Journal: bioRxiv

Article Title: Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

doi: 10.64898/2026.03.04.709551

Figure Lengend Snippet: a) Ig-like V-type domains of cancer cell surface proteins: PD-L1 crystal structure (PDB:4zqk; hotspots: I54, Y56, V68, M115, Y123), VTCN1 (B7-H4) crystal structure (PDB:4gos; hotspots: L72, L79, Y131), CD276 (B7-H3) AF2 model (hotspots: I66, L75, F123, F129), and Nectin-4 crystal structure (PDB: 4frw; hotspots: A66, L81, F132). b) Histograms summarizing the result of multiple RFdiffusion design runs for different targets. c) Block diagram presenting the evolutionary refinement algorithms described in this work. d) Schematic explaining the introduction of variations into initial AI-minibinder designs by either partial diffusion (option I) or sequence manipulation (option II). e) Boxplot showing pAE interaction scores of initial RFdiffusion designs and GA-refined (partial diffusion) designs. Median pAE scores are indicated and percentages of pAE<= 5 or pAE>5. Wilcoxon test. f) Histogram showing pAE interaction scores of the initial RFdiffusion design runs for Nectin-4. The designs that were used as inputs for the refinement algorithms are highlighted by a red box. g) Histogram showing the refinement results using option I of the evolutionary algorithm. Insert: Cartoon representation of Nectin-4 (cyan) and a set of diverse AI-minibinder designs (green).

Article Snippet: The cells were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967) and the positive population was sorted.

Techniques: Blocking Assay, Diffusion-based Assay, Sequencing

Journal: bioRxiv

Article Title: Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

doi: 10.64898/2026.03.04.709551

Figure Lengend Snippet: a) Boxplot showing differences in ipTM scores between Nectin-4 minibinder initial designs and option II GA-redefined designs. Median ipTM scores are indicated. Two-sided Wilcoxon test. b-d) Three examples of predicted structures of option II GA-refined Nectin-4 minibinders in complex with Nectin-4. Respective ipTM and pTM scores are indicated. e) Boxplot showing differences in ipTM scores between Nectin-4 minibinder initial designs and option II GA-redefined designs with implemented pTM binder penalty and with or without target isoelectric point (pI) and hydrophobicity (GRAVY score). Pairwise two-sided Wilcoxon test with Benjamini & Hochberg correction for multiple testing. f-h) Three examples (from group 3) of predicted structures of option II GA-refined Nectin-4 minibinders with pTM penalties. Respective ipTM and pTM scores are indicated.

Article Snippet: The cells were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967) and the positive population was sorted.

Techniques:

Journal: bioRxiv

Article Title: Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

doi: 10.64898/2026.03.04.709551

Figure Lengend Snippet: a) Scheme illustrating the experimental screening pipeline using the mammalian cell-surface display for AI-minibinder presentation, fluorescence-activated cell sorting and next-generation sequencing to identify candidates. b) Scatter plots showing enriched minibinders for human, murine, rat and monkey Nectin-4 (colored dots). c) Cartoon showing the experimental set-up of surface presentation of the AI-minibinder on HEK293T cells and detecting the binding to the target protein. Staining of the HA-tag was used to normalize the protein binding to the surface expression of the AI-minibinder. d) Heatmap summarizing the binding to human, murine, rat and monkey Nectin-4 Fc-fusion proteins of 40 individually validated Nectin-4 AI-minibinders using HEK293T cell-surface display. Data are shown as geometric mean fluorescence intensity (gMFI) normalized to the surface expression detected with the HA-tag signal. e) Representative histograms showing flow cytometry analyses of the binding to Nectin-4 Fc-fusion proteins from the different species for 8 AI-minibinder candidates. f) Quantification of the experiment described in e. g) Representative histograms of minibinder 2-17-59 showing selective binding to Nectin-4 Fc-fusion protein from murine, rat and monkey origin (left) and minibinder 4-21-32 demonstrating preferential binding to rat Nectin-4 Fc-fusion protein (right). h) Quantification of the experiment described in g for binder 2-17-59 (left) and 4-21-32 (right). Data are shown as mean ± SD performed in biological triplicates (n = 3). **p<0.01; ***p<0.001; ****p<0.0001; two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The cells were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967) and the positive population was sorted.

Techniques: Fluorescence, FACS, Next-Generation Sequencing, Binding Assay, Staining, Protein Binding, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

doi: 10.64898/2026.03.04.709551

Figure Lengend Snippet: a) Scatter plot showing the enriched minibinder population for human Nectin-4 (green dots). b) Scatter plots of the enriched minibinder population detected from the screen with murine, rat and monkey Nectin-4 Fc-fusion proteins overlaid into the human Nectin-4 enrichment plot (colored dots).

Article Snippet: The cells were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967) and the positive population was sorted.

Techniques:

Journal: bioRxiv

Article Title: Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

doi: 10.64898/2026.03.04.709551

Figure Lengend Snippet: a) Coomassie-stained SDS-PAGE of purified Nectin-4 AI-minibinder proteins that were overexpressed in E. coli . Left: original designs, right: biotinylated versions of minibinders including an engineered mutant cysteine residue on the ‘backside’ of the AI-minibinder, opposite of the interaction interface. b) Analytical SEC of purified AI-minibinder proteins using a Superdex Increase 75 3.2/300 column. SEC traces of a commercial standard are included as molecular weight reference. c) Coomassie-stained SDS-PAGE of E. coli supernatants after the overexpression of AI-minibinders with (+) and without (−) incubation at 94 °C for 20 min. The boiled samples were centrifuged and the cleared supernatant was loaded onto the gel. d) Nano differential scanning fluorimetry (nanoDSF) analysis showing the thermal stability of the AI-minibinder proteins. The y-axis shows the first derivative of the corresponding melting curve. The two panels show the unfolding (left) and refolding (right) phases of the experiment. e-k) SPR (surface plasmon resonance) sensorgrams showing raw binding data (colored dots) and fitted curves (black) used to determine binding affinities (K D ) of Nectin-4 AI-minibinders. The concentration series of analyte injections are indicated in the legend. l) Structural model of 3-8-16 and 3-24-26 Nectin-4 minibinders (shades of green) in complex with Nectin-4 (cyan) as determined in Chai-1 shown as overlay.

Article Snippet: The cells were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967) and the positive population was sorted.

Techniques: Staining, SDS Page, Purification, Mutagenesis, Residue, Molecular Weight, Over Expression, Incubation, Nano Differential Scanning Fluorimetry, SPR Assay, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

doi: 10.64898/2026.03.04.709551

Figure Lengend Snippet: a) Schematic illustration of the assembly of biotinylated Nectin-4 AI-minibinders with fluorophore-conjugated streptavidin as tetravalent quattrobinder. b-c) Representative histograms showing flow cytometry analyses of AF647-conjugated Nectin-4 quattrobinders in comparison to a conventional biotinylated Nectin-4 antibody with a secondary AF647-streptavidin staining on target expressing urothelial cancer cells (RT4) and non-expressing cancer cells (T24) as well as on HT-1376 WT and the corresponding knockout cell line (HT-1376 Nectin-4 KO). d-e) Quantification of the experiment described in b and c. Data are shown as mean ± SD performed in biological triplicates (n = 3). ns – non-significant; *p<0.05; ***p<0.001; ****p<0.0001; two-way ANOVA with Šídák’s multiple comparisons test.

Article Snippet: The cells were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967) and the positive population was sorted.

Techniques: Flow Cytometry, Comparison, Staining, Expressing, Knock-Out

Journal: bioRxiv

Article Title: Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

doi: 10.64898/2026.03.04.709551

Figure Lengend Snippet: a) Representative histograms showing flow cytometry analyses of AF647-conjugated Nectin-4 quattrobinders in comparison to a conventional biotinylated Nectin-4 antibody with a secondary AF647-streptavidin staining on target overexpressing CHO-Nectin-4 cells and parental CHO-K1 cells. b) Quantification of the experiment described in a. Data are shown as mean ± SD performed in biological triplicates (n = 3). c) Western blot analysis displaying Nectin-4 expression levels of various urothelial carcinoma cell lines used in this study. d) Next-generation sequencing analysis of genomic DNA from polyclonal HT-1376 WT and Nectin-4 KO cells prior and after cell sorting as well as from monoclonal HT-1376 cell lines. e) Histograms showing a concentration titration of the conventional biotinylated Nectin-4 antibody with secondary AF647-streptavidin staining and four AF647-conjugated Nectin-4 quattrobinders (3-8-16, 2-40-23, 2-36-30, 2-28-14) on HT-1376 WT and the corresponding knockout cell line (HT-1376 Nectin-4 KO). ns – non-significant; *p<0.05; ***p<0.001; ****p<0.0001; two-way ANOVA with Šídák’s multiple comparisons test.

Article Snippet: The cells were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967) and the positive population was sorted.

Techniques: Flow Cytometry, Comparison, Staining, Western Blot, Expressing, Next-Generation Sequencing, FACS, Concentration Assay, Titration, Knock-Out

Journal: bioRxiv

Article Title: Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

doi: 10.64898/2026.03.04.709551

Figure Lengend Snippet: a) Scheme illustrating the T cell engager (TCE) construct. AI-TCE constructs were transiently transfected into HEK293T cells and secreted into the supernatant. b) Western blot analysis showing the expression of TCE constructs in HEK293T cell lysates after transient transfection. c) Cartoon (left) showing the experimental set-up. Quantification of CD69 + CD8 + cells (middle) and CD25 + CD8 + cells (right) after 48 h co-culture of HT-1376 WT and Nectin-4 KO cells with human PBMCs and AI-TCEs or mock-TCE. Stimulation with an OKT3 antibody was used as positive control. d) Cartoon (left) showing the experimental set-up. Quantification of CD69 + CD8 + cells (middle) and CD25 + CD8 + cells (right) after 48 h co-culture of HT-1376 WT and Nectin-4 KO cells with human PBMCs and AI-TCEs or mock-TCE. Target cells were pre-incubated with TCEs, followed by the removal of the supernatant and an additional washing step. Stimulation with an OKT3 antibody was used as positive control. e-f) Dot plot showing the correlation between frequency of CD69 + CD8 + cells and CD25 + CD8 + cells from experiment described in d and their binding affinities (K D ) determined by SPR. g) Representative curves showing co-cultures of human PBMCs with HT-1376 WT or Nectin-4 KO cells and AI-TCEs or mock-TCE measured as cell index value. Dashed line indicates the timepoint of effector addition. h) Quantification of the cell index value of experiment described in g at timepoint 72 h after effector addition. Cell index value was normalized to the control condition (co-culture without TCE). i) Representative curves showing co-cultures of human PBMCs with HT-1376 WT or Nectin-4 KO cells and AI-TCEs or mock-TCE measured as cell index value. TCE supernatant was pre-incubated on target cells and removed before effector cells were added. Dashed line indicates the timepoint of effector addition. j) Quantification of the cell index value of experiment described in i at timepoint 72 h after effector addition. Cell index value was normalized to the control condition (co-culture without TCE). Data are shown as mean ± SD performed in biological triplicates (n = 3). ns – non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; two-way ANOVA with Šídák’s multiple comparisons test.

Article Snippet: The cells were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967) and the positive population was sorted.

Techniques: Construct, Transfection, Western Blot, Expressing, Co-Culture Assay, Positive Control, Incubation, Binding Assay, Control

Journal: bioRxiv

Article Title: Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

doi: 10.64898/2026.03.04.709551

Figure Lengend Snippet: a) Quantification of CD69 + CD4 + cells (left) and CD25 + CD4 + cells (right) after 48 h co-culture of HT-1376 WT and Nectin-4 KO cells with human PBMCs and AI-TCEs or mock-TCE. Stimulation with an OKT3 antibody was used as positive control. b) Quantification of CD69 + CD4 + cells (left) and CD25 + CD4 + cells (right) after 48 h co-culture of HT-1376 WT and Nectin-4 KO cells with human PBMCs and AI-TCEs or mock-TCE. Target cells were pre-incubated with TCEs, followed by the removal of the supernatant and an additional washing step. Stimulation with an OKT3 antibody was used as positive control. c) Curves of replicate 2 and 3 showing co-cultures of human PBMCs with HT-1376 WT or Nectin-4 KO cells and AI-TCEs or mock-TCE measured as cell index value. Dashed line indicates the timepoint of effector addition. d) Curves of replicate 2 and 3 showing co-cultures of human PBMCs with HT-1376 WT or Nectin-4 KO cells and AI-TCEs or mock-TCE measured as cell index value. TCE supernatant was pre-incubated on target cells and removed before effector cells were added. Dashed line indicates the timepoint of effector addition. Data are shown as mean ± SD performed in biological triplicates (n = 3). ns – non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; two-way ANOVA with Šídák’s multiple comparisons test.

Article Snippet: The cells were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967) and the positive population was sorted.

Techniques: Co-Culture Assay, Positive Control, Incubation

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: TIGIT but not DNAM1, CD112R or CD96—interacts with Nectin4. (A) FACS staining of IL-2 activated primary NK cells with Nectin4-Ig. Gray filled histogram represents background staining with secondary antibody only; black line histogram represents specific binding as indicated. (B–F) FACS staining of Raji cells transfected either with an empty vector as control (gray histograms) or with Nectin4 (black histograms). Cells were stained with commercial anti-Nectin4 mAb (B), TIGIT-Ig (C), DNAM1-Ig (D), CD112-Ig (E) or CD96-Ig (F). Figures show one representative experiment out of three performed. Graph depicting the mean fluorescence intensity values of the stainings appears in . (G) Direct binding of Nectin4 to TIGIT. The binding of fluorophore-labeled TIGIT-Ig and its ligands PVR-Ig (red) and Nectin4-Ig (green) was determined using MST. Measurements were repeated with at least three independent protein preparations. FACS, fluorescence-activated cell sorting; IL-2, interleukin-2; MST, microscale thermophoresis; NK, natural killer.

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Staining, Binding Assay, Transfection, Plasmid Preparation, Control, Fluorescence, Labeling, FACS, Microscale Thermophoresis

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: Nectin4 inhibits NK cytotoxicity via TIGIT. (A, C) IL2 secretion by parental BW (A, C), (A) BW-TIGIT and (C) BW-DNAM1 cells. IL2 secretion was determined by ELISA (od 650 nm) following incubation with control anti-TIGIT or anti-DNAM1 antibodies (left in a and C) or with PVR expressing cells (right in a and C). (B, D) IL2 secretion of parental BW (B, D), (B) BW-TIGIT and (D) BW-DNAM1 cells. IL2 secretion was determined by ELISA (od 650 nm) following incubation with Raji cells transfected either with an empty vector (Raji E) as a control, or with Nectin4 (Raji N4). Figure shows one representative experiment out of 3 performed. *P<0.05. (E) FACS staining of Raji cells overexpressing Nectin4 with TIGIT-Ig. TIGIT-Ig was preincubated with no antibody (left), with a control mAb (anti-CD99 mAb clone 12E7, middle) or with anti-TIGIT blocking antibody (mAb #4 generated as described previously, right). Black line histograms represent TIGIT-Ig binding. Gray filled histograms represent background staining of the secondary antibody only. (F) Mean fluorescence intensity (MFI) values of the TIGIT-Ig staining shown in (E) relative to NO antibody staining, *p<2×10 -4 . (G) [ 35 S] methionine-labeled Raji cells transfected either with an empty vector as control (Raji empty—gray) or with Nectin4 (Raji Nectin4—black), were incubated for 5 hours with NK cells. NK cells were preincubated with no antibody (left), with a control antibody (anti-CD99 mAb clone 12E7, middle) or with an anti-TIGIT antibody (mAb #4 generated as described previously, right). The effector to target (E:T) ratios are indicated on the x-axis. Figure shows one representative experiment out of three performed. Shown is the relative average killing ±SD, *p<0.05. FACS, fluorescence-activated cell sorting; IL2, interleukin-2; NK, natural killer; NS, not significant.

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Control, Expressing, Transfection, Plasmid Preparation, Staining, Blocking Assay, Generated, Binding Assay, Fluorescence, Labeling, FACS

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: Novel checkpoint inhibitor anti-Nectin4 antibodies block TIGIT binding. (A, B) FACS staining of Raji cells transfected either with an empty vector (gray histograms) or with Nectin4 over-expression (black histograms). Gray filled histograms represent background staining of secondary antibody only. Cells were stained with anti-Nectin4 mAb clone .01 (A) or clone .05 (B). (C, D) FACS staining with TIGIT-Ig of Raji overexpressing Nectin4 with (black histograms) or without (gray histograms) preincubation with anti-Nectin4 mAb clone .01 (C) or clone .05 (D). Gray, filled histograms represent background staining. Figures show one representative experiment out of three performed. Graph depicting the mean fluorescence intensity values of the stainings appears in . (E) Avidity quantification between Nectin4-Ig and anti-Nectin4 mAb clone .01 and clone .05 using MST. Measurements were repeated with at least three independent protein preparations. FACS, fluorescence-activated cell sorting; MST, microscale thermophoresis

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Blocking Assay, Binding Assay, Staining, Transfection, Plasmid Preparation, Over Expression, Fluorescence, FACS, Microscale Thermophoresis

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: Anti-Nectin4 antibodies increase NK cytotoxicity. (A) [ 35 S] methionine-labeled Raji cells transfected with Nectin4 were incubated with 1 µg/well of either mouse IgG1 as control Ab (green) or with clone .01 (purple) or clone .05 (blue) for 1 hour and then incubated with activated human NK cells for 5 hours. The effector to target (E:T) ratios are indicated on the X-axis. Figure shows one representative experiment out of three performed. Shown is the relative average killing ±SD., *p<0.05 (significance between the clones’ blocking and control antibody blocking). (B–E) FACS staining of cell lines stained with commercial anti-Nectin4 antibody (black line histograms). Gray filled histograms are background staining with secondary antibody only. Cell lines used are MDA-MB-453 (B), SK-BR-3 (C), T47D (D) and LNCaP (E). Figures show one representative experiment out of three performed. Graph depicting the mean fluorescence intensity values of the stainings appears in . (F–I) [ 35 S] methionine-labeled MDA-MB-453 (F), SK-BR-3 (G), T47D (H), and LNCaP (I) cells were incubated with either mouse IgG1 as control mAb (green) or with clone .01 (purple) or clone .05 (blue) for 1 hour and then incubated with activated human NK cell cultures for 5 hours. The E:T ratios are indicated on the X-axis. Figures show one representative experiment out of three performed. Shown is the relative average killing ±SD., *p<0.05 (significance between the clones’ blocking and the control antibody). FACS, fluorescence-activated cell sorting; NK, natural killer.

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Labeling, Transfection, Incubation, Control, Clone Assay, Blocking Assay, Staining, Fluorescence, FACS

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: Murine Nectin4 does not bind murine TIGIT. (A) Overexpression of murine Nectin4 (indicated as mNectin4) on Raji cells. Western blots were performed with antimurine Nectin4 AB and expression was compared with Raji cells expressing empty vector (indicated as empty). Staining for GAPDH was used as a loading control. (B) FACS staining of Raji cells transfected either with an empty vector as control (gray histograms), or with murine Nectin4 (black histograms). Cells were stained with murine TIGIT-Ig. Figures show one representative experiment out of three performed. Graph depicting the mean fluorescence intensity values of the stainings appears in FACS, fluorescence-activated cell sorting.

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Over Expression, Western Blot, Expressing, Plasmid Preparation, Staining, Control, Transfection, Fluorescence, FACS

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: In vivo effect of anti-Nectin4 mAb on tumors overexpressing Nectin4. (A–D) SCID-beige mice were subcutaneously implanted with 5×10 6 Raji cells either overexpressing Nectin4 (N4 oe) or empty vector (EV) either alone (A, B) or with 1×10 6 NK cells (C, D). (E, F) SCID-beige mice were subcutaneously implanted with 5×10 6 Raji cells overexpressing Nectin4 (N4 oe) and 1×10 6 NK cells. Mice were then treated with either anti-Nectin4 clone .05 mAb (clone .05) and or a control Ab. (A, C, E) Tumor growth was followed with standard caliper. Starting day of antibody treatment is marked by a black arrow. Tumor volumes were calculated by the formula: length×width 2 ×0.5. (B, D, F) Tumors ware harvested and weighed on day 21 (B, D) or 27 (F) post tumor injection. For all murine experiments n=7. *p<0.05. NK, natural killer.

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: In Vivo, Plasmid Preparation, Control, Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: The checkpoint inhibitor anti-Nectin4 mAb enhances NK killing of tumors expressing all ligands of TIGIT in vivo. (A–D) SCID-beige mice were subcutaneously implanted with 5×10 6 MDA-MB-453 cells either alone (A, B) or with 7×10 5 NK cells (C, D). Half of each group was treated with anti-Nectin4 clone .05 mAb (clone.05) or a control Ab. (A, C) Tumor growth was followed with standard caliper. starting day of antibody treatment is marked by a black arrow. Tumor volumes were calculated by the formula: length×width 2 ×0.5. (B, D) Tumors ware harvested and weighed on day 23 post-tumor injection. For all murine experiments n=7. *P<0.05. (E) Kaplan-Meier curves of lung adenocarcinoma patients (left, Geo dataset ID: GSE36471) and colon cancer patients (right, Geo dataset ID: GSE17538) after stratification by Nectin4 expression level. *P<0.05. Data were obtained from DRUGSURV. NK, natural killer

Article Snippet: Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting murine Nectin4 (MAB3116, R&D Systems (1:200)) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-32233, Santa Cruz (1:1000)) as loading control.

Techniques: Expressing, In Vivo, Control, Injection

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 1 High expression of Nectin4 and FAP in a variety of cancers. (A) Expression of Nectin4 and FAP in glioma, liposarcoma, and leiomyosarcoma was assessed by IHC. (B) Expression of Nectin4 on lung-metastasized esophageal cancer, lung-metastasized liver cancer, and bone-metastasized triple-negative breast cancer (TNBC). Also see Supplementary Figure 3. Nectin4 is mainly located in the membrane (strongly positive) and cytoplasm (weakly positive) of cancer cells; FAP is mainly located in the membrane (strongly positive) and cytoplasm (weakly positive) of stromal cells, shown in brown.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Expressing, Membrane

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 2 CAR structure and characterization of Nectin4-7.19 CAR-T cells. (A) Schematic illustration of Nectin4 CAR and Nectin4-7.19 CAR lentiviral vector. LTR: long terminal repeats; SP: CD8 signal peptide; TM: transmembrane region; P2A: 2A polypeptide element. (B) CAR expression in Nectin4 CAR-T and Nectin4-7.19 CAR-T cells was measured by flow cytometry. UTD indicates the untransduced T cells as a negative control. (C) Relative CAR expression in CD4+ and CD8+ T subsets. (D) Representative CAR-T cell phenotyping plot based on CD45RA and CCR7 in CD4+

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Plasmid Preparation, Expressing, Cytometry, Negative Control

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 3 Efficient cytotoxicity of Nectin4-7.19 CAR-T cells in vitro. (A) Expression of Nectin4 on a panel of cancer cell lines. (B) Cytotoxicity of Nectin4 CAR-T cells against ABC-1, HT1376, and MDA-MB-453 cells was detected by xCELLigence RTCA label-free technology. The left panel compares the cytotoxicity between Nectin4 CAR-T and CD19 CAR-T cells against target cells at an Effect/Target ratio of 10:1; the right panel shows the killing efficacy of Nectin4 CAR-T cells at different Effect/Target ratios. Arrows refer to the addition of CAR-T cells. The y-axis is the normalized cell index generated by the RTCA software and displayed in real time to reflect the vitality of tumor cells. The x-axis is the time of cell culture in hours. (C) Nectin4 and GFP expression in Luc. ABC-1 cells transfected with lentivirus encoding the Luciferase-T2A-GFP gene. (D) Quantified data on the specific lytic levels of Nectin4 CAR-T and Nectin4-7.19 CAR-T cells against Luc. ABC-1 cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. UTD served as a negative control. (E) Expression level of immune checkpoints was detected by flow cytometry after co-culture of Nectin4 CAR-T or Nectin4-7.19 CAR-T cells with ABC-1 cells at an Effect/Target ratio of 1:1 for 5 days. Data represent the mean ± SD of three independent experiments; ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, t-test.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: In Vitro, Expressing, Generated, Software, Cell Culture, Transfection, Luciferase, Negative Control, Cytometry, Co-Culture Assay

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 4 Therapeutic effect of Nectin4 mCAR-T cells on metastatic colorectal cancer in fully immune-competent mice. (A) The murine CAR construct was inserted upstream of an IRES-GFP marker in the MSCV retroviral plasmid pMIGR1. (B) mCAR expression of Nectin4 mCAR-T cells transfected with pMIGR1-mCAR-IRES-GFP retroviral particles. mUTD indicates the untransduced mouse T cells. (C) Nectin4 and GFP expression of Luc. MC38 cells and hNectin4-Luc. MC38 cells. (D) Quantified data on the specific lytic levels of Nectin4 mCAR-T cells against Luc. MC38 or hNectin4-Luc. MC38 cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. ***p < 0.001, t-test. (E) Secretion of IFN-g in CD4+ and CD8+ T subsets was assessed by flow cytometry after co-culture of Nectin4 mCAR-T cells or mUTD with hNectin4-Luc. MC38 cells for 12 h. ***p < 0.001, t-test. (F, G) C57BL/6 mice were s.c. inoculated with 1 × 106 hNectin4-Luc. MC38 cells on Day 0 and injected i.v. with 5.0 × 105 to 5.0 × 106 Nectin4 mCAR-T cells on Day 10. A total of 5.0 × 106 mUTD served as a negative control (N = 6 mice per group). Solid lines represent each individual mouse (F). Kaplan–Meier survival curve is shown in (G). p-values of log-rank tests were as follows: p = 0.35 (mUTD vs. 0.5 × 106 Nectin4 mCAR-T); p = 0.09 (mUTD vs. 1.5 × 106 Nectin4 mCAR-T); p = 0.0012 (mUTD vs. 5.0 × 106

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Construct, Marker, Retroviral, Plasmid Preparation, Expressing, Transfection, Luciferase, In Vitro, Cytometry, Co-Culture Assay, Injection, Negative Control

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 5 Significant anti-tumor effect of Nectin4-7.19 CAR-T therapy on metastatic lung cancer without on-target off-tumor toxicity. (A) NSG mice were i.v. inoculated with 1.0 × 106 Luc. ABC-1 cells on Day 0 and received an administration of 3 × 106 Nectin4-7.19 CAR-T cells on Day 7 (N = 3 mice per group). Mice treated with the same dosage of UTD cells served as a negative control. (B–D) Tumor xenografts were monitored via bioluminescence imaging. Representative bioluminescence images of three independent experiments are shown in (B); bioluminescence kinetics are shown in (C); solid lines represent each individual mouse. Kaplan–Meier survival curve is shown in (D), p = 0.0246 (Nectin4-7.19 CAR-T vs. UTD), N = 3, log-rank test. (E) Body weight of mice since the tumor inoculation.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Negative Control, Imaging

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 6 Synergistic effect of Nectin4-7.19 CAR-T with FAP-12 CAR-T therapy on metastatic lung cancer mouse model. (A) Schematic illustration of FAP CAR and FAP-12 CAR lentiviral vector. LS: leader signal. (B) CAR expression on FAP CAR-T and FAP-12 CAR-T cells. (C) Expression of CD45RA and CD45RO in CD4+ or CD8+ T subset to assess the subtypes of T cells. (D) 293T cells were transduced with lentivirus encoding the human FAP-Firefly-Luciferase-GFP gene or the murine FAP-Firefly-Luciferase-GFP gene to generate hFAP-Luc. 293T and mFAP-Luc. 293T cells, respectively. Expression of GFP was measured by flow cytometry. 293T cells served as negative controls. (E) Quantified data on the specific lytic levels of FAP CAR-T cells against hFAP-Luc. 293T and mFAP-Luc. 293T cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. UTD served as a negative control. (F) Cytotoxicity of FAP CAR-T and FAP-12 CAR-T cells was detected at an Effect/Target ratio of 10:1 by xCELLigence RTCA label-free technology. (G) Specific lysis of FAP CAR-T and FAP-12 CAR-T cells against hFAP- Luc. 293T was detected by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. (H) NSG mice were inoculated with 1.0 × 106 Luc. ABC-1 cells i.v. on Day 0 and received an administration of 2 × 106 FAP-12 CAR-T cells, 2 × 106 Nectin4-7.19 CAR-T cells, or an admixture of 1 × 106 Nectin4-7.19 CAR-T cells and 1 × 106 FAP-12 CAR-T cells on Day 3 (N = 3 mice per group). A total of 2.0 × 106 UTD served as a negative control. (I) Tumor xenografts were monitored via bioluminescence imaging. Representative bioluminescence images of three independent experiments in each group of mice were shown. (J) Bioluminescence kinetics of the tumor growth in the model. (K) Kaplan– Meier survival curve. p-values of log-rank tests were as follows: p = 0.0246 (Nectin4-7.19+FAP-12 vs. UTD); p = 0.0246 (Nectin4-7.19+FAP-12 vs. FAP-12); p = 0.1161 (Nectin4-7.19+FAP-12 vs. Nectin4-7.19), N = 3. (L) Body weight of mice since the tumor inoculation. Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, t-test.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Plasmid Preparation, Expressing, Transduction, Luciferase, Cytometry, In Vitro, Negative Control, Lysis, Imaging