Journal: bioRxiv

Article Title: Dual Targeting for Enhanced Tumor Immunity: Conditionally Active CD28xVISTA Bispecific Antibodies Promote Myeloid-Driven T-Cell Activation

doi: 10.1101/2025.04.07.647657

Figure Lengend Snippet: CD28xVISTA bsAb induces IL-2–luciferase reporter gene expression both in cis and in trans . A, Luciferase expression from a Jurkat IL-2-luciferase reporter cell line as a function of concentration of CD28xVISTA BS1 (green) or parental monospecific mAbs anti-VISTA (orange) or anti-CD28 (blue). B, Luciferase expression from the reporter cell line co-cultured with a HEK293 cell line expressing anti-CD3 (OKT3-scFv) alone (open symbols; left in schematic) or in combination with VISTA (filled symbols; right in schematic) enabling co-stimulation by a CD28xVISTA bsAb in cis . C, Luciferase expression from the IL-2 reporter cell line as a function of CD28xVISTA BS1 when co-cultured with the HEK293 cell line expressing anti-CD3 (OKT3-scFv) in combination with a CHO cell line overexpressing VISTA (orange) enabling co-stimulation by a CD28xVISTA bsAb in trans . A parallel negative control with the parental CHO cells substituting for CHO-VISTA cells (blue) or a positive control with the HEK293 cell line co-expressing anti-CD3 (OKT3-scFv) and VISTA (allowing for cis co-stimulation; green) are indicated.

Article Snippet: Human T-cell mediated killing of LNCaP prostate cancer cells was analyzed on the xCELLigence real-time cell analysis platform (Agilent) by co-culturing LNCaP cells with PBMCs and VISTA + Kasumi-3 cells in the presence of a CD3xPSMA bispecific T-cell engager (BPS Bioscience; Cat. # 101242-2 with a CD3-binding domain from the CD3xCD20 TCE Mosunetuzumab) alone or in combination with CD28xVISTA BS2.

Techniques: Luciferase, Gene Expression, Expressing, Concentration Assay, Cell Culture, Negative Control, Positive Control

Journal: bioRxiv

Article Title: Dual Targeting for Enhanced Tumor Immunity: Conditionally Active CD28xVISTA Bispecific Antibodies Promote Myeloid-Driven T-Cell Activation

doi: 10.1101/2025.04.07.647657

Figure Lengend Snippet: In vitro human T-cell co-stimulation in trans by CD28xVISTA BS2 (non-pH sensitive) potentiates LNCaP killing by a CD3xPSMA T-cell engager. A, Schematic representation of LNCaP prostate cancer cells co-cultured with Kasumi-3 and T-cells from human PBMCs. LNCaP killing by a CD3xPSMA TCE can potentially be enhanced through CD28 trans co-stimulation by CD28xVISTA BS2 B, Signal resulting from LNCaP cells in the Agilent xCELLigence assay platform in the absence of mAbs (green squares), in the presence of increasing sub-optimal concentrations of CD3xPSMA alone (4 ng/ml (orange), 12 ng/ml (light green) or 37 ng/ml (blue)); or same concentrations of CD3xPSMA in the presence of 0.1 μg/ml CD28xVISTA BS2 (purple, black, and blue triangles, respectively). A control with lysis buffer added to co-culture wells is indicated by gray. Only growth of the adherent LNCaP cells caused a change in the impedance signal measured by the xCELLigence platform. C, IL-2 or TNF-α cytokine release measured Day 1, ( D ) T-cell activation measured as %CD25 + of total T-cells on Day 6 and ( E ) T-cell proliferation enumerated as T-cell count per volume on Day 6 in the absence (blue) or presence of 0.1 μg/ml CD28xVISTA BS2 (orange) (mean values +/-SD; n=3 technical replicates).

Article Snippet: Human T-cell mediated killing of LNCaP prostate cancer cells was analyzed on the xCELLigence real-time cell analysis platform (Agilent) by co-culturing LNCaP cells with PBMCs and VISTA + Kasumi-3 cells in the presence of a CD3xPSMA bispecific T-cell engager (BPS Bioscience; Cat. # 101242-2 with a CD3-binding domain from the CD3xCD20 TCE Mosunetuzumab) alone or in combination with CD28xVISTA BS2.

Techniques: In Vitro, Cell Culture, Control, Lysis, Co-Culture Assay, Activation Assay, Cell Counting