tag protein  (R&D Systems)

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: Next-generation short-read DNA sequencing of CDR3 amplicons. A Purified GPA33 was used as antigen in immunoplate ELISA wells and screened using four rounds of biopanning. These phage pools were validated using two independent assays: NGS CDR3 sequencing (this figure) and ELISA (Fig. ). B After the final round of biopanning, each phage pool was used directly in PCR reactions with barcoded primers that aim to measure enriched CDR3 sequences as a function of rounds of biopanning . B The position and sequence of the ‘old’ Illumina sequences (in brown, O-Illumina forward primer and, in orange, O-Illumina reverse barcoded primer), the 3-bp adapter position (in violet) and the sequences of the forward (pelB) and reverse primers (one forward primer and four reverse primers capturing FR4). C Isolation of monoclonal bacteriophage with anti-GPA33 activity using the pGEMP library. Following four rounds of biopanning, polyclonal bacteriophage pools were assayed for anti-GPA33 activity by ELISA. R2–R4 indicate biopanning rounds, pools named ‘F’ (FNL and F25) were raised against Fc-tagged GPA33. Those named ‘H’ (H25 and HNL) were raised against His-tagged GPA33. The labels ‘25’ and ‘NL’ refer to two different preparations of the pGEMP bacteriophage library from the master stock. The data are plotted as relative scFV phage binding (in relative light units (RLU)) as a function of the biopanning round and target. D The phage pools from C were processed using short-read NGS to measure the efficiency of biopanning. An example of the amplicons generated with these primers using phage pools R1–R4 from GPA33 screening with the ~ 500-bp amplicon highlighted. E Pooled amplicons were subjected to QC analysis using fluorometric methods involving the Invitrogen Qubit dsDBNA assay and quantified using an Agilent Bioanalyzer 2100 to define the amplicon molecular mass. The major amplicon of ~ 500 bp was quantified along with higher molecular mass DNA adducts that co-purified during gel electrophoresis. The amplicon was blunt end ligated to the new generation of Illumina adaptor primers and subjected to DNA sequencing by Source Bioscience (Cambridge, UK). The top 10,000 sequences were selected and analysed for DNA reads as a function of biopanning rounds with the parental library as the negative control (Supplementary Table 1 shows the top 10,000 sequences). F Examples of the ten most dominant antibody sequences enriched by round 4. The most highly enriched CDR3 domain isolated appeared in round 4 with over 500,000 sequence reads after PCR and NGS. Additional clones with different CDR3 sequences that enriched in round 1 but did not over-dominate by round 4 are also shown. The data are presented as the number of reads as a function of each round of biopanning and include the number of reads detected in the parental library. The data do not directly reflect the actual abundance of any one antibody sequence because data generation required over 30 cycles of PCR to generate the amplicons for NGS sequencing, which in turn required additional amplification stages. Thus, the relative read number provided a qualitative gauge of abundance with the caveat that there would be a relatively low sequence bias affecting amplicon production during the PCR cycles

Article Snippet: Human Fc-tagged GPA33 protein (Sino Biologics, cat. no. 11277-H08H) and His-tagged human GPA33 protein (Acro Biosystems, cat. no. GP3-H5224) were used for antibody selection.

Techniques: DNA Sequencing, Purification, Enzyme-linked Immunosorbent Assay, Sequencing, Isolation, Activity Assay, Binding Assay, Generated, Amplification, Nucleic Acid Electrophoresis, Negative Control, Clone Assay

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: Isolation of a GPA33-specific antibody from a scFV phage display library. A Relative purity of GPA33 proteins used as bait in antibody screening. SDS gel electrophoresis was used to separate FC-tagged GPA33 and His-tagged GPA33 without and with DTT. The gel was stained with Coomassie Blue; lanes are markers, on the far left; lane 1, Fc-GPA33; lane 2, Fc-GPA33 + DTT; lane 3, His-GPA33; lane 4, His-GPA33 + DTT. B Monoclonal bacteriophage from phage pools (Fig. C) were isolated and propagated by picking single colonies of ER2738 infected with bacteriophage from agar plates, and tested for anti-GPA33 activity by high-throughput ELISA. FNL4 refers to round 4 phage from the FNL screen using FC-tagged GPA33, and F253 screen refers to the F25 screen from round 3 (as in Fig. C). C Numbered monoclonal scFvs from two biopanning pools (labelled FNL4 and F253, B and Fig. C) were surveyed for binding activity versus recombinant GPA33, from two different sources (His and Fc tagged). BSA was used as a negative control for antigen-binding activity. D Sequence structure of the synthetic scFV. The sequence of CDR3 from all active phage scFV monoclonals from round 4 matched the dominant sequence in Fig. F (top row) with 523,246 sequencing reads. E , F Production in ExpiCHO cells and ELISA. A plasmid with a single cistronic mRNA encoding light and heavy chains of this abundant active clone, separated by the T2A translation termination sequence ( E ), was used to produce ( F ) either an immunoblot developed with ECL showing the heavy and light chain from an IgG encoding plasmid (lanes 1 and 2) or scfv-Fc fusion protein encoding plasmid (lanes 3 and 4). G Activity of the RSE-05 antibody as either a scFv-FC fusion or IgG. ELISA was used to measure the activity of the ExpiCHO-produced antibodies. Fc-GPA33 or his-GPA33 were coated onto immunoplates, and binding was measured using ECL. H ELISA was used to estimate the affinity of RSE-05 for the extracellular domain of GPA33 bound to a solid phase. Binding of RSE-05 to GPA33 was measured across a concentration range of RSE-05, to determine the concentration of RSE-05 which gives 50% of maximum binding, and thereby an estimate of dissociation constant. Triplicate measurements are in purple, with fitting performed as described

Article Snippet: Human Fc-tagged GPA33 protein (Sino Biologics, cat. no. 11277-H08H) and His-tagged human GPA33 protein (Acro Biosystems, cat. no. GP3-H5224) were used for antibody selection.

Techniques: Isolation, SDS-Gel, Electrophoresis, Staining, Infection, Activity Assay, High Throughput Screening Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Negative Control, Sequencing, Plasmid Preparation, Western Blot, Produced, Concentration Assay

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: Development of antibodies to detect captured GPA33 in an ELISA format. Evaluation of RSE-05 in capture-sensor ELISA format using three distinct polyclonal antibodies to GPA33. A The indicated sera (from peptide 1 and peptide 2 derived from potential GPA33 surface loops; Supplementary Fig. 6) were validated for presence of peptide specific IgG; the sera was affinity purified on a peptide 1 or peptide 2 column, respectively, and after elution with 0.1 M glycine buffer (pH 2.5), fractions were reduced and separated on an SDS gel and stained with Coomassie Blue to validate the presence and amounts of peptide-specific IgG. B Dot-blotting of transfected HEK293T cell lysates without or with GPA33. 7.5 μg of protein lysate was used in each dot. Left, empty-vector transfections. Right, GPA33-gene transfections. Polyclonal fractions 1.5 and 2.6 were used for peptide 1 and peptide 2 respectively. C Anti-GPA33 activity of polyclonal sera by ELISA format, fractions 1.5 and 2.6. 200 ng of tagged GPA33 was coated, or with pre-treatment using 50 mM DTT to reduce di-sulphide bonds, with uncoated wells as negative control. D Sandwich ELISAs using three different polyclonal antibodies in conjunction with RSE-05 to determine whether RSE-05 can function in a capture-sensor format. The indicated antibodies were coated to the immunoplate wells as capture agents (RSE-05, Atlas polyclonal antibody, CPF1.5 P1 polyclonal antibody or CEG 2.6 P2 polyclonal antibody). Following this, 200 ng of recombinant GPA33 protein (FC tagged) was added then complementary antibodies were added in sensor format, labelled as ‘detection’. Anti-mouse or anti-rabbit HRP secondary antibodies were then added, and signal measured using HRP and luminescence

Article Snippet: Human Fc-tagged GPA33 protein (Sino Biologics, cat. no. 11277-H08H) and His-tagged human GPA33 protein (Acro Biosystems, cat. no. GP3-H5224) were used for antibody selection.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Affinity Purification, SDS-Gel, Staining, Transfection, Plasmid Preparation, Activity Assay, Negative Control, Recombinant

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: The anti-GPA33 IgG RSE-05 and scFv-FC fusion antibody was purified from ExpiCHO cells and as bait to define a potential linear epitope component using peptide phage display. Purified RSE-05 IgG or the scFv-FC fusion was captured on protein A beads and incubated with the peptide-phage library, followed by one round of biopanning and next-generation sequencing. The sequencing data was used to derive images from a de novo analysis using Hammock software . A , B The three presented images are derived from Hammock software as the three best motifs, left to right. The web logo (relative amino acid frequencies) shows the most enriched sequence clusters for the GPA33 antibodies. A , B The size of each amino acid corresponds to its relative frequency at a given position within the cluster. A Bits are plotted a function of amino acid at each position to measure how much information (in bits) a given position carries and how ‘certain’ or ‘variable’ this information is. The data are expressed in terms of Shannon information as reported previously . B Probability plotted as a function of amino acid at each position, which indicates the relative frequency (probability of occurrence) of a particular amino acid at a given position in a multiple sequence alignment (MSA) as reported previously . C The graph shows the number of sequences ( y -axis) aligned to the amino acid sequence of the GPA33 protein ( x -axis). The x -axis represents the GPA33 protein sequence (aa 41–60), and the y -axis represents the number of sequences aligned to the GPA33 sequence at each position that are identical to the GPA33 protein sequence. The RSE-05 IgG binding results are plotted with red bars (S024), and the RSE-05 scFv-FC fusion binding data are plotted with blue bars (S014)

Article Snippet: Human Fc-tagged GPA33 protein (Sino Biologics, cat. no. 11277-H08H) and His-tagged human GPA33 protein (Acro Biosystems, cat. no. GP3-H5224) were used for antibody selection.

Techniques: Purification, Incubation, Next-Generation Sequencing, Sequencing, Software, Derivative Assay, Binding Assay

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: Expression of key transmembrane receptors in oesophageal adenocarcinoma. Identification of tumour specific transmembrane receptors in oesophageal adenocarcinoma. A previous study comprising the largest multi-OMIC characterization of oesophageal adenocarcinoma applied mass spectrometry to matched tumour, normal adjacent oesophageal squamous mucosa and normal adjacent gastric mucosa to identify targets that were over-expressed in tumour relative to two related normal tissues. These data are plotted as the ratio of protein expression in tumour versus normal gastric and as a function of tumour versus normal oesophagus. We highlight GPA33 and Claudin 18, as well as other therapeutic antibody targets in OAC, including EGFR and ERBB2. The upper right quadrant contains the tumour elevated targets, where we highlight the transmembrane receptors that might form future diagnostic or therapeutic targets. We label this as Claudin 18 as the tryptic peptides did not cover the 18.2 isoform. B – G Representative expression of GPA33 and Claudin 18.2 in a n = 106 patient TMA. Serial slices of the TMA were taken to compare expression of GPA33 and Claudin 18.2 in the same tumour region. B , C A representative cancer that is GPA33 positive and Claudin 18.2 negative ( n = 38 out of 106); D – E A representative cancer that is positive for GPA33 and Claudin 18.2 ( n = 10 out of 106). Red and blue arrows in D and E highlight, within the same tumour field, membrane positive and membrane negative receptor expression, respectively. F , G A representative expression of GPA33 and Claudin 18.2 in normal gastric epithelium. The brown colour in the images reflects outer membrane expression. Positive GPA33 or Claudin 18.2 membrane expression in the cores used a similar inclusion criterion as described for PD-L1 using IHC, viz. partial or complete linear membrane staining (as described in pharmDx Interpretation Manual—Esophageal Cancer (Dako/Agilent). H A summary of the number of tumours (out of n = 106) with defined GPA33 and Claudin 18.2 expression status. The statistical data are as follows: chi-squared test (without correction): χ 2 = 4.340, df = 1, p = 0.0372; Fisher’s exact test: p = 0.0571; odds ratio = 0.407 (95% confidence interval (CI) 0.163–0.960). The conclusion is that there is a negative association between GPA33 and Claudin 18.2 in cancer cores and their generally mutual exclusive expression is statistically significant

Article Snippet: Human Fc-tagged GPA33 protein (Sino Biologics, cat. no. 11277-H08H) and His-tagged human GPA33 protein (Acro Biosystems, cat. no. GP3-H5224) were used for antibody selection.

Techniques: Expressing, Mass Spectrometry, Diagnostic Assay, Biomarker Discovery, Membrane, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: The RSE-05 MAB binds to full-length GPA33 protein after transfection into human cells. A Validation of GPA33 expression plasmids in human cells. MCF7 cells were transfected with either HA-tagged (T) (lanes 1 and 3) or non-tagged (nT) full length GPA33 containing the transmembrane domain (lanes 2 and 4) expression plasmids, and after acquiring the cell lysates using urea lysis buffer (8 M urea in PBS), a denaturing immunoblot was processed in lanes that either contain DTT (D) (lanes 1 and 2) or without DTT in the sample loading buffer (lanes 3–5). Lysates from non-transfected MCF7 cells are in lane 5. Samples were immunoblotted with the Atlas pAb (commercial antibody), which can detect all isoforms of GPA33. The presumed monomeric form of GPA33 has a faster mobility in the absence of DTT, which presumably reflects SDS resistance of the two IgG-like lobes to denaturation due to the three di-sulphide bonds. B Analysis of RSE-05 MAB binding to GPA33 in fixed cells using immunofluorescence. C , D Analysis of RSE-05 MAB binding to cell surface localized GPA33 in living cells. MCF7 cells were mock transfected ( C ) or transfected with the GPA33 expression plasmid ( D ) and processed by flow cytometry. The data are plotted as: left panels, FITC-A is plotted as a function of FSC-A; right panels, cell count is plotted as a function of FITC-A (left panels and right panels). E MCF7 cells were stably transfected with the GPA33 expression plasmid and flow cytometry was used to isolate the positive cells (in grey) separated from the GPA33 negative cells (in red). The data are plotted as: FITC-A as a function of FSC-A stable GPA33-positive cells and GPA33-negative negative cells. F Analysis of individual GPA33 + cell clones, 3, 7 and 11. Three GPA33 stably expressing cell clones were processed by flow cytometry to measure GPA33-positive staining with the RSE-05 MAB. The data are plotted as: GPA33-positive cells as a function of FITC-A

Article Snippet: Human Fc-tagged GPA33 protein (Sino Biologics, cat. no. 11277-H08H) and His-tagged human GPA33 protein (Acro Biosystems, cat. no. GP3-H5224) were used for antibody selection.

Techniques: Transfection, Biomarker Discovery, Expressing, Lysis, Western Blot, Binding Assay, Immunofluorescence, Plasmid Preparation, Flow Cytometry, Cell Characterization, Stable Transfection, Clone Assay, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: The epitope of the RSE-05 IgG is sensitive to reduction. A The indicated antibodies were used in immunoblots using purified His-tagged GPA33 protein without or with DTT in the SDS loading buffer. After incubating with the primary antibodies and adding anti-mouse or anti-rabbit HRP-conjugated secondary antibodies, membranes were stained using TMB. B ELISA was used to measure the binding activity of the RSE-05 MAB compared with the commercially available rabbit polyclonal antibody (Atlas pAb). Using GPA33 treated with the indicated chemicals (0.5% v/v SDS and/or 50 mM DTT), at different temperatures, RSE-05 and Atlas pAb (commercial antibody) binding was evaluated using anti-mouse or anti-rabbit HRP-conjugated secondary antibody, respectively. The binding activity is measured in luminescence, relative light units. C ELISA performed as in B , in this case with DTT titrated

Article Snippet: Human Fc-tagged GPA33 protein (Sino Biologics, cat. no. 11277-H08H) and His-tagged human GPA33 protein (Acro Biosystems, cat. no. GP3-H5224) were used for antibody selection.

Techniques: Western Blot, Purification, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: Characterization of redox-sensitive epitope using cysteine mutants. A The di-sulphide bonds of GPA33 (green, yellow and magenta) indicated on a ColabFold -generated GPA33 structure (cyan). The Ig-like V type (left) and Ig-like C2-type (right) are shown separately. B Immuno-dot-blots of HEK293T cell lysates following transfection with wild-type GPA33/cysteine-mutant GPA33 expression vectors. Lysates were first reduced with 50 mM or 0 mM DTT. Sample ‘X’ is untransfected control. Membranes shown are representative from three biological replicates. C Denaturing SDS-PAGE immunoblots of HEK293T cell lysates. As in B , lysate was treated with 50 mM or 0 mM DTT prior to separation by SDS-PAGE

Article Snippet: Human Fc-tagged GPA33 protein (Sino Biologics, cat. no. 11277-H08H) and His-tagged human GPA33 protein (Acro Biosystems, cat. no. GP3-H5224) were used for antibody selection.

Techniques: Generated, Transfection, Mutagenesis, Expressing, Control, SDS Page, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform

doi: 10.1186/s11658-025-00852-1

Figure Lengend Snippet: XL-MS identifies sites of interaction between CDR2-FR2 of the RSE-05 IgG and lobe 1 of GPA33. A . Overall analysis of the cross-linked adducts between the IgG and antigen identified using XL-MS . B Detailed sequence analysis of the peptide adducts identified containing the DSS cross link. XL-1, 2, 3 and 4 are marked using green arrows. Yellow highlights FR2 sequences, and green highlights CDR2 sequences. C Position of Ser17 and Lys65 (in magenta colour) cross-linked amino acid sites on GPA33 lobe 1 in relation to the Cys22–Cys96 di-sulphide bond (in yellow)

Article Snippet: Human Fc-tagged GPA33 protein (Sino Biologics, cat. no. 11277-H08H) and His-tagged human GPA33 protein (Acro Biosystems, cat. no. GP3-H5224) were used for antibody selection.

Techniques: Structural Proteomics, 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) 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: Per sample, 4 × 10 6 cells were blocked with human Fc receptor blocking solution (Biolegend, #422302) diluted 1:200 in PBS and afterwards incubated with 10 μg/mL of recombinant, Fc-tagged Nectin-4 protein from human, murine, rat, and cynomolgus origin (ACROBiosystems, Supplementary Table 1) 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: Per sample, 4 × 10 6 cells were blocked with human Fc receptor blocking solution (Biolegend, #422302) diluted 1:200 in PBS and afterwards incubated with 10 μg/mL of recombinant, Fc-tagged Nectin-4 protein from human, murine, rat, and cynomolgus origin (ACROBiosystems, Supplementary Table 1) 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: Per sample, 4 × 10 6 cells were blocked with human Fc receptor blocking solution (Biolegend, #422302) diluted 1:200 in PBS and afterwards incubated with 10 μg/mL of recombinant, Fc-tagged Nectin-4 protein from human, murine, rat, and cynomolgus origin (ACROBiosystems, Supplementary Table 1) 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: Per sample, 4 × 10 6 cells were blocked with human Fc receptor blocking solution (Biolegend, #422302) diluted 1:200 in PBS and afterwards incubated with 10 μg/mL of recombinant, Fc-tagged Nectin-4 protein from human, murine, rat, and cynomolgus origin (ACROBiosystems, Supplementary Table 1) 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: Per sample, 4 × 10 6 cells were blocked with human Fc receptor blocking solution (Biolegend, #422302) diluted 1:200 in PBS and afterwards incubated with 10 μg/mL of recombinant, Fc-tagged Nectin-4 protein from human, murine, rat, and cynomolgus origin (ACROBiosystems, Supplementary Table 1) 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: Per sample, 4 × 10 6 cells were blocked with human Fc receptor blocking solution (Biolegend, #422302) diluted 1:200 in PBS and afterwards incubated with 10 μg/mL of recombinant, Fc-tagged Nectin-4 protein from human, murine, rat, and cynomolgus origin (ACROBiosystems, Supplementary Table 1) 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: Per sample, 4 × 10 6 cells were blocked with human Fc receptor blocking solution (Biolegend, #422302) diluted 1:200 in PBS and afterwards incubated with 10 μg/mL of recombinant, Fc-tagged Nectin-4 protein from human, murine, rat, and cynomolgus origin (ACROBiosystems, Supplementary Table 1) 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: Per sample, 4 × 10 6 cells were blocked with human Fc receptor blocking solution (Biolegend, #422302) diluted 1:200 in PBS and afterwards incubated with 10 μg/mL of recombinant, Fc-tagged Nectin-4 protein from human, murine, rat, and cynomolgus origin (ACROBiosystems, Supplementary Table 1) 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: Per sample, 4 × 10 6 cells were blocked with human Fc receptor blocking solution (Biolegend, #422302) diluted 1:200 in PBS and afterwards incubated with 10 μg/mL of recombinant, Fc-tagged Nectin-4 protein from human, murine, rat, and cynomolgus origin (ACROBiosystems, Supplementary Table 1) in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA).

Techniques: Co-Culture Assay, Positive Control, Incubation