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: Cells of the polyclonal HT-1376 Nectin-4 KO cell line were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967).

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: Cells of the polyclonal HT-1376 Nectin-4 KO cell line were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967).

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: Cells of the polyclonal HT-1376 Nectin-4 KO cell line were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967).

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: Cells of the polyclonal HT-1376 Nectin-4 KO cell line were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967).

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: Cells of the polyclonal HT-1376 Nectin-4 KO cell line were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967).

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: Cells of the polyclonal HT-1376 Nectin-4 KO cell line were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967).

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: Cells of the polyclonal HT-1376 Nectin-4 KO cell line were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967).

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: Cells of the polyclonal HT-1376 Nectin-4 KO cell line were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967).

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: Cells of the polyclonal HT-1376 Nectin-4 KO cell line were stained 1:100 with APC-labelled anti-human Nectin-4 antibody (Miltenyi Biotec, #130-116-028, clone REA967).

Techniques: Co-Culture Assay, Positive Control, Incubation

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 samples were stained with 75 nM of quattrobinder solution or as control with a conventional biotinylated anti-Nectin-4 antibody (Miltenyi Biotec, #130-116-100, clone REA967, 1:100 dilution) and a secondary staining step with 75 nM of Alexa FluorTM 647-conjugated streptavidin (Invitrogen, #S21374) in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA).

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 samples were stained with 75 nM of quattrobinder solution or as control with a conventional biotinylated anti-Nectin-4 antibody (Miltenyi Biotec, #130-116-100, clone REA967, 1:100 dilution) and a secondary staining step with 75 nM of Alexa FluorTM 647-conjugated streptavidin (Invitrogen, #S21374) in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA).

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 samples were stained with 75 nM of quattrobinder solution or as control with a conventional biotinylated anti-Nectin-4 antibody (Miltenyi Biotec, #130-116-100, clone REA967, 1:100 dilution) and a secondary staining step with 75 nM of Alexa FluorTM 647-conjugated streptavidin (Invitrogen, #S21374) in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA).

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 samples were stained with 75 nM of quattrobinder solution or as control with a conventional biotinylated anti-Nectin-4 antibody (Miltenyi Biotec, #130-116-100, clone REA967, 1:100 dilution) and a secondary staining step with 75 nM of Alexa FluorTM 647-conjugated streptavidin (Invitrogen, #S21374) in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA).

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 samples were stained with 75 nM of quattrobinder solution or as control with a conventional biotinylated anti-Nectin-4 antibody (Miltenyi Biotec, #130-116-100, clone REA967, 1:100 dilution) and a secondary staining step with 75 nM of Alexa FluorTM 647-conjugated streptavidin (Invitrogen, #S21374) in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA).

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 samples were stained with 75 nM of quattrobinder solution or as control with a conventional biotinylated anti-Nectin-4 antibody (Miltenyi Biotec, #130-116-100, clone REA967, 1:100 dilution) and a secondary staining step with 75 nM of Alexa FluorTM 647-conjugated streptavidin (Invitrogen, #S21374) in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA).

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 samples were stained with 75 nM of quattrobinder solution or as control with a conventional biotinylated anti-Nectin-4 antibody (Miltenyi Biotec, #130-116-100, clone REA967, 1:100 dilution) and a secondary staining step with 75 nM of Alexa FluorTM 647-conjugated streptavidin (Invitrogen, #S21374) in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA).

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 samples were stained with 75 nM of quattrobinder solution or as control with a conventional biotinylated anti-Nectin-4 antibody (Miltenyi Biotec, #130-116-100, clone REA967, 1:100 dilution) and a secondary staining step with 75 nM of Alexa FluorTM 647-conjugated streptavidin (Invitrogen, #S21374) in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA).

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 samples were stained with 75 nM of quattrobinder solution or as control with a conventional biotinylated anti-Nectin-4 antibody (Miltenyi Biotec, #130-116-100, clone REA967, 1:100 dilution) and a secondary staining step with 75 nM of Alexa FluorTM 647-conjugated streptavidin (Invitrogen, #S21374) in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA).

Techniques: Co-Culture Assay, Positive Control, Incubation

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: Phenotype was screened by flow cytometry staining of human Nectin-4 (Miltenyi Biotec, #130-116-028, clone REA967) with a BD FACS CantoTM II analyzer and genotyping was performed as described above for the polyclonal culture.

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: Phenotype was screened by flow cytometry staining of human Nectin-4 (Miltenyi Biotec, #130-116-028, clone REA967) with a BD FACS CantoTM II analyzer and genotyping was performed as described above for the polyclonal culture.

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: Phenotype was screened by flow cytometry staining of human Nectin-4 (Miltenyi Biotec, #130-116-028, clone REA967) with a BD FACS CantoTM II analyzer and genotyping was performed as described above for the polyclonal culture.

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: Phenotype was screened by flow cytometry staining of human Nectin-4 (Miltenyi Biotec, #130-116-028, clone REA967) with a BD FACS CantoTM II analyzer and genotyping was performed as described above for the polyclonal culture.

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: Phenotype was screened by flow cytometry staining of human Nectin-4 (Miltenyi Biotec, #130-116-028, clone REA967) with a BD FACS CantoTM II analyzer and genotyping was performed as described above for the polyclonal culture.

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: Phenotype was screened by flow cytometry staining of human Nectin-4 (Miltenyi Biotec, #130-116-028, clone REA967) with a BD FACS CantoTM II analyzer and genotyping was performed as described above for the polyclonal culture.

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: Phenotype was screened by flow cytometry staining of human Nectin-4 (Miltenyi Biotec, #130-116-028, clone REA967) with a BD FACS CantoTM II analyzer and genotyping was performed as described above for the polyclonal culture.

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: Phenotype was screened by flow cytometry staining of human Nectin-4 (Miltenyi Biotec, #130-116-028, clone REA967) with a BD FACS CantoTM II analyzer and genotyping was performed as described above for the polyclonal culture.

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: Phenotype was screened by flow cytometry staining of human Nectin-4 (Miltenyi Biotec, #130-116-028, clone REA967) with a BD FACS CantoTM II analyzer and genotyping was performed as described above for the polyclonal culture.

Techniques: Co-Culture Assay, Positive Control, Incubation

Journal: bioRxiv

Article Title: Ex vivo drug testing in metastatic biopsies reveals patient-specific vulnerabilities to cancer targeting and immune activating drugs

doi: 10.64898/2026.02.06.704037

Figure Lengend Snippet: ( A ) Schematic illustration of the drug response assay. Created with BioRender.com. TME, tumor microenvironment; IF, immunofluorescence. ( B ) Cellular composition of malignant pleural effusions (MPEs) from five NSCLC patients as determined by flow cytometry. ( C ) ATP-based viability of a MPE in different culture media. Each dot represents the mean ± standard deviation (shaded envelope) of n = 5 technical replicates from one representative donor (P179). ( D ) Flow cytometric analysis of MPE viability in HPLM medium. Each dot represents an individual donor (n = 5). P-values were calculated using Wilcoxon matched-pairs signed rank test. ( E ) Log2-fold change (Log2FC) in cellular composition of MPEs after 5 days in HPLM medium compared to baseline, analyzed by flow cytometry. Each dot represents an individual donor. Samples from n = 5 donors were measured. To illustrate fold changes, cell fractions below the 2%-detection limit were excluded from analysis. P-values were determined using a one-sample Wilcoxon test against zero. ( F ). As for (E), but for marker expression of cancer cells (PD-L1, Nectin-4, TROP2) and T cells (PD-1). ( G ) Cytokine secretion and immune checkpoint expression of MPEs (n = 5) after 5 days of ex vivo culture. Color scale represents Z-scores normalized across patients for each cytokine or marker. nMFI, mean fluorescence intensity normalized to fluorescence minus one (FMO) control. ( H ) Relative cell loss of two non-adherent cell lines following the optimized IHC liquid handling protocol. Dots represent outliers among technical replicate wells across n = 3 biological replicates (384-well plates). P-values were calculated using a one-sample Wilcoxon test. ( I ) Pseudocolor plot depicting MFIs of CD45 and EpCAM signal for all segmented masks of a spike-in of MCF-7 cells in lymph node cells. Cell populations were classified based on manual gating. ( J ) Number of cancer (left) and immune cells (right) detected using our image analysis pipeline. Box plots represent data of n = 10 technical replicate wells. ( K ) Cancer cell fractions in malignant pleural effusions (MPEs) from NSCLC patients (n = 5), determined by EpCAM-based immunocytochemistry. The dashed line indicates the detection limit (10 cells per well) of the drug response assay.

Article Snippet: Cells were resuspended in 100 μL of FACS buffer (1% (w/v) BSA, 2 mM EDTA in DPBS) per 1 x 10 6 cells and stained using the following antibodies: Alexa Fluor® 488 anti-human CD3, clone HIT3a (BioLegend, #300319, RRID:AB_493690), PerCP/Cyanine5.5 anti-human CD14, clone HCD14 (BioLegend, # 325621, RRID:AB_893252), PE anti-human CD279 (PD-1), clone EH12.2H7 (BioLegend, # 329905, RRID:AB_940481), PE/Cyanine7anti-human CD11c, clone Bu15 (BioLegend, # 337215, RRID:AB_2129791), APC-Vio770 anti-human CD19, clone LT19 (1:50; Miltenyi Biotec, # 130-098-073, RRID:AB_2661296), Brilliant VioletTM 421 anti-human CD45, clone 2D1 (BioLegend, # 368522, RRID:AB_2687375), Brilliant VioletTM 605 anti-human HLA-DR, clone L243 (BioLegend, # 307639, RRID:AB_11219187), Brilliant VioletTM 785 anti-human CD56 (NCAM), clone 167 (BioLegend, # 362549, RRID:AB_2566058), Alexa Fluor® 488 anti-human CD326 (EpCAM), clone Co17-1A (BioLegend, # 369808, RRID:AB_2650905), PE anti-human TROP2, REAfinityTM (1:50; Miltenyi Biotec, # 130-115-097, RRID:AB_2726914), APC anti-human Nectin-4, REAfinityTM (1:50; Miltenyi Biotec, # 130-116-103, RRID:AB_2727350), Brilliant VioletTM 421 anti-human CD274 (B7-H1, PD-L1), clone 29E.2A3 (BioLegend, # 329714, RRID:AB_2563852), Brilliant VioletTM 785 anti-human CD45, clone HI30 (BioLegend, # 304048, RRID:AB_2563129), Zombie AquaTM (1:1,000; BioLegend, # 423102), Zombie NIRTM (1:500; BioLegend, # 423106).

Techniques: Immunofluorescence, Flow Cytometry, Standard Deviation, Marker, Expressing, Ex Vivo, Fluorescence, Control, Immunocytochemistry

Journal: Frontiers in Immunology

Article Title: Osteoclasts affect the anti-cancer activity of NK cells

doi: 10.3389/fimmu.2026.1730283

Figure Lengend Snippet: Expression of NK cell receptor ligands by OCs by flow cytometry. The graph shows the expression levels of PVR and Nectin-2, respectively ligands of DNAM-1 and TIGIT on OCs and on OC+NK cells after 3 days (A) . Characterization of NID-1 ligand of NKp44 and B7H6 ligand of NKp30 on OCs and on OCs+NK cells, with A549s and with both NK cells+A549s (B) . N = 6 experiments. Groups were compared using a one-way repeated measures analysis of variance with post hoc Tukey test (* p < 0.05; ** p < 0.01).

Article Snippet: After 10 days, spheroid-like structures were originated, then dissociated by ACCUTASE and analyzed by flow cytometry through the staining with the following anti-human monoclonal antibodies CD133 PE-Vio770, Nectin-2 PE, Nectin-4 Vio Bright V423 (Miltenyi Biotec, Bergisch Gladbach, Germany), CXCR4 APC (BD Bioscence, Franklin Lakes, New Jersey, USA), PVR BV510 (Biolegend, San Diego, USA).

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Osteoclasts affect the anti-cancer activity of NK cells

doi: 10.3389/fimmu.2026.1730283

Figure Lengend Snippet: Analysis of CD133 + /CXCR4 + CSC subset. Evaluation of the CSC CD133 + /CXCR4 + subset of A549s, in co-culture with NK cells, OCs and with both OCs and NK cells (A) . Evaluation of expression levels of Nectin-2 (ligand of TIGIT and DNAM-1) (B) and Nectin-4 (ligand of TIGIT) (C) on CD133 + cells, in co-culture with NK cells, OCs and with NK cells+OCs (B, C) . N = 5 experiments. Groups were compared using a one-way repeated measures analysis of variance with post hoc Tukey test (* p < 0.05; ** p < 0.01).

Article Snippet: After 10 days, spheroid-like structures were originated, then dissociated by ACCUTASE and analyzed by flow cytometry through the staining with the following anti-human monoclonal antibodies CD133 PE-Vio770, Nectin-2 PE, Nectin-4 Vio Bright V423 (Miltenyi Biotec, Bergisch Gladbach, Germany), CXCR4 APC (BD Bioscence, Franklin Lakes, New Jersey, USA), PVR BV510 (Biolegend, San Diego, USA).

Techniques: Co-Culture Assay, Expressing