA).

Techniques: Virus, Infection

Journal: Clinical and Translational Medicine

Article Title: The dual role of CD70 in B‐cell lymphomagenesis

doi: 10.1002/ctm2.1118

Figure Lengend Snippet: CD70 genetic aberrations predict inferior overall survival in Chinese diffuse large B‐cell lymphoma (DLBCL) patients. (A) DLBCL patients with CD70 genetic aberrations had a shorter OS than those with wild‐type CD70 . The P ‐value was calculated by the log‐rank test. HR (95% CI): 1.849 (1.139–3.002). (B–E) Kaplan–Meier survival curve of overall survival in the CD70 wild‐type group and CD70 gene‐altered group within the indicated cohorts. The P ‐value was calculated by the log‐rank test. HR (95% CI): GCB: 0.980 (0.430–2.238); non‐GCB: 2.878 (1.564–5.294); R‐CHOP, 1.577 (0.771–3.224); CHOP, 1.988 (0.815–4.852).

Article Snippet: The cells were lysed 48 h after transfection, and CD70 expression was detected by Western blotting using a rabbit anti‐human CD70 monoclonal antibody (orb213695, Biorbyt, recognising amino acids 76–115, Cambridge, UK). β‐Actin was served as a loading control (#4967; Cell Signaling, Danvers, MA).

Techniques:

Journal: Clinical and Translational Medicine

Article Title: The dual role of CD70 in B‐cell lymphomagenesis

doi: 10.1002/ctm2.1118

Figure Lengend Snippet: Higher CD70 expression is observed in advanced‐stage Swedish diffuse large B‐cell lymphoma (DLBCL) patients and correlates with inferior overall survival. (A) Representative images of CD70 staining in DLBCL patients with early‐ or advanced‐stage disease. (B) Higher CD70 protein expression was detected in DLBCL biopsies from patients with advanced‐stage disease. Early stage, median CD70 score = 3, range: 0–8; advanced stage, median CD70 score = 4, range: 0–8. Mann–Whitney U ‐test, P = 0.0117. (C) Kaplan–Meier survival curve of overall survival in the low and high CD70 groups. The P value was determined by the log‐rank test. HR (95% CI): 1.472 (1.083–2.000). (D,E) The overall survival curves in the low and high CD70 groups within the CHOP‐treated (D) and R‐CHOP‐treated (E) DLBCL patients. The P value was determined by the log‐rank test. HR (95% CI): CHOP‐treated: 1.637 (1.121–2.392); R‐CHOP‐treated: 1.163 (0.538–2.515). (F) Kaplan–Meier survival curve of overall survival for DLBCL patients with high and low CD70 mRNA expression in the GSE117556 dataset. The P ‐value was determined by the log‐rank test, P = 0.0219. HR (95%CI): 1.453 (1.054–2.003). (G) Kaplan–Meier survival curve of overall survival for DLBCL patients with high and low CD70 mRNA expression in the TCGA DLBCL database. The data were generated by OSdlbcl online consensus survival analysis web server. The P ‐value was determined by the log‐rank test, P = 0.0085. (H) Higher CD70 protein expression was detected in the EBV + DLBCL, not otherwise specified (NOS). Mann–Whitney U ‐test, P = 0.0202.

Article Snippet: The cells were lysed 48 h after transfection, and CD70 expression was detected by Western blotting using a rabbit anti‐human CD70 monoclonal antibody (orb213695, Biorbyt, recognising amino acids 76–115, Cambridge, UK). β‐Actin was served as a loading control (#4967; Cell Signaling, Danvers, MA).

Techniques: Expressing, Staining, MANN-WHITNEY, Generated

Journal: Clinical and Translational Medicine

Article Title: The dual role of CD70 in B‐cell lymphomagenesis

doi: 10.1002/ctm2.1118

Figure Lengend Snippet: Single‐cell RNA sequencing revealed T‐cell exhaustion in diffuse large B‐cell lymphoma (DLBCL) with high levels of CD70. (A) Single‐cell RNA sequencing analyses of 11 DLBCL samples. Uniform manifold approximation and projection (UAMP) plot showing the sample origins and DLBCL subtypes of each cell cluster (left and middle panels). Clusters were assigned to the indicated cell types by canonical markers (right panel). CAFs, cancer‐associated fibroblasts. NK cells, natural killer cells. (B) Quantification of CD70 on the malignant B cells of each sample. Samples were grouped (low and high) based on the CD70 expression on malignant B‐cells. (C) UAMP plot showing T‐cell subclusters (left panel). Sample origins are indicated in the middle panel. Subclusters were assigned to CD70 high and low groups based on the CD70 expression on the corresponding malignant B cells (right panel). (D) Expression of functional status markers of T cells. (E) The percentages of CD8cyto‐2 and CD8cyto‐3 cells per sample are shown. * P = 0.0495, one‐tailed Mann–Whitney U ‐test. (F) The percentages of T cells per sample are shown for the indicated subcluster. Not significant, one‐tailed Mann–Whitney U ‐test.

Article Snippet: The cells were lysed 48 h after transfection, and CD70 expression was detected by Western blotting using a rabbit anti‐human CD70 monoclonal antibody (orb213695, Biorbyt, recognising amino acids 76–115, Cambridge, UK). β‐Actin was served as a loading control (#4967; Cell Signaling, Danvers, MA).

Techniques: RNA Sequencing, Expressing, Functional Assay, One-tailed Test, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: The dual role of CD70 in B‐cell lymphomagenesis

doi: 10.1002/ctm2.1118

Figure Lengend Snippet: CD70/CD27 and PD‐1/PD‐L1 coinhibition rescues T‐cell exhaustion and reduces lymphoma growth in vivo. (A,B) T‐cell‐cytotoxic score and T‐cell‐exhausted score (calculated based on transcriptomic data) for diffuse large B‐cell lymphoma (DLBCL) patients with low ( n = 64) and high ( n = 63) CD70 mRNA expression, Student's t ‐test; ns, not significant. (C) The mRNA expression of PDCD1 , CTLA‐4 , TIGIT , LAG3 and HAVCR2 in DLBCL patients with low ( n = 64) and high ( n = 63) CD70 mRNA expression; the fold changes in the mRNA expression of PDCD1 and LAG3 were 1.9 and 1.3, respectively. Student's t ‐test, PDCD1: P = 0.0242; LAG3 : P = 0.0223; ns, not significant. (D) Representative immunohistochemical (IHC) images of CD70, CD3, PD‐1 and PD‐L1 in DLBCL patients. Original magnification, 20×; scale bar, 100 μm. (E) CD3 + T‐cell infiltration was similar between the different CD70 expression groups. n = 115. Student's t ‐test; NS, not significant. (F) PD‐1 expression was higher in the high CD70 group. n = 115. Mann–Whitney U ‐test, ** P = 0.0004. (G) PD‐L1 expression was higher in the high CD70 group. n = 114. Mann–Whitney U ‐test, ** P = 0.0030. (H–M) A20 lymphoma cell‐bearing BALB/c mice were treated with isotype control antibody or the indicated blocking antibody every three days, starting 7 days after inoculation. n = 5 mice/group. (H) Representative IHC staining for CD8, PD‐1, and granzyme B in tumour tissues from the indicated treated groups. Original magnification, 40×; scale bar, 50 μm. (I) CD8 + T‐cell counts (left panel), PD‐1 + (middle panel) and granzyme B + cell (right panel) numbers under a 40× field for the indicated groups. One‐way ANOVA with Tukey's test; NS, not significant. (J) When the mice were sacrificed, the tumours were harvested. (K,L) Tumour volumes were monitored every 3 days and calculated as the length×width 2 ×0.5. One‐way ANOVA with Tukey's test. (M) The tumour weight in the indicated groups. One‐way ANOVA with Tukey's test.

Article Snippet: The cells were lysed 48 h after transfection, and CD70 expression was detected by Western blotting using a rabbit anti‐human CD70 monoclonal antibody (orb213695, Biorbyt, recognising amino acids 76–115, Cambridge, UK). β‐Actin was served as a loading control (#4967; Cell Signaling, Danvers, MA).

Techniques: In Vivo, Expressing, Immunohistochemical staining, MANN-WHITNEY, Control, Blocking Assay, Immunohistochemistry

Journal: Cancer Science

Article Title: CD70 antibody‐drug conjugate: A potential novel therapeutic agent for ovarian cancer

doi: 10.1111/cas.15027

Figure Lengend Snippet: Correlation between CD70 expression levels and clinicopathological characteristics in serous ovarian carcinoma

Article Snippet: The cells were washed in phosphate‐buffered saline (PBS) (Nacalai Tesque) and were detached with 0.02% ethylenediaminetetraacetic acid solution (Nacalai Tesque)., , The cells were washed twice with fluorescence‐activated cell sorting (FACS) buffer (PBS with 1% FBS and 0.1% sodium azide) and then incubated with mouse monoclonal antihuman CD70 antibody (1:50, clone Ki‐24; BD Biosciences) as the primary antibody.

Techniques: Expressing

Journal: Cancer Science

Article Title: CD70 antibody‐drug conjugate: A potential novel therapeutic agent for ovarian cancer

doi: 10.1111/cas.15027

Figure Lengend Snippet: Confirmation of cluster of differentiation 70 (CD70) expression in serous ovarian carcinoma cells and surgical specimens. A, Representative CD70 staining in clinical samples. The immunohistochemistry score divided clinical samples into three groups: high (>4 points), low (1‐3 points), and negative (0 points). Scale bar: 100 μm. B, Among the 63 samples, 22 (34.9%), 21 (33.3%), and 20 (31.7%) represented the CD70‐high, CD70‐low, and CD70‐negative groups, respectively. C, The comparison of CD70 expression before and after neoadjuvant chemotherapy is shown. CD70 staining was assessed according to the intensity score. The black bar indicates the median score of intensity. Significantly higher scores are observed in the after neoadjuvant chemotherapy (NACT) group compared to those in the before NACT group ( P =.03). (Mann‐Whitney U test was used to compare the groups). D, CD70 expression was determined using western blotting analysis in four serous ovarian carcinoma cell lines and three ovarian clear cell carcinoma cell lines. Strong CD70 expression was observed in A2780cisR and SKOV3cisR cells, moderate CD70 expression was observed in SKOV3 cells, and no CD70 expression was observed in A2780, OVTOKO, OVISE, and RMG‐I cells. E, In fluorescence‐activated cell sorting analysis, CD70 expression was detected in A2780cisR, SKOV3, and SKOV3cisR cells using an anti‐CD70 monoclonal antibody. The gray‐shaded areas show isotype control

Article Snippet: The cells were washed in phosphate‐buffered saline (PBS) (Nacalai Tesque) and were detached with 0.02% ethylenediaminetetraacetic acid solution (Nacalai Tesque)., , The cells were washed twice with fluorescence‐activated cell sorting (FACS) buffer (PBS with 1% FBS and 0.1% sodium azide) and then incubated with mouse monoclonal antihuman CD70 antibody (1:50, clone Ki‐24; BD Biosciences) as the primary antibody.

Techniques: Expressing, Staining, Immunohistochemistry, MANN-WHITNEY, Western Blot, Fluorescence, FACS

Journal: Cancer Science

Article Title: CD70 antibody‐drug conjugate: A potential novel therapeutic agent for ovarian cancer

doi: 10.1111/cas.15027

Figure Lengend Snippet: Silencing cluster of differentiation 70 (CD70) expression was not associated with platinum resistance and proliferation. A, CD70 expression was silenced by CD70 small interfering RNA (siRNA) transfection in A2780cisR and SKOV3cisR cells. Silencing of CD70 expression was confirmed by western blotting analysis. B, IC 50 values for cisplatin were determined in A2780cisR and SKOV3cisR cells. IC 50 values of cisplatin in A2780cisR‐siCD70‐1 and A2780cisR‐siCD70‐3 cells were similar to those in A2780cisR‐C cells. IC 50 values of cisplatin were not significantly different among SKOV3cisR‐C, SKOV3cisR‐siCD70‐1, and SKOV3cisR‐siCD70‐3 cells. Kruskal‐Wallis H test was used to compare groups. C, Proliferation assays were performed in mock cells and two types of CD70‐silenced cells in A2780cisR, SKOV3, and SKOV3cisR cells. No significant difference in proliferation between mock cells and CD70‐silenced cells was observed in each cell line. Kruskal‐Wallis H test was used to compare groups. Abbreviations: No treat, no treatment; NS, not significant

Article Snippet: The cells were washed in phosphate‐buffered saline (PBS) (Nacalai Tesque) and were detached with 0.02% ethylenediaminetetraacetic acid solution (Nacalai Tesque)., , The cells were washed twice with fluorescence‐activated cell sorting (FACS) buffer (PBS with 1% FBS and 0.1% sodium azide) and then incubated with mouse monoclonal antihuman CD70 antibody (1:50, clone Ki‐24; BD Biosciences) as the primary antibody.

Techniques: Expressing, Small Interfering RNA, Transfection, Western Blot

Journal: Cancer Science

Article Title: CD70 antibody‐drug conjugate: A potential novel therapeutic agent for ovarian cancer

doi: 10.1111/cas.15027

Figure Lengend Snippet: Cluster of differentiation 70 (CD70) expression after cisplatin and paclitaxel exposure. A, The expression of AP‐1 (c‐Jun) was determined in groups with no treatment and after 1, 4, 8, and 12 h of cisplatin exposure. c‐Jun expression was not observed in any of the groups. B, mRNA level of CD70 expression was examined in A2780 mock cells and A2780‐NF‐κB‐p65‐silenced cells 1 and 2 h after cisplatin exposure. The expression levels of CD70 mRNA were corrected with those of GAPDH mRNA. CD70 expression was induced by short‐term exposure to cisplatin in A2780 mock cells, whereas CD70 was not induced on cisplatin exposure in A2780‐NF‐κB‐p65‐silenced cells. C, The expression of CD70, NF‐κB‐p65, and phospho‐NF‐κB‐p65 was determined in A2780 mock cells and A2780‐NF‐κB‐p65‐silenced cells at 0 h and after 1, 4, 8, and 12 h of cisplatin exposure. Knockdown of NF‐κB‐p65 and phospho‐NF‐κB‐p65 was observed in A2780‐NF‐κB‐p65‐silenced cells. In A2780 mock cells, the expression of NF‐κB‐p65 and phospho‐NF‐κB‐p65 was induced after cisplatin exposure and CD70 expression was also induced after cisplatin exposure. In A2780cisR cells, NF‐κB‐p65 suppression led to a substantial decrease in CD70 expression. D, The expression of CD70 was determined at 0 h and after 1, 4, 8, and 12 h of paclitaxel exposure in A2780 cells and at 0 h in A2780pacR cells. The expression of CD70 was not observed in any of the cells. Abbreviations: CDDP, cisplatin; mRNA, messenger ribonucleic acid; pacR, paclitaxel resistant cells; P‐NF‐κB, phospho‐NF‐κB; PTX, paclitaxel

Article Snippet: The cells were washed in phosphate‐buffered saline (PBS) (Nacalai Tesque) and were detached with 0.02% ethylenediaminetetraacetic acid solution (Nacalai Tesque)., , The cells were washed twice with fluorescence‐activated cell sorting (FACS) buffer (PBS with 1% FBS and 0.1% sodium azide) and then incubated with mouse monoclonal antihuman CD70 antibody (1:50, clone Ki‐24; BD Biosciences) as the primary antibody.

Techniques: Expressing

Journal: Cancer Science

Article Title: CD70 antibody‐drug conjugate: A potential novel therapeutic agent for ovarian cancer

doi: 10.1111/cas.15027

Figure Lengend Snippet: In vitro cell growth activity. A, A2780, A2780cisR, SKOV3, and SKOV3cisR cells were exposed to anti‐cluster of differentiation 70 (CD70) monoclonal antibody or anti‐human‐IgG 1 ‐isotype control antibody for 144 h. Cytotoxicity assays showed no significant growth inhibition in these cell lines. The y axis shows relative growth compared with that in the no treatment group. B, The structure of CD70‐antibody‐drug conjugate (ADC) consists of an anti‐CD70 antibody conjugated to the monomethyl auristatin F (MMAF) payload. Reused data from Nakae et al , Figure Copyright (2020) with permission from Elsevier. C, Cells were exposed to CD70‐ADC and human‐IgG 1 ‐control‐ADC. Compared with the control ADC, CD70‐ADC significantly inhibited the growth of CD70‐positive A2780cisR, SKOV3, and SKOV3cisR cells. Cytotoxicity assays showed no significant growth inhibition in the CD70‐negative A2780 cell line. The y axis shows relative growth compared with that in the no treatment group. Abbreviations: ADC, antibody‐drug conjugate, mAb; monoclonal antibody

Article Snippet: The cells were washed in phosphate‐buffered saline (PBS) (Nacalai Tesque) and were detached with 0.02% ethylenediaminetetraacetic acid solution (Nacalai Tesque)., , The cells were washed twice with fluorescence‐activated cell sorting (FACS) buffer (PBS with 1% FBS and 0.1% sodium azide) and then incubated with mouse monoclonal antihuman CD70 antibody (1:50, clone Ki‐24; BD Biosciences) as the primary antibody.

Techniques: In Vitro, Activity Assay, Inhibition

Journal: Cancer Science

Article Title: CD70 antibody‐drug conjugate: A potential novel therapeutic agent for ovarian cancer

doi: 10.1111/cas.15027

Figure Lengend Snippet: Antitumor activity of cluster of differentiation 70‐antibody‐drug conjugate (CD70‐ADC) in vivo. Antitumor efficacy of CD70‐ADC was investigated in the A2780cisR (A‐C) and SKOV3 cisR (D‐F) xenograft mouse models ( n = 6). Tumor‐bearing mice were intravenously administered PBS, control‐ADC (3 mg/kg), or CD70‐ADC (3 mg/kg) twice weekly (days 0, 4, 8, and 12) four times. Dots represent observed values and bars represent SEM. * P < .05, ** P < .01; Kruskal‐Wallis H test was used to compare the groups). In (A) and (D), * or ** indicates the significant difference between control‐ADC group vs PBS treatment group. A‐C, The average tumor volume at each time point is shown in the graph. Similar tumor sizes were observed between the control ADC and PBS groups ( P = .92). Tumor size in the CD70‐ADC treatment group was significantly suppressed relative to that in the control ADC group. Tumors were resected and weighed 28 days after implantation in the A2780cisR xenograft mouse model. The bar graph shows the average tumor weight with SEM. Relative bodyweight changes are shown in the right panel. D‐F, The average tumor volume at each time point is shown in the graph. The average tumor volume at each time point is shown in the graph. Similar tumor sizes were observed between the control ADC and PBS groups ( P = .2). Tumor size in the CD70‐ADC treatment group was significantly suppressed relative to that in the control ADC group. Tumors were resected and weighed on day 28 after implantation in the SKOV3cisR xenograft mouse model. The bar graph shows the average tumor weight with SEM. Relative bodyweight changes are shown in the right panel. G, A single dose of PBS, control‐ADC, or CD70‐ADC was administered to A2780cisR xenograft mice. The percentage of mitotic cells after CD70‐ADC administration significantly increased relative to that after control‐ADC administration. Scale bar: 100 μm. H, Phospho‐histone H3 (Ser10) staining was assessed quantitatively as the ratio of mitotic cells to the total number of tumor cells in five fields (×400 magnification). The black bar indicates the median rate of mitotic cells. ** P < .01; Kruskal‐Wallis H test was used to compare groups. Abbreviations: ADC, antibody‐drug conjugate; PBS, phosphate‐buffered saline; NS, not significant; SEM, standard error of the mean

Article Snippet: The cells were washed in phosphate‐buffered saline (PBS) (Nacalai Tesque) and were detached with 0.02% ethylenediaminetetraacetic acid solution (Nacalai Tesque)., , The cells were washed twice with fluorescence‐activated cell sorting (FACS) buffer (PBS with 1% FBS and 0.1% sodium azide) and then incubated with mouse monoclonal antihuman CD70 antibody (1:50, clone Ki‐24; BD Biosciences) as the primary antibody.

Techniques: Activity Assay, In Vivo, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Rationalized inhibition of mixed lineage kinase 3 and CD70 enhances life span and antitumor efficacy of CD8 + T cells

doi: 10.1136/jitc-2019-000494

Figure Lengend Snippet: Loss of MLK3 promotes CD70-mediated apoptosis in CD8 + T cells. (A and B) Representative contour plots (left) and quantification (right) of CD4 + CD70 + and CD8 + CD70 + T cell populations, gated on splenic CD4 + and CD8 + T cells, respectively. Values are mean±SEM, *p<0.05 for knockout versus WT by unpaired t test (n=4 mice/group). (C) Protein expression of M2-MLK3 in lysates of vector control and MLK3 (WT) overexpressing Jurkat cells. GAPDH was taken as loading control. (D) Representative contour plots (left) and quantification (right) of CD70 expression on vector control and MLK3 (WT) overexpressing Jurkat cells. Values are the mean±SEM, **p<0.001 for MLK3 (WT) versus vector control by unpaired t test (n=3). (E) Experimental plan for figure F and G. (F and G) Representative contour plots (left) and quantification (right) of splenic CD4 + Annexin V + T cells and CD8 + Annexin V + T cell population. Values are the mean±SEM, p values (*p<0.05) for αCD70 mAb versus isotype control by unpaired t test (n=5 mice/group). mAb, monoclonal antibody; MLK3, mixed lineage kinase 3; WT, wild type.

Article Snippet: The in vivo blocking of CD70 was achieved using anti-mouse CD70 monoclonal antibody (mAb; 100 μg/mouse; BioXcell).

Techniques: Knock-Out, Expressing, Plasmid Preparation, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Rationalized inhibition of mixed lineage kinase 3 and CD70 enhances life span and antitumor efficacy of CD8 + T cells

doi: 10.1136/jitc-2019-000494

Figure Lengend Snippet: Loss of MLK3 promotes activation-induced apoptosis in CD8 + T cells. (A) Representative contour plots (left) and quantification (right) of cell death and apoptosis in activated vector control and MLK3 (WT) overexpressing Jurkat cells. Values are the mean±SEM, ***p<0.0001 for MLK3 (WT) versus vector control by unpaired t test (n=3). (B) Caspase-3 activity in cell lysates of vector control and MLK3 (WT) overexpressing Jurkat cells on activation with ImmunoCult for 72 hours. DEVD-AFC was taken as substrate for caspase-3 activity. Values are the mean±SD, **p<0.001 for MLK3 (WT) versus vector control by unpaired t test (n=3). (C) Representative contour plots (upper panel) and quantification (lower panel) of CD70 expression on activated vector control and MLK3 (WT) overexpressing Jurkat cells. Values are the mean±SEM, **p<0.001 for MLK3 (WT) versus vector control by unpaired t test (n=3). (D) Representative contour plots (left) and quantification (right) of cell death and apoptosis, gated on activated T cells from WT and MLK3 −/− mice. Values are the mean±SEM, p value for knockout versus WT by unpaired t test (n=6 mice/group). (E) Caspase-3 activity in cell lysates of activated pan T cells from WT and MLK3 −/− mice. Values are the mean±SEM, *p<0.05 for knockout versus WT by unpaired t test (n=4 mice/group). (F) Representative histogram plots (left) and quantification (right) of annexin V expression, gated on CD8 + T cells isolated from WT and MLK3 −/− mice. Values are the mean±SEM, p values (*p<0.05) for knockout versus WT by unpaired t test (n=3 mice/group). (G) Representative contour plots (upper panel) and quantification (lower panel) of CD25 + Annexin V + , CD38 + Annexin V + and CD69 + Annexin V + population, gated on CD8 + T cells from WT and MLK3 −/− mice. Values are the mean±SD, *p<0.05, **p<0.001 and ***p<0.0001 for knockout versus WT by unpaired t test (n=3 mice/group). MLK3, mixed lineage kinase 3; WT, wild type.

Article Snippet: The in vivo blocking of CD70 was achieved using anti-mouse CD70 monoclonal antibody (mAb; 100 μg/mouse; BioXcell).

Techniques: Activation Assay, Plasmid Preparation, Control, Activity Assay, Expressing, Knock-Out, Isolation

Journal: Journal for Immunotherapy of Cancer

Article Title: Rationalized inhibition of mixed lineage kinase 3 and CD70 enhances life span and antitumor efficacy of CD8 + T cells

doi: 10.1136/jitc-2019-000494

Figure Lengend Snippet: CD70 blockade increases life and effector function of CD8 + T cells deficient in MLK3. (A) Experimental plan of CD70 blockade and activation of T cells in vivo (for figure (B–D)). (B) Representative images for protein expression of TNFα (left) and quantification (right) in splenocytes isolated from WT and MLK3 −/− mice. For quantification of TNFα, fluorescence in single cell from three different images was quantified by Image J software. Values are the mean±SEM, p values (***p<0.0001) for αCD70 mAb treatment versus isotype control by unpaired t test (n=30 cells/group). (C) Representative contour plots (left) and quantification (right) of splenic Annexin V + CD8 + T cells on in vivo CD70 blockade and T cell activation. Values are the mean±SEM, *p<0.05 for αCD70 mAb versus isotype control by unpaired t test (n=3 mice/group). (D) CD8 + T cell chemotaxis in the presence of CCL3 or CCL4 (each, 100 ng/mL) for 3 hours. Values are the mean±SEM, p values (*p<0.05, **p<0.001) for CCL3 or CCL4 versus control by one-way analysis of variance, Bonferroni's multiple comparison test (upper panel) or αCD70 mAb versus isotype control by unpaired t test (lower panel) (n=3 mice/group). (E) Experimental approach for figure F. (F) Histogram plot of intracellular GZMB expression (left) and quantification (right), gated on CD8 + T cells. Values are the mean±SD, *p<0.05 values for αCD70 mAb treatment versus isotype control by unpaired t test (n=4 mice/group). DAPI, 4′, 6-diamidino-2-phenylindole; DCs, dendritic cells; FITC, fluorescein isothiocyanate; MLK3, mixed lineage kinase 3; OVA, ovalbumin; RFU, relative fluorescence units; WT, wild type.

Article Snippet: The in vivo blocking of CD70 was achieved using anti-mouse CD70 monoclonal antibody (mAb; 100 μg/mouse; BioXcell).

Techniques: Activation Assay, In Vivo, Expressing, Isolation, Fluorescence, Software, Control, Chemotaxis Assay, Comparison

Journal: Journal for Immunotherapy of Cancer

Article Title: Rationalized inhibition of mixed lineage kinase 3 and CD70 enhances life span and antitumor efficacy of CD8 + T cells

doi: 10.1136/jitc-2019-000494

Figure Lengend Snippet: Combined blockade of MLK3 and CD70 increases tumor infiltration of cytotoxic CD8 + T cells. (A) Experimental approach for figure B. (B) Representative contour plots (left) and quantification (right) of CD8 + CD70 + T cells, gated on T cells derived from dLN of tumor-bearing mice treated either with control or URMC-099. Values are the mean±SEM, ***p<0.0001 for URMC-099 versus control by unpaired t test (n=4 mice/group). (C) Experimental plan for combination therapy in 4T1 breast cancer model. (D) Representative contour plots (left) and quantification (right) of intracellular TNFα expression in splenocytes from control and treated tumor-bearing mice. Values are the mean±SD, *p<0.05 for combination of URMC-099 and αCD70 mAb versus URMC-099 alone treatment by unpaired t test (n=3 mice/group). (E) Representative IHC images of TNFα expressions in tumor sections from control, isotype, anti-CD70 mAb, URMC-099 and combination of URMC-099 and anti-CD70 mAb-treated mice (n=3 mice/group). Size bar=20 µm. (F) Representative confocal microscopy images of CD8 and GZMB expressions in tumor sections from control and treated mice (n=3 mice/group). Co-localization data were generated from confocal microscopy images. Size bar=20 µm. (G) Protein expressions of GZMB in tumor lysates isolated from control and treated mice. GAPDH was taken as loading controls. dLN, draining lymph node; IHC, immunohistochemistry; MLK3, mixed lineage kinase 3; TNFα, tumor necrosis factor-α.

Article Snippet: The in vivo blocking of CD70 was achieved using anti-mouse CD70 monoclonal antibody (mAb; 100 μg/mouse; BioXcell).

Techniques: Derivative Assay, Control, Expressing, Confocal Microscopy, Generated, Isolation, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Rationalized inhibition of mixed lineage kinase 3 and CD70 enhances life span and antitumor efficacy of CD8 + T cells

doi: 10.1136/jitc-2019-000494

Figure Lengend Snippet: Combined blockade of MLK3 and CD70 induces mitochondrial apoptosis in tumor cells. (A) Representative confocal microscopy images of Bid protein expression in tumor sections from control, isotype, anti-CD70 mAb, URMC-099 and combination of URMC-099 and anti-CD70 mAb-treated mice (n=3 mice/group). Co-localization data are generated from confocal microscopy images. Size bar=5 µm. (B) Protein expressions of Bax and Bcl-2 in tumor lysates isolated from control and treated mice and (C and D) quantification by densitometry using Image Lab software. Vinculin was taken as loading controls. Values are the mean±SEM (n=2 mice/group). (E) In situ terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) assay for determination of apoptosis in tumor sections from control and treated mice (n=3 mice/group). (F) For quantification of FITC positive (TUNEL) cells, Image J software was used. Magnification=20×. Values are the mean±SEM, ***p<0.0001 values treatment versus control by one-way analysis of variance, Bonferroni's multiple comparison test (n=3 mice/group). (G) Change in tumor volume (day 13 to day 0) in control and treated mice. Values are the mean±SEM, p value (*p<0.05 and **p<0.001) for combination of URMC-099 and αCD70 mAb versus URMC-099 alone or anti-CD70 alone treatment by unpaired t test (n=5 mice/group, except isotype treatment where n=4 mice/group). (H) Change in tumor volume (ie, day 13 to day 0) in CD8 + T cell depleted mice, control or treated with: isotype, anti-CD70, URMC-099 and combination of anti-CD70+URMC−099. Values are the mean±SEM (n=3 mice/group). (I) Protein expressions of Bax and Bcl-2 in tumor lysates, isolated from control, and treated (as indicated in figure 7H above) mice. Vinculin was taken as loading controls (n=3 mice/group). MLK3, mixed lineage kinase 3.

Article Snippet: The in vivo blocking of CD70 was achieved using anti-mouse CD70 monoclonal antibody (mAb; 100 μg/mouse; BioXcell).

Techniques: Confocal Microscopy, Expressing, Control, Generated, Isolation, Software, In Situ, TUNEL Assay, Comparison

Journal: Neuro-Oncology

Article Title: CD70, a novel target of CAR T-cell therapy for gliomas

doi: 10.1093/neuonc/nox116

Figure Lengend Snippet: Overexpression of CD70 on primary and recurrent gliomas, but not on tumor infiltrating T cells. (A–B) Differential CD70 gene expression in normal brain, primary and recurrent LGG, and GBM. The gene expression level was defined as RSEM (RNA-seq by expectation-maximization), and patient’s informations were culled from TCGA RNA-seq datasets. (C) Protein level of CD70 was evaluated by immunohistochemistry using tumors from primary LGGs (n = 41), GBMs (n = 44), and normal brains (n = 7). (D) CD70 protein expression between primary and recurrent GBMs. Tumors from 7 paired GBMs before and after recurrence were evaluated. (E) The CD70 positivity within each CD70-expressing primary GBM. (F) Determination of CD70 expression on tumor and T cells. Seven CD70+ GBMs were co-stained with fluorescent conjugated CD3 (green) and CD70 (red) antibodies; 4′,6′-diamidino-2-phenylindole was used for nuclear staining. Representative pictures (with an enlarged overlaid area) from one tumor are shown. Mann–Whitney U test was used.

Article Snippet: CD70 was assessed using the mouse monoclonal anti-CD70 antibody (Santa Cruz).

Techniques: Over Expression, Gene Expression, RNA Sequencing, Immunohistochemistry, Expressing, Staining, MANN-WHITNEY

Journal: Neuro-Oncology

Article Title: CD70, a novel target of CAR T-cell therapy for gliomas

doi: 10.1093/neuonc/nox116

Figure Lengend Snippet: CD70 positivity and CD3+ infiltration in resected tumor from primary and recurrent gliomas a

Article Snippet: CD70 was assessed using the mouse monoclonal anti-CD70 antibody (Santa Cruz).

Techniques: Expressing

Journal: Neuro-Oncology

Article Title: CD70, a novel target of CAR T-cell therapy for gliomas

doi: 10.1093/neuonc/nox116

Figure Lengend Snippet: CD70 gene expression is inversely correlated with overall survival in patients with primary gliomas. (A–B) CD70 expression among different subgroups of LGGs and GBMs. (C–F) Predicted median overall survival decreases with increasing CD70 expression in primary LGGs, IDH wild-type LGGs, GBMs, and GBMs in the mesenchymal subgroup (CD70 RNA-seq expression was considered as a continuous predictor of survival, adjusted for gender and age in all subgroup models using Cox proportional hazards (PH) regression; further adjustment was made for tumor subtype in the GBM subgroup model). (G) Summary of differential median overall survival between CD70 high and low groups defined by mean values of CD70 RSEM in LGGs and GBMs (subgroup median survival estimates adjusted for age, gender and GBM tumor subtype via Cox PH regression).

Article Snippet: CD70 was assessed using the mouse monoclonal anti-CD70 antibody (Santa Cruz).

Techniques: Gene Expression, Expressing, RNA Sequencing

Journal: Neuro-Oncology

Article Title: CD70, a novel target of CAR T-cell therapy for gliomas

doi: 10.1093/neuonc/nox116

Figure Lengend Snippet: CD70 involved in cytokine/chemokine productions in GBM. (A–B) CD70 overexpression influences the cytokine/chemokine pathway in a primary GBM line. Triplicated RNA samples from pGBM#3 (CD70+) were transduced with CD70 (Over-exp) or lentiviral vector control (blank), confirmed by gene (fragments per kilobase of transcript per million mapped reads, FPKM) and protein expression (flow cytometry). Gene enrichment of the upregulated gene list (Over-exp vs blank) was performed by PANTHER-Pathway; a volcano plot and the top-ranked pathways are shown. (C) Primary GBM lines secrete various chemokines. Culture supernatants from the primary lines were collected 8 days after the tumors were seeded, and chemokine array was performed. (D–G) Gene expression changes between overexpression and silence of CD70 in pGBM#3 were evaluated by real-time quantitative PCR. (H) IL-8 secretion was measured by enzyme-linked immunosorbent assay. Unpaired t-test was used.

Article Snippet: CD70 was assessed using the mouse monoclonal anti-CD70 antibody (Santa Cruz).

Techniques: Over Expression, Transduction, Plasmid Preparation, Control, Expressing, Flow Cytometry, Gene Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Neuro-Oncology

Article Title: CD70, a novel target of CAR T-cell therapy for gliomas

doi: 10.1093/neuonc/nox116

Figure Lengend Snippet: CD70 is associated with T-cell infiltration and CD8+ T-cell death in GBM. (A) Relative higher numbers of infiltrating CD3+ T cells in CD70+ compared with CD70− tumors. Paraffin-embedded GBM tumor samples described in Fig. 1 were analyzed by immunohistochemistry for CD70 and CD3, respectively, using serial slides. (B) CD70 predominantly induces CD8+ T-cell death after engaging tumor cells. CD70-manipulated pGBM#3 cells were cocultured with 3 allogeneic PBMCs obtained from healthy donors. Fluorescence activated cell sorting (FACS) analysis was performed 14 days after the coculture (left). The cell death of T-cell subsets was quantitated by propidium iodide (PI) staining for both CD4+ and CD8+ T cells (right). These results were representative of 4 individual experiments. Mann–Whitney test was used to assess statistical significance. (C) The ratio of CD4+ to CD8+ T cells among the cocultured T cells. FACS analysis from (B) was also evaluated by CD4+ to CD8+ ratio, and the gating strategy is shown in the upper panel. Paired t-test was used to assess statistical significance.

Article Snippet: CD70 was assessed using the mouse monoclonal anti-CD70 antibody (Santa Cruz).

Techniques: Immunohistochemistry, Fluorescence, FACS, Staining, MANN-WHITNEY

Journal: Neuro-Oncology

Article Title: CD70, a novel target of CAR T-cell therapy for gliomas

doi: 10.1093/neuonc/nox116

Figure Lengend Snippet: Tumor recognitions of gliomas by human and mouse CD70 CAR T cells. (A) Human CAR construct and its transduction efficiency in human T cells. The transduction efficiency was measured by tdTomato positive cells and the frequency of CD4+/CD8+ T cells 4 days posttransduction are shown. (B) GBM targets for hCAR T cells. GBM lines naturally expressing various levels of CD70 were tested for the CD70 surface expression by FACS. (C–D) Recognition of these lines by hCAR T cells. The recognitions were measured by IFN-γ release and cytotoxic killing assay. (E–G) Manipulation of tumor CD70 expression alters the CAR T cells recognition. The tumor lines were cocultured with manipulated GBM lines by either overexpressing or silencing CD70. (H) Irradiation enhances tumor CD70 expression and CAR T-cell recognition. The U87 line was irradiated with 6 Gy, CD70 expression was determined by FACS on day 3 and then cocultured with CAR T cells. (I) Transduction of mCD70 CAR to mouse T cells. Construct of mouse CD70-specific CAR (mCD70-CAR), transduction efficiency determined by tdTomato and frequency of CD4+ /CD8+ T cells in the mCAR T cells 4 days posttransduction are shown. (J–M) CD70-overexpressed murine glioma lines were recognized by mCD70-CAR T cells. The tumors (104) were cocultured with (104) mCD70-CAR T cells or vector/NT control T cells, respectively. For the killing assay, various ratios of effector to target (E:T) were respectively cocultured. Unpaired t-test was used.

Article Snippet: CD70 was assessed using the mouse monoclonal anti-CD70 antibody (Santa Cruz).

Techniques: Construct, Transduction, Expressing, Irradiation, Plasmid Preparation, Control

Journal: Neuro-Oncology

Article Title: CD70, a novel target of CAR T-cell therapy for gliomas

doi: 10.1093/neuonc/nox116

Figure Lengend Snippet: Antitumor response of CD70 CAR T cells against CD70+ gliomas in vivo. (A) Antitumor response of hCAR T cells in human xenograft glioma model. CD70+ (U87.Luc, 5 × 104/mouse) tumor-bearing (confirmed by imaging 6 days after tumor inoculation) mice were adoptively transferred through tail-vein injection with NT or various doses (105–107/mouse, 8/group) of hCAR T cells 7 days after the tumor implantation. (B–C) A complete response and prolonged survival induced by mCAR T cells in syngeneic glioma models. Two models were used: groups of 6–8 weeks C57BL/6J mice (10/group) were intracranially inoculated with 1 × 105 KR70 tumor cells derived from a tumor clone, designated as KR70-C, and then adoptively transferred (1 × 107) NT or mCAR T cells on days 5 and 7 post tumor implantation. Luciferase imaging was carried out 10 and 17 days after the tumor implantation. The same experiment described in (B) was performed, except the inoculated tumor cells were derived from bulk tumors, KR70-B. All experiments were repeated at least 2 times. Mann–Whitney test and the log-rank test were used.

Article Snippet: CD70 was assessed using the mouse monoclonal anti-CD70 antibody (Santa Cruz).

Techniques: In Vivo, Imaging, Injection, Tumor Implantation, Derivative Assay, Luciferase, MANN-WHITNEY