Journal: bioRxiv

Article Title: A targetable dependency on nonsense-mediated decay for proteostasis and immune control in small cell lung cancer

doi: 10.64898/2026.03.31.715503

Figure Lengend Snippet: A) DNA Sanger sequencing of the amplified B2M locus in representative H841 CRISPR/Cas9 WT and B2M-KO clones aligned to the reference sequence (NCBI, NG_012920.2) with sgRNA and the identified insertion mutation in H841 B2M-KO cells highlighted. B-D) Pharmacological NMD inhibition with either KVS0001 or SMG1i-11j compounds in healthy PBMC donors upon T cell artificial activation. ( B ) Proportion of viable CD45+ cells (left) and T cells (right) within PBMC populations after 4 days treatment with a T cell artificial activation cocktail (Act = CD3+CD28+IL-2) vs unstimulated conditions (Naive). ( C ) Proliferation of CD4+ (left) and CD8+ (right) T cells derived from T cell counts expressed as fold change (FC) for activated (Act) conditions relative to the unstimulated (Naive) control. ( D ) Proportion of T cells (CD3+), B cells (CD19+), NK cells (CD56+) and Myeloid cells (CD11b+) within PBMC populations (CD45+) from healthy donors directly after thawing (d-1), at the beginning of stimulation (CD3+CD28+IL-2) (d0) 4 days post-stimulation (d4). Graphs represent mean + SEM (n = 7). ***P<0,001; **P<0.01; *P<0.05; ns=non-significant (One-way ANOVA). E) Representative flow cytometry dot plots for the staining of CD45 vs CD56 (for NK cells) and CD8 vs CD4 (both for T cells) following 4 days co-culture of healthy donor PBMCs with WT H841 tumor cells. F) In vivo tumor growth in immunocompetent C57BL/6J mice transplanted with murine RP1380 TetO-shCTRL fed with normal or doxycycline-containing diet (n ≥ 4). G) Flow cytometry immunophenotyping of RP1380 TetO-shCTRL tumors harvested at the end of experiments shown in panel F (n ≥ 4). H-I) MHC-I surface expression quantified by flow cytometry in the indicated human and murine cell lines following genetic NMD inhibition via siRNA-mediated SMG1-KD and UPF1-KD ( H ) or doxycycline-inducible SMG1-KD ( I ). J) In vivo assessment of MHC-I (H2-Kb) surface expression in control vs NMD-inhibited murine RP1380 TetO-shSMG1 tumors (following doxycycline diet, DOXY) grown subcutaneously in C57BL/6 mice. K) Flow cytometry data for murine RP1380 TetO-shSMG1 allograft models grown in C57BL/6 mice significantly correlating tumor-specific H2-Kb surface expression with levels of immune cell infiltration (CD45+ infiltration). L) In vivo levels of tumor-specific MHC-I (H2-Kb) surface expression in control vs NMD-inhibited RP1380 TetO-shSMG1 tumors (DOXY) grown subcutaneously in RAG1-KO C57BL/6 mice. K) Flow cytometry data for murine RP1380 TetO-shSMG1 allograft models grown in RAG1-KO C57BL/6 mice correlating tumor-specific H2-Kb surface expression with levels of immune cell (CD45+) infiltration. L) Representative flow cytometry contour plot showing T cells (CD45+/CD3+) and B cells (CD45+/CD19+) in the blood from WT C57BL/6 and RAG1-KO C57BL/6 mice.

Article Snippet: Murine primary antibodies used were CD45 (Miltenyi #130110665), CD3 (Miltenyi #130119793), Nkp46 (BD Biosciences #3122669), CD19 (Miltenyi #130112037), CD11b (Miltenyi #1301138063), CD14 (Miltenyi #130115559), H2-Kb (Miltenyi #130115586), IFN-γ (ThermoFisher #48731182), KI-67 (Miltenyi #130120418).

Techniques: Sequencing, Amplification, CRISPR, Clone Assay, Mutagenesis, Inhibition, Activation Assay, Derivative Assay, Control, Flow Cytometry, Staining, Co-Culture Assay, In Vivo, Expressing

Journal: bioRxiv

Article Title: A targetable dependency on nonsense-mediated decay for proteostasis and immune control in small cell lung cancer

doi: 10.64898/2026.03.31.715503

Figure Lengend Snippet: A) DNA Sanger sequencing of the amplified B2M locus in representative H841 CRISPR/Cas9 WT and B2M-KO clones aligned to the reference sequence (NCBI, NG_012920.2) with sgRNA and the identified insertion mutation in H841 B2M-KO cells highlighted. B-D) Pharmacological NMD inhibition with either KVS0001 or SMG1i-11j compounds in healthy PBMC donors upon T cell artificial activation. ( B ) Proportion of viable CD45+ cells (left) and T cells (right) within PBMC populations after 4 days treatment with a T cell artificial activation cocktail (Act = CD3+CD28+IL-2) vs unstimulated conditions (Naive). ( C ) Proliferation of CD4+ (left) and CD8+ (right) T cells derived from T cell counts expressed as fold change (FC) for activated (Act) conditions relative to the unstimulated (Naive) control. ( D ) Proportion of T cells (CD3+), B cells (CD19+), NK cells (CD56+) and Myeloid cells (CD11b+) within PBMC populations (CD45+) from healthy donors directly after thawing (d-1), at the beginning of stimulation (CD3+CD28+IL-2) (d0) 4 days post-stimulation (d4). Graphs represent mean + SEM (n = 7). ***P<0,001; **P<0.01; *P<0.05; ns=non-significant (One-way ANOVA). E) Representative flow cytometry dot plots for the staining of CD45 vs CD56 (for NK cells) and CD8 vs CD4 (both for T cells) following 4 days co-culture of healthy donor PBMCs with WT H841 tumor cells. F) In vivo tumor growth in immunocompetent C57BL/6J mice transplanted with murine RP1380 TetO-shCTRL fed with normal or doxycycline-containing diet (n ≥ 4). G) Flow cytometry immunophenotyping of RP1380 TetO-shCTRL tumors harvested at the end of experiments shown in panel F (n ≥ 4). H-I) MHC-I surface expression quantified by flow cytometry in the indicated human and murine cell lines following genetic NMD inhibition via siRNA-mediated SMG1-KD and UPF1-KD ( H ) or doxycycline-inducible SMG1-KD ( I ). J) In vivo assessment of MHC-I (H2-Kb) surface expression in control vs NMD-inhibited murine RP1380 TetO-shSMG1 tumors (following doxycycline diet, DOXY) grown subcutaneously in C57BL/6 mice. K) Flow cytometry data for murine RP1380 TetO-shSMG1 allograft models grown in C57BL/6 mice significantly correlating tumor-specific H2-Kb surface expression with levels of immune cell infiltration (CD45+ infiltration). L) In vivo levels of tumor-specific MHC-I (H2-Kb) surface expression in control vs NMD-inhibited RP1380 TetO-shSMG1 tumors (DOXY) grown subcutaneously in RAG1-KO C57BL/6 mice. K) Flow cytometry data for murine RP1380 TetO-shSMG1 allograft models grown in RAG1-KO C57BL/6 mice correlating tumor-specific H2-Kb surface expression with levels of immune cell (CD45+) infiltration. L) Representative flow cytometry contour plot showing T cells (CD45+/CD3+) and B cells (CD45+/CD19+) in the blood from WT C57BL/6 and RAG1-KO C57BL/6 mice.

Article Snippet: Murine primary antibodies used were CD45 (Miltenyi #130110665), CD3 (Miltenyi #130119793), Nkp46 (BD Biosciences #3122669), CD19 (Miltenyi #130112037), CD11b (Miltenyi #1301138063), CD14 (Miltenyi #130115559), H2-Kb (Miltenyi #130115586), IFN-γ (ThermoFisher #48731182), KI-67 (Miltenyi #130120418).

Techniques: Sequencing, Amplification, CRISPR, Clone Assay, Mutagenesis, Inhibition, Activation Assay, Derivative Assay, Control, Flow Cytometry, Staining, Co-Culture Assay, In Vivo, Expressing

Journal: Cancer Research Communications

Article Title: Enhancement of CD117-Targeted Bispecific T-cell Engagement by CD33-Targeted Bispecific T-cell Costimulation in Acute Myeloid Leukemia

doi: 10.1158/2767-9764.CRC-25-0672

Figure Lengend Snippet: Characterization of CD33xCD28 IgG4-scFv 2 TCE. A, Illustration of a combination of a bispecific TCE targeting TAA1 (CD117) on tumor cells and CD3ε on T cells with a bispecific TCE targeting TAA2 (CD33) on tumor cells and CD28 on T-cells. B, Plasmid map of the CD33xCD28 IgG4-scFv 2 construct. C, Protein structure of the CD33xCD28 IgG 4 -scFv 2 construct. D–F, CD33xCD28 IgG4-scFv 2 analysis by mass spectrometry in nonreduced ( D ) and reduced ( E and F ) conditions. G, Size-exclusion chromatography of CD33xCD28 IgG4-scFv 2 . H, SDS-page analysis of CD33xCD28 IgG4-scFv 2 under nonreducing (NR) and reducing (R) conditions M = protein ladder indicating the molecular size (kDa). I, Binding of CD33xCD28 IgG4-scFv 2 to CD33 on MOLM-14 CD117 High GFP + Luc + cells. J, Binding of CD33xCD28 IgG4-scFv 2 to CD28 on peripheral blood T cells. Binding capacity was assessed by the titration of the bispecific antibody and detected by anti-human IgG antibody. MFI was normalized to background fluorescence. Apparent K D was calculated by nonlinear regression. Mean ± SD from three independent experiments, each plated in duplicates.

Article Snippet: In selected cases, a CD3 + /CD19 + double depletion was performed using human CD3 and CD19 MicroBeads (Miltenyi Biotec, cat. #130-136-718), following the manufacturer’s protocol.

Techniques: Plasmid Preparation, Construct, Mass Spectrometry, Size-exclusion Chromatography, SDS Page, Binding Assay, Titration, Fluorescence

Journal: Cancer Research Communications

Article Title: Enhancement of CD117-Targeted Bispecific T-cell Engagement by CD33-Targeted Bispecific T-cell Costimulation in Acute Myeloid Leukemia

doi: 10.1158/2767-9764.CRC-25-0672

Figure Lengend Snippet: CD33xCD28 IgG4-scFv 2 in combination with CD117xCD3 TCE mediates more effective lysis of primary AML cells. A, Representative flow cytometry plots showing CD117 and CD33 expression on four different primary human AML blast populations (CD45dim) upon coculture with healthy donor–derived T cells at an E:T ratio of approximately 1:1. Cells were incubated with antibody constructs as indicated (CD117xCD3 at the indicated concentrations, in combination with 0.5 nmol/L CD33xCD28 IgG4-scFv 2 or 0.5 nmol/L CD28 IgG4). Plots are shown for 0 and 48 hours. B, Percentage of specific lysis of CD45 dim CD3 − AML blasts of the individual patient samples after 48 hours. C, Combined percentage of CD25 + T cells. D, Combined IFNγ in supernatants of coculture. E, Combined proliferation of T cells. Data represent the mean ± SEM from two independent healthy donor–derived T-cell samples, each plated in duplicate. Statistical significance was determined using two-way ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: In selected cases, a CD3 + /CD19 + double depletion was performed using human CD3 and CD19 MicroBeads (Miltenyi Biotec, cat. #130-136-718), following the manufacturer’s protocol.

Techniques: Lysis, Flow Cytometry, Expressing, Derivative Assay, Incubation, Construct