Journal: Blood Advances

Article Title: TCRVβ-targeting antibody-drug conjugates as a novel strategy to eliminate malignant T cells in T cell cancers

doi: 10.1182/bloodadvances.2026020282

Figure Lengend Snippet: Anti-TCR Vβ2-PNU ADC inhibits tumor growth in vivo. (A) Representative flow cytometric plot showing percentage of viable MOLT-16 (T-ALL) cells after treatment for 72 hours with 1 μg/mL of either anti-TCR Vβ2-PNU or hIgG1-PNU or an unconjugated anti-TCR Vβ2 antibody. (B) Graph showing percentage of viable MOLT-16 (T-ALL) cells after treatment for 72 hours with 1 μg/mL of either anti-TCR Vβ2-PNU or hIgG1-PNU or an unconjugated anti-TCR Vβ2 antibody. n = 3 biological replicates. Ordinary 1-way ANOVA was performed, and data are shown as mean ± s.e.m.; P < .001. (C) Schematic showing timeline of mouse experiment. Treatments were administered IV. Figure created with biorender.com . Vadivel, C. K. (2026) https://BioRender.com/csk02dd . (D) NSG mice were injected subcutaneously with MOLT-16 cells (day 0). Mice were randomized into 4 treatment groups: PBS (n = 7), anti-TCR Vβ2 antibody (n = 8), hIgG1-PNU (n = 8), or anti-TCR Vβ2-PNU ADC (n = 8), all dosed at 0.75 mg/kg. Treatments were administered as IV injections in the tail on day 7, day 14, and day 21. Representative images show tumor growth in the treatment groups (white-dotted circles), except treatment with anti-TCR Vβ2-PNU ADC (black-dotted region). (E) Kaplan-Meier survival curves of MOLT-16–bearing NSG mice, n = 8 (TCR Vβ2-PNU, hIgG1-PNU, anti-TCR Vβ2 antibody) and n = 7 for PBS. The median survival was as follows: 20 days (PBS), 19 days (anti-TCR Vβ2 antibody), 18 days (hIgG1-PNU), and end point not reached (undefined; anti-TCR Vβ2-PNU ADC). Statistical analysis was performed using the log-rank Mantel-Cox test; P < .0001. (F) Cells were extracted from tumors of MOLT-16–bearing NSG mice. Flow cytometric plots showing percentage of viable extracted tumor cells after treatment for 72 hours with either anti-TCR Vβ2-PNU or mIgG1-PNU or an unconjugated anti-TCR Vβ2 antibody at the indicated concentration. ANOVA, analysis of variance; s.e.m., standard error of the mean; T-ALL, T-cell acute lymphoblastic leukemia.

Article Snippet: We purchased anti-TCR Vβ2 custom antibody (REA654/MPB2D5) from Miltenyi Biotec.

Techniques: In Vivo, Injection, Concentration Assay

Journal: Blood Advances

Article Title: TCRVβ-targeting antibody-drug conjugates as a novel strategy to eliminate malignant T cells in T cell cancers

doi: 10.1182/bloodadvances.2026020282

Figure Lengend Snippet: Generated anti-TCR Vβ2-PNU ADC kills malignant T cells in vitro. (A) Schematic overview of the generation of anti-TCR Vβ2-PNU ADC (detailed in “Methods”). The cytotoxic drug PNU-159682 conjugated to the antibody is highlighted in yellow. Chemical structure created with molview.org PubChem Identifier: CID 145712370. Figure created with biorender.com . Vadivel, C. K. (2026) ( https://BioRender.com/2nax8xf ). (B) Efficiency of the PNU conjugation was assessed on a Coomassie-stained SDS-PAGE gel. The complete upward shift in MW of the heavy chain (∼50 kDa) of the ADCs (IgG1-PNU and TCR Vβ2-PNU) compared to the azide-activated antibodies (IgG1-az and TCR Vβ2-az) confirms that both ADCs are homogeneous, with a DAR of 2. (C) Flow cytometric plot showing percentage of CD3 + CD4 + TCR Vβ2 + malignant cells in PBMCs from a patient with leukemic CTCL (L-CTCL). (D) Flow cytometric plot showing viable malignant cells from a patient with TCR Vβ2 + L-CTCL after treatment with either anti-TCR Vβ2-PNU or IgG1-PNU or an unconjugated anti-TCR Vβ2 antibody at the indicated concentration for 72 hours. (E) Representative flow cytometric plots showing the percentage of viable malignant cells in an additional patient with TCR Vβ2 + L-CTCL after treatment with 2 μg/mL of either anti-TCR Vβ2-PNU or hIgG1-PNU or an unconjugated anti-TCR Vβ2 antibody for 72 hours. (F) Graph showing percentage of viable cells relative to PBS control in malignant cells from 3 patients with TCR Vβ2 + L-CTCL after treatment with 2 μg/mL of either anti-TCR Vβ2-PNU or hIgG1-PNU or an unconjugated anti-TCR Vβ2 antibody for 72 hours. Ordinary 1-way ANOVA was performed, and data are shown as mean ± s.e.m.; P < .001. (G) Representative flow cytometric plots showing percentage of viable malignant cells in a patient with TCR Vβ2 - CTCL after treatment with 2 μg/mL of either anti-TCR Vβ2-PNU or hIgG1-PNU or an unconjugated anti-TCR Vβ2 antibody for 72 hours. (H) Graph showing percentage of viable cells relative to PBS control in malignant cells from 2 patients with TCR Vβ2 - L-CTCL after treatment with 2 μg/mL of either anti-TCR Vβ2-PNU or hIgG1-PNU or an unconjugated anti-TCR Vβ2 antibody for 72 hours. Ordinary 1-way ANOVA was performed, and data are shown as mean ± s.e.m.; p = ns. ANOVA, analysis of variance; DAR, drug-to-antibody ratio; kDa, kilodalton; MW, molecular weight; ns, nonsignificant; s.e.m., standard error of the mean.

Article Snippet: We purchased anti-TCR Vβ2 custom antibody (REA654/MPB2D5) from Miltenyi Biotec.

Techniques: Generated, In Vitro, Conjugation Assay, Staining, SDS Page, Concentration Assay, Control, Molecular Weight

Journal: Blood Advances

Article Title: TCRVβ-targeting antibody-drug conjugates as a novel strategy to eliminate malignant T cells in T cell cancers

doi: 10.1182/bloodadvances.2026020282

Figure Lengend Snippet: TCRVβ2 is internalized by TCR Vβ2 + cells. (A) Bar plot showing the frequency of TCR Vβ2 (TRBV20-1), the most common TCRVβ (TRBV) among others in malignant T cells, in publicly available single-cell RNA sequencing data from 104 patients with CTCL. (B) Anti-TCR Vβ2-pHrodo antibody (1 μg/mL) was added to MOLT-16 (TCR Vβ2 + cells) or MyLa 2059 (TCR Vβ2 − cells). Quantification of red fluorescence over time after the addition of anti-TCR Vβ2-pHrodo antibodies to indicated cells. Data are mean ± s.e.m. of 3 technical replicates. n = 2 biological replicates. (C) MOLT-16 cells were incubated with anti-TCR Vβ2 antibody (1 μg/mL) and then analyzed using confocal microscopy. LAMP1 antibodies and DAPI (4′,6-diamidino-2-phenylindole) staining mark lysosomes and nuclei, respectively. Antihuman IgG–Alexa Fluor 488 was used to detect the location of anti-TCR Vβ2 antibodies. Scale bars, 200 μm. n = 2 biological replicates. h, hour; LAMP1, lysosomal-associated membrane protein 1; min, minutes; s.e.m, standard error of the mean.

Article Snippet: We purchased anti-TCR Vβ2 custom antibody (REA654/MPB2D5) from Miltenyi Biotec.

Techniques: Single Cell, RNA Sequencing, Fluorescence, Incubation, Confocal Microscopy, Staining, Membrane

Journal: Science Advances

Article Title: Reprogramming of valine metabolism mediated by abnormally low ALDH6A1 expression promotes invasive metastasis of gastric cancer

doi: 10.1126/sciadv.aeb2892

Figure Lengend Snippet: ( A and B ) Heatmaps (A) and average profiles (B) of H3K4me2 ChIP-seq signals within ±1 kb (A) or ± 3 kb (B) of TSSs are shown for control and MMA-treated AGS cells (4 mM, 10 days). ( C ) Venn diagram intersecting genes significantly up-regulated by RNA-seq ( P < 0.05, log 2 FC ≥ 1) with genes exhibiting increased H3K4me2 after MMA treatment. ( D ) Integrative Genomics Viewer tracks illustrating H3K4me2 enrichment (red, MMA; blue, control) around the MMP7 and ANGPT2 promoters; bar charts on the right show the corresponding mRNA fold changes. ( E and F ) ChIP-qPCR confirms elevated H3K4me2 at the ANGPT2 and MMP7 promoters after ALDH6A1-KO (E) or MMA exposure (F) ( n = 3). ( G and H ) RT-qPCR showing that ALDH6A1 loss (G) or MMA treatment (H) up-regulates ANGPT2 and MMP7 transcripts ( n = 3). ( I ) Western blot analysis demonstrating changes in ANGPT2 and MMP7 proteins after ALDH6A1-KO (KO) or ALDH6A1-OE (OE). ( J ) Dose-dependent induction of ANGPT2 and MMP7 proteins by increasing MMA concentrations. Data are means ± SD. Statistical significance was assessed by the two-tailed Student’s t test. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For rescue experiments, α-KG (MedChemExpress, catalog no. HY-W013636) was added to MMA-treated cultures at a final concentration of 5 mM and coincubated for the same 10-day period.

Techniques: ChIP-sequencing, Control, RNA Sequencing, ChIP-qPCR, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Science Advances

Article Title: Reprogramming of valine metabolism mediated by abnormally low ALDH6A1 expression promotes invasive metastasis of gastric cancer

doi: 10.1126/sciadv.aeb2892

Figure Lengend Snippet: ( A ) RT-qPCR analysis of representative H3K4 histone methyltransferases (H3K4 HMTs; KMT2A , KMT2B , and SETD1A ) in AGS cells with ALDH6A1-KO or MMA treatment (10 mM, 10 days) ( n = 3). ( B ) Global H3K4 HMT activity under the same conditions ( n = 3). ( C ) RT-qPCR analysis of H3K4 HDMs ( KDM1A , KDM5A , KDM5B , and KDM5C ). ( D ) Global H3K4 HDM activity under the same conditions ( n = 3). ( E to I ) Representative Transwell migration and Matrigel invasion images (E) and quantification of invaded and migrated AGS (F and G) and HGC-27 cells (H and I) in WT, ALDH6A1-KO, and ALDH6A1-KO cells reexpressing KDM5A, KDM5B, or KDM5C. Scale bars, 100 μm. ( J ) Western blot analysis of EMT markers and H3K4me2 in the same cells. ( K to O ) Representative Transwell migration and Matrigel invasion images (K) and quantification of invaded and migrated AGS (L and M) and HGC-27 cells (N and O) after MMA treatment with reexpression of KDM5A, KDM5B, or KDM5C. Scale bars, 100 μm. ( P ) Western blot analysis showing that KDM5C, but not KDM5A or KDM5B, reduced EMT markers and H3K4me2 in MMA-treated cells. Data are means ± SD. Statistical significance was determined by the one-way ANOVA with post hoc multiple comparisons. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For rescue experiments, α-KG (MedChemExpress, catalog no. HY-W013636) was added to MMA-treated cultures at a final concentration of 5 mM and coincubated for the same 10-day period.

Techniques: Quantitative RT-PCR, Activity Assay, Migration, Western Blot

Journal: Science Advances

Article Title: Reprogramming of valine metabolism mediated by abnormally low ALDH6A1 expression promotes invasive metastasis of gastric cancer

doi: 10.1126/sciadv.aeb2892

Figure Lengend Snippet: ( A to C ) ITC analysis of KDM5C binding to MMA (A), α-KG (B), and a premixed MMA + α-KG solution (C). MMA bound KDM5C with a higher affinity ( K d = 7.22 × 10 −5 M) than α-KG ( K d = 6.69 × 10 −4 M), and the presence of MMA altered the apparent α-KG–binding profile ( K d = 2.03 × 10 −4 M). CI, confidence interval. ( D ) Docking model based on the AlphaFold 3 showing the overlap of MMA and α-KG within the KDM5C catalytic pocket (E516 and N616). ( E to I ) Representative Transwell migration and Matrigel invasion images (E) and quantification of invaded and migrated AGS (F and G) and HGC-27 cells (H and I) in WT, ALDH6A1-KO, and ALDH6A1-KO cells supplemented with α-KG (5 mM, 10 days). Scale bars, 100 μm. ( J ) Western blot analysis showing that α-KG supplementation reversed the enhanced EMT and H3K4me2 signature induced by ALDH6A1-KO. ( K to O ) Representative Transwell migration and Matrigel invasion images (K) and quantification of invaded and migrated AGS (L and M) and HGC-27 cells (N and O) treated with MMA (10 mM) in the absence or presence of α-KG supplementation (5 mM) for 10 days. Scale bars, 100 μm. ( P ) Western blot analysis showing that α-KG abolished the pro-invasive phenotype and H3K4me2 accumulation induced by MMA. ( Q and R ) ChIP-qPCR analysis showing that α-KG reduced H3K4me2 enrichment at the ANGPT2 and MMP7 promoters in ALDH6A1-KO and MMA-treated cells ( n = 3). Data are means ± SD. Statistical significance was determined by one-way ANOVA with post hoc multiple comparisons for (F) to (I) and (L) to (O) and by two-way ANOVA for (Q) and (R). ** P < 0.01 and *** P < 0.001.

Article Snippet: For rescue experiments, α-KG (MedChemExpress, catalog no. HY-W013636) was added to MMA-treated cultures at a final concentration of 5 mM and coincubated for the same 10-day period.

Techniques: Binding Assay, Migration, Western Blot, ChIP-qPCR

Journal: Science Advances

Article Title: Reprogramming of valine metabolism mediated by abnormally low ALDH6A1 expression promotes invasive metastasis of gastric cancer

doi: 10.1126/sciadv.aeb2892

Figure Lengend Snippet: ( A and B ) Representative bioluminescence images (A), gross liver morphology, and hematoxylin and eosin (H&E) staining (B) from mice injected with gastric cancer cells of the indicated genotypes or treatments, including WT, ALDH6A1-OE, ALDH6A1-KO, ALDH6A1-KO and dm-α-KG, and ALDH6A1-KO and l -carnitine. Boxed regions indicate higher-magnification views. ( C and D ) Representative bioluminescence images (C), gross liver morphology, and H&E staining (D) from mice injected with control or MMA-treated cells with or without dm-α-KG. ( E and F ) Quantification of total bioluminescence signals from (A) and (C), respectively ( n = 6). ( G ) LC-MS/MS measurement of circulating MMA in mouse serum from the indicated groups ( n = 5 to 6). ( H and I ) Western blot analysis of EMT/invasion-associated markers and H3K4me2 in metastatic tissues from the groups in (A) and (C), respectively. ( J and K ) ChIP-qPCR analysis showing reduced H3K4me2 enrichment at the ANGPT2 and MMP7 promoters after dm-α-KG or l -carnitine treatment in ALDH6A1-KO (J) and MMA-treated (K) liver metastases. ( L to N ) Representative IVIS images (L), gross liver morphology, H&E staining, H3K4me2 IHC (M), and photon flux quantification (N) from mice treated with Alda-1 or l -carnitine after splenic implantation of WT AGS cells ( n = 6 per group). DMSO, dimethyl sulfoxide. ( O ) LC-MS/MS measurement of serum MMA after Alda-1 or l -carnitine treatment ( n = 6). ( P ) ChIP-qPCR analysis of H3K4me2 enrichment at ANGPT2 and MMP7 promoters in metastatic tumors ( n = 6). ( Q ) Western blot analysis of EMT markers and H3K4me2 in liver metastases. Data are means ± SD. Statistical significance was determined by the one-way ANOVA with post hoc multiple comparisons. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For rescue experiments, α-KG (MedChemExpress, catalog no. HY-W013636) was added to MMA-treated cultures at a final concentration of 5 mM and coincubated for the same 10-day period.

Techniques: Staining, Injection, Control, Liquid Chromatography with Mass Spectroscopy, Western Blot, ChIP-qPCR