Journal: mBio

Article Title: Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

doi: 10.1128/mBio.01624-16

Figure Lengend Snippet: Murine and human stalk-specific IgG antibodies activate the antibody-dependent cellular phagocytosis (ADCP) pathway in an epitope-specific manner. (A and B) ADCP reporter assays were performed on Cal09-infected (A) or X-31-infected (B) A549 cells using a panel of human (A) or murine (B) monoclonal antibodies. (A) The broadly neutralizing stalk-specific antibodies h6F12 and hKB2 were assessed for their ability to activate FcγRIIA. (B) The same assay was repeated using the broadly neutralizing HA stalk binding antibody 9H10 and head binding antibody XY102. The means and standard errors of means are shown for three independent experiments. (C) Bead-based phagocytosis assays were performed using fluorescent microspheres sequentially coated with protein L, followed by either the HA stalk-specific antibody h6F12 or the strain-specific head antibody h29E3. Preparation of the beads in this way leaves the antibody Fc region accessible. (D) Freshly isolated human neutrophils were incubated with microspheres. Following incubation, unbound microspheres were removed by centrifugation and washing; uptake was measured by fluorescence. Means and standard errors of means are shown. Data are representative of three independent experiments ( n = 8 donors in total). Values that are significantly different ( P < 0.0001) by Kruskal-Wallis test with Dunn’s multiple comparison are indicated by the bar and four asterisks. Values that are not significant (ns) are also indicated.

Article Snippet: Antibodies and infected cells were incubated at 37°C for 30 min. At the completion of incubation, Jurkat effector cells (Promega) stably expressing FcγRIIA were resuspended in assay buffer and added to plates at a concentration of 1.5 × 10 4 cells per well.

Techniques: Infection, Bioprocessing, Binding Assay, Isolation, Incubation, Centrifugation, Fluorescence, Comparison

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Binding affinities to human HER2 and CD3 at 37 °C by surface plasmon resonance. Data are K D ( n = 12 technical replicates of a single experiment for HER2-XPAT protein and HER2(1x-N); n = 8 for HER2(1x-C) and uTCE). Surface plasmon resonance sensorgrams for these data are provided in Supplementary Figs. – . b – d , In vitro tumor cytotoxicity of HER2-XPAT protein and its metabolites following a 48-h incubation with co-cultures of huPBMCs and the high HER2-expressing human tumor cell lines (1:1 effector–target ratio) SKOV3 ( b ), BT-474 ( c ) or the medium-low HER2-expressing MCF7 cell line ( d ). e , In vitro cytotoxicity of HER2-XPAT protein and its metabolites against BT-474 cells co-cultured with huPBMCs (1:1 effector–target ratio). f , Impact of the protease-cleavable linker on in vitro cytotoxicity versus BT-474 cells co-cultured with huPBMCs. g , h , CD69-positive T cells ( g ) and IL-2 secretion ( h ) following 72-h incubation of huPBMC/SKOV3 co-cultures with HER2-XPAT protein or its unmasked form (uTCE). i , Target-dependent T-cell activation with HER2-XPAT protein and its metabolites. CD3-expressing Jurkat reporter T cells were incubated with BT-474 cells at a 5:1 effector–target ratio for 6 h, followed by quantification of NFAT -induced luciferase activity and measured in relative luminescence units (RLUs). Mean data for n = 2 technical replicates within one single experiment ( b – i ). Extended Data Table provides a summary of EC 50 values for the different forms of the HER2-XPAT proteins in the cytotoxicity and reporter T-cell activation assays.

Article Snippet: The activation of CD3 + Jurkat T cells (Promega, J1625, J1601) by HER2-XPAT protein and unmasked HER2-TCE was assessed.

Techniques: Binding Assay, SPR Assay, In Vitro, Incubation, Expressing, Cell Culture, Activation Assay, Luciferase, Activity Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: CD3 + Jurkat reporter T cells were incubated with or without BT-474 cells at a 5ː1 effector-target ratio for 6 hours, followed by quantification of NFAT -induced luciferase activity. The graph shows individual data points from a single representative experiment. There were n = 2 biological repeats at each concentration tested within the same experiment for each cell line. HER2, human epidermal growth factor receptor 2; RLU, relative luminescence unit; uTCE, unmasked T-cell engager; XPAT proteins, TCE fused to XTEN polypeptides.

Article Snippet: The activation of CD3 + Jurkat T cells (Promega, J1625, J1601) by HER2-XPAT protein and unmasked HER2-TCE was assessed.

Techniques: Incubation, Luciferase, Activity Assay, Concentration Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , An XPAT protein comprises a TCE core with two scFvs, one targeting CD3 and the other, a TAA. Each scFv is masked by a protease-releasable XTEN mask, unstructured, hydrophilic polypeptides that act as modular, tunable masks, in addition to extending the half-life of the TCE. Each XTEN mask connects to the TCE core via a protease-cleavable linker, designed to be cleavable by any of eight proteases from three different classes (matrix metalloproteinases, serine proteases and cysteine proteases) involved in cancer progression . b , Predicted structure of HER2-XPAT protein visualized using AlphaFold2 v.2.0, a machine-learning-based computational method for predicting protein structures with reasonable accuracy . Colors indicate anti-HER2 domain, pale green; anti-CD3 domain, light orange; XTEN masks, blue; protease-cleavable linker, red; linkers, gray. The model represents a static picture showing a plausible conformation of the unstructured XTEN masks and the length of unstructured XTEN relative to the folded antibody domains in an XPAT protein. c , XPAT proteins are expected to remain largely intact in healthy tissues, where protease activity is well controlled by protease inhibitors. XPAT protein unmasking occurs in two steps via one of two potential paths to the fully unmasked state. The two requisite cleavage events can occur in either order and each sequence (either the top or bottom paths shown) is equally likely. In aggregate, both 1x-N and 1x-C partially unmasked forms will exist, depending on the cleavage path. Removal of both XTEN masks liberates the unmasked HER2-TCE (uTCE). d , XPAT proteins are designed to exploit the dysregulated protease activity present in tumors versus healthy tissues and expand the therapeutic index of TCEs through preferential unmasking in the TME. The active uTCE promotes the formation of immunologic synapses between tumor and T cells, resulting in potent cytotoxicity. Notably, the uTCE has a short half-life and should be rapidly cleared, thereby sparing healthy tissues when the uTCE diffuses away from the TME. By design, the molecular weight of the uTCE (∼59 kDa) is sufficiently small to allow rapid kidney filtration .

Article Snippet: The activation of CD3 + Jurkat T cells (Promega, J1625, J1601) by HER2-XPAT protein and unmasked HER2-TCE was assessed.

Techniques: Activity Assay, Sequencing, Molecular Weight, Filtration