sheep anti 5t4 antibody r d systems  (R&D Systems)

Journal: Molecular Cancer Therapeutics

Article Title: The Bispecific Tumor Antigen-Conditional 4–1BB x 5T4 Agonist, ALG.APV-527, Mediates Strong T-Cell Activation and Potent Antitumor Activity in Preclinical Studies

doi: 10.1158/1535-7163.MCT-22-0395

Figure Lengend Snippet: Molecular design and optimization. A, ALG.APV-527 in the bispecific ADAPTIR format. ALG.APV-527 comprises two different sets of binding domains (scFv) targeting 4–1BB (shown in dark green) and 5T4 (shown in light green) linked to an IgG1 hinge and an IgG1 Fc-domain (shown in gray). The Fc-domain has been engineered to reduce interaction with FcγRs, thereby avoiding depletion of target cells by antibody-dependent cellular cytotoxicity/antibody-dependent cellular phagocytosis (ADCC/ADCP) or complement-dependent cytotoxicity (CDC) and restricting activation of 4–1BB only when simultaneously engaged to 5T4. B, The mode of action of ALG.APV-527: ALG.APV-527 directs the stimulation of T cells and NK cells to 5T4 + tumors and is designed to minimize the toxicity observed with other 4–1BB therapeutics. (i) ALG.APV-527 localizes to tumors where both targets, 4–1BB and 5T4, are highly expressed. (ii) ALG.APV-527 binds simultaneously to 5T4 on tumor cells and 4–1BB on tumor-infiltrating immune cells that activates the immune cells (e.g., enhances secretion of cytolytic molecules such as IFNγ and granzyme B and promotes proliferation). (iii) Activated immune cells induce cytolysis of tumor cells. C, X-ray structure of the parental 4–1BB–binding domain (Fab1618) in ribbon (blue) bound to 4–1BB (green shades, surface projection) showing that the binding epitope includes residues in domain 1 (lemon green) and domain 2 (forest green) of 4–1BB. Eleven hydrogen bonds were found between CDRH1 (yellow), CDRH2 (orange), CDRH3 (magenta), CDRL1 (red), and 4–1BB as shown in the close-up picture to the left. The 4–1BBL (trimeric 4–1BBL, in surface projection, light purple) binding to 4–1BB has been superimposed to illustrate the binding sites of ALG.APV-527, and the 4–1BBL on 4–1BB are distinct.

Article Snippet: For IHC detection of 5T4 expression in human and cynomolgus tissue, two α-5T4 antibodies [Mouse α-5T4 (524731, R&D Systems) and rabbit α-5T4 antibody (EPR5529, Abcam)] were used.

Techniques: Binding Assay, Activation Assay

Journal: Molecular Cancer Therapeutics

Article Title: The Bispecific Tumor Antigen-Conditional 4–1BB x 5T4 Agonist, ALG.APV-527, Mediates Strong T-Cell Activation and Potent Antitumor Activity in Preclinical Studies

doi: 10.1158/1535-7163.MCT-22-0395

Figure Lengend Snippet: ALG.APV-527 binding to target and target expression. A, Binding of ALG.APV-527 to human 5T4-expressing tumor cells using flow cytometry. Secondary fluorescently labeled Fc-targeting Ab was used for detecting bsAb binding and quantified using flow cytometry plotted at MFI. Quantification of 5T4 protein expression was done using BD Quantibrite beads. Antibodies bound per cell (ABC) were calculated using the lots’ standard of PE molecules bound per cell. B and C, Primary human PBMCs were stimulated for 48 hours with or without α-CD3, biotinylated ALG.APV-527, or isotype ADAPTIR. Control was added in a serial dilution followed by streptavidin-APC and cell surface markers for T cells. Cells were then gated on CD3 + CD8 + cells and analyzed for binding using flow cytometry and plotted as ( B ) MFI (background subtracted). C, Human and cynomolgus α-CD3–activated PBMCs were also plotted using nonlinear regression log (agonist) versus normalized response—variable slope. Data normalized pooled data from two experiments are shown, total n = 6. D, Formalin-fixed paraffin-embedded TMAs were stained for 5T4 expression. 5T4 expression was not detected in the liver (1). 5T4 expression was detected in the placenta (2; positive control) and in multiple tumor indications with variable incidence, frequency (percentage of positive cells), and intensity in staining (0–3+), as exemplified in images of NSCLC (3; 2–3+, >75%), malignant epithelial mesothelioma (4; 1–3+, >75%), pancreatic duct adenocarcinoma (5; 1–3+; >75%), and squamous cell carcinoma of the tongue (6; 1–3+ >50–75%).

Article Snippet: For IHC detection of 5T4 expression in human and cynomolgus tissue, two α-5T4 antibodies [Mouse α-5T4 (524731, R&D Systems) and rabbit α-5T4 antibody (EPR5529, Abcam)] were used.

Techniques: Binding Assay, Expressing, Flow Cytometry, Labeling, Control, Serial Dilution, Formalin-fixed Paraffin-Embedded, Staining, Positive Control

Journal: Molecular Cancer Therapeutics

Article Title: The Bispecific Tumor Antigen-Conditional 4–1BB x 5T4 Agonist, ALG.APV-527, Mediates Strong T-Cell Activation and Potent Antitumor Activity in Preclinical Studies

doi: 10.1158/1535-7163.MCT-22-0395

Figure Lengend Snippet: Agonist function of ALG.APV-527 on T cells is dependent on 5T4 engagement resulting in T-cell activation and proliferation. A, 4–1BB reporter cells were stimulated with serial dilutions of ALG.APV-527 or urelumab analogue in the presence of hu5T4 (left), empty vector (left center) transfected CHO-K1 cells, FcγR1-tranfected CHO cells (right center) or different 5T4-expressing tumor cells (right) for 5 hours. The 5T4 receptor levels were evaluated on TF1 (17530/cell), H1975 (43153/cell), and MDA-MB-231 (32670/cell) using QuantiBright beads. B, PBMC were cocultured with MDA-MB-231 cells, 10 ng/mL OKT3, and a titration of ALG.APV-527 or huIgG1 controls for 6 days. The graphs show the number of CD8 T cells that had undergone at least one cell division and are represented as cell counts. C, Primary cell trace–labeled PBMCs were stimulated with α-CD3 Ab in solution and serial dilutions of ALG.APV-527 in the presence of 5T4-expressing CHO-K1 cells. The number of CD8 + T cells (left) and a representation of the proliferation of CD8 + T cells (right) were evaluated at 96 hours via flow cytometry. Average of three healthy donors is graphed. D and E, Primary PBMCs were stimulated with 0, 10 or 100 ng/mL of α-CD3 (OKT-3) in solution and serial dilutions of ALG.APV-527 in the presence of CT26-hu5T4 or CT26-WT cells for 48 hours. D, Expression of 4–1BB on CD4 T cells, CD8 T cells and NK cells. E, Intracellular IFNγ levels were evaluated using flow cytometry in CD8 T cells (left) or CD4 T cells (right). F, Purified T cells and sub-lethally UV-irradiated CT26-WT cells or CT26-hu5T4 tumor cells transfected to express 5T4 (1×10 6 /cell) were incubated with a dilution of ALG.APV-527. The secretion of IFNγ was measured in the supernatant after 72 hours of culture using ELISA. G, Purified CD8 T cells were co-cultured with HCT 116 tumor cells, α-CD3 (OKT-3) on beads, and serial dilutions of ALG.APV-527. IFNγ was assessed by ELISA. H, PBMCs were cultured with either MDA-MB-231 or H1975 tumor cell lines, α-CD3 OKT3 at 10 ng/mL and titrated ALG.APV-527, or the negative control ADAPTIR. OX40 surface expression was evaluated via flow cytometry at 72 hours.

Article Snippet: For IHC detection of 5T4 expression in human and cynomolgus tissue, two α-5T4 antibodies [Mouse α-5T4 (524731, R&D Systems) and rabbit α-5T4 antibody (EPR5529, Abcam)] were used.

Techniques: Activation Assay, Plasmid Preparation, Transfection, Expressing, Titration, Labeling, Flow Cytometry, Purification, Irradiation, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture, Negative Control

Journal: Molecular Cancer Therapeutics

Article Title: The Bispecific Tumor Antigen-Conditional 4–1BB x 5T4 Agonist, ALG.APV-527, Mediates Strong T-Cell Activation and Potent Antitumor Activity in Preclinical Studies

doi: 10.1158/1535-7163.MCT-22-0395

Figure Lengend Snippet: ALG.APV-527 enhances NK cell activation and proliferation. A and B, Primary IL2-stimulated (10 ng/mL) NK cell assays and Mitomycin C–treated HCT116 tumor cells expressing endogenous levels of 5T4 (6.2×10 4 /cell) were incubated with a dilution of ALG.APV-527. A, The secretion of Granzyme B and IFNγ was measured in the supernatant after 72 hours of culture using ELISA. B, The expression of CD25 was measured by flow cytometry after 24 hours in culture. C, Primary NK cells were stimulated with IL2 (8 ng/mL) and serial dilutions of ALG.APV-527 in the presence of irradiated CHO-hu5T4 cells. The number of CD335 + NK cells were assessed in 2 healthy samples on day 6 by flow cytometry ( C , left) and representative dilution of CellTrace proliferation ( C , right).

Article Snippet: For IHC detection of 5T4 expression in human and cynomolgus tissue, two α-5T4 antibodies [Mouse α-5T4 (524731, R&D Systems) and rabbit α-5T4 antibody (EPR5529, Abcam)] were used.

Techniques: Activation Assay, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Irradiation

Journal: Molecular Cancer Therapeutics

Article Title: The Bispecific Tumor Antigen-Conditional 4–1BB x 5T4 Agonist, ALG.APV-527, Mediates Strong T-Cell Activation and Potent Antitumor Activity in Preclinical Studies

doi: 10.1158/1535-7163.MCT-22-0395

Figure Lengend Snippet: ALG.APV-527 localizes to 5T4-expressing tumors in vivo and induces tumor rejection and memory responses. A, Twin tumor model experimental set-up: On day 0, each mouse received one 5T4-negative (B16.WT) and one 5T4-positive (B16-hu5T4) tumor injected (subcutaneous, 1×10 5 cells) at each side of the hind flank/back. Intraperitoneal treatment of ALG.APV-527 (100 μg) was given on days 6 and 13, and mice were sacrificed on day 14 (24 hours after the final treatment). Tumors were collected, and the level of binding of ALG.APV-527 to cells was assessed by IHC using an antibody-detecting human IgG. B, IHC images of ALG.APV-527 or vehicle control localized to 5T4-negative tumors (left) or 5T4-positive tumors (right). C, Detection of ALG.APV-527 binding to single-cell suspensions of 5T4-positive or -negative tumors using anti-human IgG by flow cytometry. The graph shows the mean frequency ± SEM of human IgG + cells among live CD45 − tumor cells after two doses of ALG.APV-527 ( n = 5). As control of maximal binding to the single-cell suspension, reagents (ALG.APV-527 and anti-human IgG) were also added ex vivo to only vehicle-treated mice ( n = 2). D, Experimental set-up to assess antitumor efficacy and memory responses. E and F, Day 0, 4×10 6 MB49 cells expressing human 5T4 were injected subcutaneously into hu4–1BB KI mice. Starting on day 7, treatments of ALG.APV-527 or urelumab analogue were administered intraperitoneally twice weekly until day 24, and tumor volume and survival were assessed over time. Tumor volumes and survival from three studies were combined; n = 8–16 mice per group. The dose schedule was the same for all studies with treatments on days 7, 10, 13, 17, 20, and 24 post tumor challenge. E, Mean ± SEM of tumor volume for each group, pooled from the three separate studies is presented. For animals that reached tumor endpoints, that last recorded value was carried forward. F, Survival events were recorded each time a mouse reached the endpoint (tumor volume ≥1,500 mm 3 ). Pooled survival data from three studies are presented as median survival and statistical significance were calculated for the combined three studies using JMP survival analysis with a log-rank test and Wilcoxon test for comparison of survival curves. G, Complete responder mice that had cleared their primary tumors were re-challenged with 4×10 6 MB49–5T4 or 0.5×10 6 MB49-WT tumor cells on day 80. Naïve mice served as controls. No additional therapy was given. Seven to eight mice/treatment group. E and G, Mean tumor volume +SEM for each group is plotted until the first mouse in group reached the endpoint. Differences in mean tumor volume from day 3 through day 26 for the study groups were determined using JMP-repeated measures’ analysis with the Tukey's multiple comparison test. All treatment groups ( P < 0.05) were considered significant compared with controls.

Article Snippet: For IHC detection of 5T4 expression in human and cynomolgus tissue, two α-5T4 antibodies [Mouse α-5T4 (524731, R&D Systems) and rabbit α-5T4 antibody (EPR5529, Abcam)] were used.

Techniques: Expressing, In Vivo, Injection, Binding Assay, Control, Flow Cytometry, Suspension, Ex Vivo, Comparison

Journal: Avicenna Journal of Medical Biotechnology

Article Title: Antibody-Drug Conjugates: Possibilities and Challenges

doi:

Figure Lengend Snippet: Current ADCs in clinical development based on targeting antigens with an overview of their properties

Article Snippet: PF-06263507, anti-5T4 ADC , Phase I, for treatment of lung and breast cancer with 5T4 expression , huIgG1 anti-5T4 , MMAF , Native cysteine residues, MC noncleavable linker , n/a,4.34 mg/kg , no , Pfizer ( ) .

Techniques: Activity Assay, Expressing, Transduction, Migration, Coagulation, Infection, Cell Differentiation, Inhibition, Transformation Assay