Journal: bioRxiv

Article Title: L1CAM-CAR T cells with enhanced potency overcome low-density antigen expression in rhabdomyosarcoma

doi: 10.64898/2026.02.27.707949

Figure Lengend Snippet: (A) Western blot of L1CAM (∼200 kDa) in RMS PDXs and FN-/FP-RMS cell lines; α -tubulin served as loading control. Numbers below lanes indicate relative intensities normalized to α-tubulin. (B) Flow cytometry of FN- and FP-RMS cell lines and controls (gray isotype). Antigen density estimated with Quantibrite-PE calibration. Data representative of three independent experiments. (C) IHC of orthotopic xenografts (RD, Rh30) with L1CAM staining (brown) and hematoxylin counterstain (blue). Negative-control sections without primary antibody. Scale bars, 50 µm. (D-E) Tissue microarray of 34 primary RMS PDXs stained for L1CAM (brown), with triplicate tumor cores per case. The array included 36 cores from 34 xenografts; cores A9/C7 and A6/B2 were duplicates. (E) H-score quantification of L1CAM expression per case. Expression categorized as High (≥150), Medium (51-149), or Low (≤50). Subtypes color-coded (aRMS, green; eRMS, blue).

Article Snippet: 48 h later, cells were sorted for mouse L1CAM expression using PE- conjugated anti-mouse CD171 (L1CAM) antibody (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany) to have a homogenous population.

Techniques: Western Blot, Control, Flow Cytometry, Staining, Negative Control, Microarray, Expressing

Journal: bioRxiv

Article Title: L1CAM-CAR T cells with enhanced potency overcome low-density antigen expression in rhabdomyosarcoma

doi: 10.64898/2026.02.27.707949

Figure Lengend Snippet: (A) Flow cytometry of Rh30 and RD cells (wild-type, L1CAM knockout, and isotype controls) stained with CE7 antibody to detect tumor-associated L1CAM. (B) Schematic of CE7-derived CAR constructs with varied hinge and costimulatory domains. Domains shown include antigen-binding domain (ABD), hinge, transmembrane (TM), costimulatory (CSD), signaling domain (SD), and reporter elements. (C-D) Tumor cell killing by CAR T cells against fLuc + Rh30 and RD cells (C) or their L1CAM knockout counterparts (D) after 48 h coculture at E:T ratios of 1:8, 1:4, 1:1, and 2:1, measured by luciferase-based survival assay. Data represent CAR T cells generated from four independent donors (n = 4), each tested in technical triplicates. (E) IFN-γ release by CAR T cells cocultured with fLuc + Rh30 and RD cells and corresponding L1CAM knockout targets, measured by ELISA in culture supernatants 24 h post coculture. Data represent CAR T cells generated from three independent donors (n = 3), each tested in technical triplicates. Statistical analysis for panels C-E was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test against L1CAM.III as reference. P values are denoted as: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; ** p < 0.0001.

Article Snippet: 48 h later, cells were sorted for mouse L1CAM expression using PE- conjugated anti-mouse CD171 (L1CAM) antibody (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany) to have a homogenous population.

Techniques: Flow Cytometry, Knock-Out, Staining, Derivative Assay, Construct, Binding Assay, Luciferase, Clonogenic Cell Survival Assay, Generated, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: L1CAM-CAR T cells with enhanced potency overcome low-density antigen expression in rhabdomyosarcoma

doi: 10.64898/2026.02.27.707949

Figure Lengend Snippet: (A) Flow cytometry analysis of CD69 expression on CAR T cells transduced with L1CAM.CT, L1CAM.I, L1CAM.II, L1CAM.III, L1CAM.IV, CD19 CAR, or left untransduced (UTD). Representative histograms are shown. (B) Quantification of CD69 expression (% positive CAR T cells) across constructs. Data represent mean ± SD from three independent donors (n=3). (C) CAR T cell expansion over 13 days in culture. Cell counts (left y-axis, bars) and viability (right y-axis, lines) are shown. Data represent mean ± SD from three independent donors (n=3). (D) CD4/CD8 ratio of CAR T cells determined by flow cytometry on day 14. Data represent mean ± SD from three independent donors (n=3). (E) Representative flow cytometry plots showing CD4 and CD8 distribution among UTD, CD19, and L1CAM CAR T cells (CT, I, II, III, IV) on day 14.

Article Snippet: 48 h later, cells were sorted for mouse L1CAM expression using PE- conjugated anti-mouse CD171 (L1CAM) antibody (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany) to have a homogenous population.

Techniques: Flow Cytometry, Expressing, Transduction, Construct

Journal: bioRxiv

Article Title: L1CAM-CAR T cells with enhanced potency overcome low-density antigen expression in rhabdomyosarcoma

doi: 10.64898/2026.02.27.707949

Figure Lengend Snippet: (A) Tumor cell killing by L1CAM.III- and B7-H3-CAR T cells against fLuc + Rh30 and RD cells after 48 h coculture at E:T ratios of 1:8, 1:4, 1:1, and 2:1, measured by luciferase-based survival assay. UTD T cells served as negative controls. Data from three independent donors (n = 3), each tested in technical triplicates. (B) IFN-γ release from L1CAM.III- and B7-H3-CAR T cells cocultured with Rh30 and RD cells for 24 h, measured by ELISA. Data represent mean ± SD from three independent donors (n = 3), each tested in technical triplicates. (C) Flow cytometry showing B7-H3 but not L1CAM expression on MRC-5 fibroblasts. RD and Rh30 cells were used as positive controls, while UTD T cells served as the negative control. Isotype controls are colored in gray. (D) Killing of fLuc + MRC-5 fibroblasts by L1CAM.III- and B7-H3-CAR T cells after 48 h coculture, measured by luciferase-based survival assay. Data from three independent donors (n = 3), each tested in technical triplicates. (E) IHC of representative healthy human tissues (stomach, lungs, liver, prostate, kidney, cerebellum) stained for L1CAM or B7-H3 (brown) with hematoxylin counterstain (blue). Scale bar = 50 µm. (F) Quantitative analysis of IHC staining of TMAs. DAB-positive signal was quantified, with the tissue showing strongest staining set to 100% and all others expressed relative to this maximum. Statistical analysis for panels A, B and D was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test against UTD as reference. P values are denoted as: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; ** p < 0.0001.

Article Snippet: 48 h later, cells were sorted for mouse L1CAM expression using PE- conjugated anti-mouse CD171 (L1CAM) antibody (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany) to have a homogenous population.

Techniques: Luciferase, Clonogenic Cell Survival Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Negative Control, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: L1CAM-CAR T cells with enhanced potency overcome low-density antigen expression in rhabdomyosarcoma

doi: 10.64898/2026.02.27.707949

Figure Lengend Snippet: Immunohistochemical staining of L1CAM and B7-H3 across a panel of normal human tissues. Formalin-fixed paraffin-embedded sections were stained with anti-L1CAM antibody (upper rows) or B7-H3 antibody (lower rows) and counterstained with hematoxylin. Scale bars: 50 μm.

Article Snippet: 48 h later, cells were sorted for mouse L1CAM expression using PE- conjugated anti-mouse CD171 (L1CAM) antibody (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany) to have a homogenous population.

Techniques: Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded

Journal: bioRxiv

Article Title: L1CAM-CAR T cells with enhanced potency overcome low-density antigen expression in rhabdomyosarcoma

doi: 10.64898/2026.02.27.707949

Figure Lengend Snippet: (A) Schematic representation of CAR constructs (CD19, L1CAM.III, L1CAM.CT, B7-H3), each co-expressing Antares via a P2A linker. (B) Experimental setup for Rh30 xenograft study: 2.5×10 5 fLuc + Rh30 cells injected at day 0, followed by two intravenous doses of 5×10 6 Antares + CAR T cells on days 5 and 12. (C) Representative in vivo bioluminescence imaging of Rh30 tumor-bearing mice treated with the indicated CAR T cells (n=5 per group). (D) Longitudinal Rh30 tumor growth monitored by bioluminescence flux (photons/s) in individual mice. (E) Kaplan-Meier survival analysis of Rh30-bearing mice treated with each CAR T cell group. (F) Body weights (g) of Rh30-bearing mice during treatment. (G) Experimental setup for RD model: 5×10 5 fLuc + RD cells injected at day 0, followed by two intravenous doses of 5×10 6 Antares + CAR T cells on days 5 and 12. (H) Representative in vivo bioluminescence imaging of RD tumor-bearing mice treated with the indicated CAR T cells (n=5 per group). (I) Longitudinal RD tumor growth monitored by bioluminescence flux (photons/s) in individual mice. (J) Kaplan-Meier survival analysis of RD-bearing mice treated with the indicated CAR T cells. (K) Body weights (g) of RD-bearing mice during treatment.

Article Snippet: 48 h later, cells were sorted for mouse L1CAM expression using PE- conjugated anti-mouse CD171 (L1CAM) antibody (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany) to have a homogenous population.

Techniques: Construct, Expressing, Injection, In Vivo, Imaging

Journal: bioRxiv

Article Title: L1CAM-CAR T cells with enhanced potency overcome low-density antigen expression in rhabdomyosarcoma

doi: 10.64898/2026.02.27.707949

Figure Lengend Snippet: Formalin-fixed paraffin-embedded sections were prepared from brain, heart, lung, kidney, liver, and skeletal muscle of mice treated with CD19-, L1CAM.III-, L1CAM.CT-, or B7-H3-CAR T cells. Sections were stained with hematoxylin to assess general tissue morphology and cellular architecture. Across all treatment groups, no histological abnormalities, necrosis, or inflammatory infiltrates were observed. Representative images are shown for each organ and CAR T cell condition. Scale bars: 50 µm.

Article Snippet: 48 h later, cells were sorted for mouse L1CAM expression using PE- conjugated anti-mouse CD171 (L1CAM) antibody (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany) to have a homogenous population.

Techniques: Formalin-fixed Paraffin-Embedded, Staining

Journal: bioRxiv

Article Title: L1CAM-CAR T cells with enhanced potency overcome low-density antigen expression in rhabdomyosarcoma

doi: 10.64898/2026.02.27.707949

Figure Lengend Snippet: (A-B) Flow cytometry analysis of human and mouse L1CAM expression in Rh30 (A) and RD (B) cells. L1CAM knockout (KO) cells were transduced to express truncated mouse L1CAM. Gray histograms represent isotype controls. (C) Tumor cell killing by L1CAM.III- and B7-H3-CAR T cells against fLuc⁺ Rh30 (left) and RD (right) cells after 48 h co-culture at E:T ratios of 1:2, 1:1, 5:1, and 10:1, measured by luciferase-based survival assay. CD19-CAR T cells served as negative controls. Data represent CAR T cells from three independent donors (n=3), each tested in technical triplicates. Statistical analysis for panels A, B and D was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test against CD19-CAR T cells as reference. P values are denoted as: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; ** p < 0.0001.

Article Snippet: 48 h later, cells were sorted for mouse L1CAM expression using PE- conjugated anti-mouse CD171 (L1CAM) antibody (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany) to have a homogenous population.

Techniques: Flow Cytometry, Expressing, Knock-Out, Co-Culture Assay, Luciferase, Clonogenic Cell Survival Assay

Journal: Molecular cancer therapeutics

Article Title: Development of antibody-drug conjugates targeting L1CAM to treat metastatic cancer

doi: 10.1158/1535-7163.MCT-25-1184

Figure Lengend Snippet: L1CAM as a prognostic marker and a therapeutic target in various cancer types. A , L1CAM transcript levels in primary and metastatic tumors from different tissues. Bar graph, mean ± S.E.M. Sample sizes are indicated in the figure. Data were sourced from TNMplot. Scheme created with BioRender. Tumor L1CAM expression is likely to be underestimated in stroma-dense tumors (e.g. Ovarian, Pancreatic). B , The overall survival rate for various cancer types with respect to L1CAM transcript levels. Overall survival, individual data points in grey; median survival in black and red. Sample sizes are indicated in the figure. Data were sourced from KM-plotter which integrates datasets from GEO, EGA, and TCGA. C , L1CAM IHC staining of LUAD primary tumor and metastases from patient tissue sections. Scale bar, 50 μm. D , Clinical profiles of LUAD PDX cohort ranked by L1CAM H-score. Each column represents a single patient from whom the xenograft was derived. Sample sizes are indicated in the figure. E , L1CAM IHC staining of LUAD PDX tissue section, followed by immunofluorescence imaging of L1CAM staining and UMAP analysis of L1CAM transcript levels from scRNA-seq data (4,017 cells) from PDX tumoroids. Scale bar, 30 μm (IHC); 10 μm (IF). Selected PDX line is indicated by the black arrow from panel D. F , L1CAM mRNA levels from a bulk RNA-seq in various LUAD cell lines and their KRAS mutation status. Each column represents a single cell line. Sample sizes are indicated in the figure. A.U., arbitrary unit. G , Western immunoblot analysis of L1CAM in selected LUAD cell lines from panel F , as indicated by the colored arrows. Statistical significance was assessed using a two-tailed Mann-Whitney test ( A,B ). ns, not significant; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Antibodies used include mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), and human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Marker, Expressing, Immunohistochemistry, Derivative Assay, Immunofluorescence, Imaging, Staining, RNA Sequencing, Mutagenesis, Single Cell, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Molecular cancer therapeutics

Article Title: Development of antibody-drug conjugates targeting L1CAM to treat metastatic cancer

doi: 10.1158/1535-7163.MCT-25-1184

Figure Lengend Snippet: Generation of L1CAM antibodies binding to the extracellular portion. A , Schematic representation of the L1CAM molecule and its extracellular domains as binding sites for the generated antibodies. B , Workflow for hit selection of hybridoma supernatants from mice immunized with L1CAM immunogens. C , Molecular characterization of the L1CAM antibodies derived from the experimental steps outlined in panel B .

Article Snippet: Antibodies used include mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), and human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Binding Assay, Generated, Selection, Derivative Assay

Journal: Molecular cancer therapeutics

Article Title: Development of antibody-drug conjugates targeting L1CAM to treat metastatic cancer

doi: 10.1158/1535-7163.MCT-25-1184

Figure Lengend Snippet: Payload optimization for L1CAM ADCs. Cytotoxicity curves for L1CAM ADCs generated from either the 14A10 (purple) or 143G03 (red) antibody conjugated to PNU-159682 ( A ), MMAE ( B ), ABZ038 ( C ), Duocarmycin ( D ), or α-Amanitin ( E ) using a Val-Cit linker. Cell viability was measured as a function of ADC concentration (0.1 pM to 100 nM) after 96 h incubation with HEK293T cells overexpressing L1CAM (293T-L1CAM) or MCF-7 and MDA-MB-231 human breast cancer cell lines. A mouse IgG1 isotype control antibody conjugated to the corresponding payload (black) and the corresponding free drug (dotted grey) were included as controls.

Article Snippet: Antibodies used include mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), and human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Generated, Concentration Assay, Incubation, Control

Journal: Molecular cancer therapeutics

Article Title: Development of antibody-drug conjugates targeting L1CAM to treat metastatic cancer

doi: 10.1158/1535-7163.MCT-25-1184

Figure Lengend Snippet: Linker optimization for L1CAM ADCs. Schematic representations of the ADC with cleavable Glu-Val-Cit ( A ) or non-cleavable tri-glycine ( B ) linkers and the PNU-159682 payload ( 50 ). Cytotoxicity curves for 143G03 (red) and 14A10 (blue) ADCs conjugated to PNU-159682 with either a cleavable ( C ) or non-cleavable ( D ) linker in 293T-L1CAM, MCF-7, and MDA-MB-231 cell lines. An ADC generated from a mouse IgG1 isotype control antibody (IgG1-ADC; black) and free PNU-159682 (dotted grey) were included as controls.

Article Snippet: Antibodies used include mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), and human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Generated, Control

Journal: Molecular cancer therapeutics

Article Title: Development of antibody-drug conjugates targeting L1CAM to treat metastatic cancer

doi: 10.1158/1535-7163.MCT-25-1184

Figure Lengend Snippet: In vivo efficacy of L1CAM ADCs against a triple negative breast cancer model. Athymic mice were transplanted subcutaneously ( A, B ) or via tail vein injection to establish orthotopic lung metastasis ( C, D ) with MDA-MB-231 LM2 breast cancer cells. Once tumors had grown, mice were administered 4 weekly doses (shown in red arrows) of ADCs generated with the 143G03 or 14A10 ADC conjugated to PNU-159682 with a cleavable (C) linker, (0.3 mg/kg) or non-cleavable (NC) linker (1 mg/kg), and IgG1-ADC or vehicle control. Tumor volume ( A ), Weekly lung metastasis ( C ) and overall survival ( B, D ) were monitored. n = 6 mice per condition. Statistical significance was assessed using the Kolmogorov-Smirnov test ( A, C ) or log-rank (Mantel-Cox) test ( B, D ). ns, not significant; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Antibodies used include mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), and human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: In Vivo, Injection, Generated, Control

Journal: Molecular cancer therapeutics

Article Title: Development of antibody-drug conjugates targeting L1CAM to treat metastatic cancer

doi: 10.1158/1535-7163.MCT-25-1184

Figure Lengend Snippet: In vitro efficacy of L1CAM ADC across various cancer types. Cytotoxicity curves for 13F04 ADC (red) conjugated to PNU-159682 with a non-cleavable linker in the following cell lines: 293T-L1CAM ( A ), MCF-7 ( B ), MDA-MB-231 ( C ), H524 ( D ), H2030 ( E ), H23 parental ( F ), and H23 with L1CAM knockout ( G ). Efficacy was also evaluated in PDX-derived Ru631 tumoroids with either shControl ( H ) or shL1CAM knockdown ( I ). An ADC generated from a mouse IgG1 isotype control antibody (IgG1-ADC; black) was used as a negative control.

Article Snippet: Antibodies used include mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), and human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: In Vitro, Knock-Out, Derivative Assay, Knockdown, Generated, Control, Negative Control

Journal: Molecular cancer therapeutics

Article Title: Development of antibody-drug conjugates targeting L1CAM to treat metastatic cancer

doi: 10.1158/1535-7163.MCT-25-1184

Figure Lengend Snippet: In vivo efficacy of L1CAM ADC against triple negative breast cancer and lung adenocarcinoma (LUAD) metastasis. A,B , Single-cell suspensions of MDA-MB-231 LM2 cells transduced with luciferase were injected into NSG mice via the tail vein. After 1 week of metastatic engraftment and outgrowth in the lungs, the mice received control ADC (1 mg/kg) or 13F04 ADC (1 mg/kg) by intraperitoneal injection once weekly for four weeks at the indicated times ( red arrows ). Tumor burden was monitored by BLI weekly. A , Metastatic burden of mice inoculated with MDA-MB-231 cells and treated with control ADC or 13F04 anti-L1CAM ADC. n = 5 mice per group. *** P = 0.0006. B , Overall survival plots of mice in the experiment of panel ( A ) ** P = 0.0035. C , NSG mice were inoculated via the tail vein with GFP-expressing Ru631 LUAD PDX cells. One week later, mice were treated with a single dose of 13F04 ADC or IgG control ADC, and lungs were harvested and analyzed by IF staining for GFP (cancer cells) and L1CAM in one week after ADC treatment. White arrows, cytoplasmic L1CAM staining upon anti-L1CAM ADC treatment. Scale bar, 10 μm. D , Quantification of L1CAM − or L1CAM + fraction of Ru631 PDX treated with ADC in the experiment of panel ( C ). n = 3,195 cells (Control); 2,110 cells (13F04). ** P = 0.0079. E , H&E staining of lung sections before or one week after a single dose of ADC treatment, administered four weeks after inoculation of Ru631 PDX cells into NSG mice via the tail vein. Magnified regions are shown ( red boxes ). Scale bar, 200 μm. F , Bar graph showing the percent area of metastatic lesions per lung in the experiments of panel ( E ). n = 3 per experimental condition. * P = 0.0239. G , IF staining for human pan-cytokeratin (cancer cells) and cleaved caspase 3 in lung metastatic colonies one week after a single dose of ADC treatment, administered four weeks post-inoculation of Ru631 PDX cancer cells into NSG mice via the tail vein. Scale bar, 50 μm. H , Fraction of cleaved caspase 3 + (apoptotic) cells detected by IF in the experiment of panel ( G ). n = 3,257 cells (Control); 788 cells (13F04). **** P < 0.0001. I , IF staining for GFP (cancer cells) and SOX2 one week after a single dose of ADC treatment, administered one week after inoculation of Ru631 PDX cancer cells into NSG mice via the tail vein. Scale bar, 10 μm. J , Percentage of SOX2 + cancer cells detected by IF in the experiment of panel ( I ). n = 5 per experimental condition. * P = 0.0317. K-N , Therapeutic assessment of LUAD metastasis treated with L1CAM or IgG ADCs. Single-cell suspensions from LUAD PDX tumoroids transduced with luciferase were injected into NSG mice via the tail vein. After 4 to 5.5 weeks of metastatic engraftment and outgrowth in the lungs, the mice received control ADC (1 mg/kg) or 13F04 ADC (1 mg/kg) by intraperitoneal injection once weekly for four weeks at the indicated times ( red arrows ). Tumor burden was monitored by BLI weekly. K , Metastatic burden of mice inoculated with Ru323 PDX tumoroid cells and treated with control ADC or 13F04 anti-L1CAM ADC. n = 5 mice per group. **** P < 0.0001. L , Overall survival plots of mice in the experiment of panel ( K ) ** P = 0.0018. M , Metastatic burden of mice inoculated with Ru631 PDX tumoroid cells and treated with control ADC or 13F04 anti-L1CAM ADC. Control, n = 14 mice; 13F04, n = 15 mice. **** P < 0.0001. N , Overall survival plots of mice in the experiment of panel ( M ). **** P < 0.0001. The bar graph indicates mean ± S.E.M. ( D,F,J ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( H ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( D,H,J ), two-tailed t test after passing the Shapiro-Wilk normality test ( F ), Kolmogorov-Smirnov test ( A,K,M ) or log-rank (Mantel-Cox) test ( B,L,N ).

Article Snippet: Antibodies used include mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), and human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: In Vivo, Single Cell, Transduction, Luciferase, Injection, Control, Expressing, Staining, Whisker Assay, Two Tailed Test, MANN-WHITNEY