Anti psma antibody clone lni 17

To verify the presence of the OX40L, 4-1BBL, and anchored GM-CSF and LD ligands on the surface of the VLPs, we performed flow cytometry of VLPs bound to micrometric polystyrene particles (Polybead microspheres, Polysciences, Warrington, PA, USA). For the detection of OX40L and 4-1BBL, 5 × 10⁴ beads were previously loaded with 1 μg capture anti-4-1BBL antibodies (eBioscience clone TKS-1) or anti-OX40L (eBioscience clone OX89) and incubated to 10⁹ 4-1BBL + OX40L bivalent VLPs or CON VLPs. These beads with antibodies and VLPs were stained with anti-4-1BBL PE or anti-OX40L PE and flow cytometry was performed. To verify the LD ligand, beads were previously loaded with the PSMA protein. Briefly, 5 × 10⁴ beads were incubated with anti-PSMA antibody (clone LNI-17, BioLegend, San Diego, CA, USA) and then incubated with a lysate of PSMA-expressing NIH-3T3 cells (Figure S10C). The VLPs LD + GM-CSF + 4-1BBL, LD + GM-CSF + OX40L and LD-CON VLPs were added to the PSMA beads labeled with anti-4-1BBL PE, anti-OX40L PE, and anti-GM-CSF PE antibodies following flow cytometry.

CAR T cells were stained with a Live/Dead Fixable Near-IR stain (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min, followed by a phosphate-buffered saline (PBS) wash and incubation with a ChromPure Human IgG blocking solution (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 5 min. An antibody cocktail containing anti-PSMA antibody (clone LNI-17; BioLegend, San Diego, CA, USA), anti-EGFRt (Erbitux; Lilly, Indianapolis, IN, USA), anti-CD4 (clone OKT4; BioLegend), anti-CD8 (clone RPA-T8; BioLegend), and an in-house–generated anti-idiotypic antibody to detect the CAR was added for 20 min. Cells were then washed twice with staining buffer (BioLegend) and analyzed on a NovoCyte cytometer (ACEA Biosciences, San Diego, CA, USA). Data analysis was performed using FlowJo v10.1 software (Tree Star, Ashland, OR, USA).

Tumor cells were either isolated from fresh prostate tumor tissue procured from LF’s lab at UCSF (San Francisco, California, USA) or received as frozen dissociated aliquots along with donor matched PBMCs from Discovery Life Sciences (Newtown, Pennsylvania, USA). The fresh prostate tissue was dissociated as previously described.31 (link) For the cytotoxicity assay, exogenous human PBMCs were co-cultured with dissociated tumor cells at an E:T of 1:2 for 24 hours at 37°C, 8% CO₂. The death of PSMA⁺ cells was analyzed by flow cytometry on the BD FACSCelesta. An anti-CD45 antibody (BioLegend) was used to distinguish hematopoietic cells, and a non-epitope competing anti-PSMA antibody (clone LNI17, BioLegend) was used to identify PSMA⁺ cells. The percentage of PSMA⁺ live cells was determined using eBioscience Fixable Viability Dye eFluor 780 (Invitrogen, Carlsbad, California, USA). Culture supernatant from each well was used to measure IL-2, IFNγ, and TNFα by MSD.