KPMW135, a Biosuperior CD3 Bispecific Version of Rituximab Created By a Novel Chemical Conjugation Technology Demonstrates Increased Anti-Tumor Activity By Adding T Cell-Mediated Cytotoxicity Activity to the Existing Mechanisms of Rituximab

• Published in: Blood 2020-11, Vol.136 (Supplement 1), p.9-10
• Main Authors: Vidal, Christian, Cukan, Michael, Vaill, Ada, Bunin, Anna, Rossi, Ann Marie, Iben, Lawrence, Trinh, David, McGrath, Katy, Berbasova, Tanya, Romee, Rizwan, Alvarez, Enrique, Rastelli, Luca, Welsch, Matthew
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2020-142113

Background We describe KPMW135, a novel CD20 x CD3 bispecific molecule, in which a CD3-directed ScFv (SP34) is conjugated to rituximab (RTX). RTX is a chimeric monoclonal antibody (mAb) specific for CD20, used to treat B-cell lymphomas and lymphocytic leukemias. Monoclonal Ab therapy enhancer (MATE™) enables efficient site-directed chemical conjugation to “off-the-shelf” therapeutic mAbs and allows the development of bispecific therapeutic candidates. Our MATE platform allows for the chemical engineering of existing Abs without the need to create new DNA vectors or genetic engineering of master cell lines. This approach is superior to previous conjugation methods that 1) lack site-directed conjugation specificity by indiscriminately binding to available amino acid residues or 2) require genetic engineering to create conjugate tags. Methods SP34 scFv was constructed using published CDRs. The expression was in FreeStyle 293 cells, followed by affinity purification and SEC to obtain monomeric scFv. The binding of CD20 to KPMW135 was measured by Bio-Layer Interferometry using Octet (Fortebio). Equilibrium dissociation constant (Kd) was calculated using the Octet HT software. The binding of CD3εδ, CD16a, and FcRn were determined by ELISA using neutravidin-coated plates. Biotinylated Human CD3ε & CD3δ heterodimer protein (Avi tag), CD16a (Avi Tag) and human CD16a were used. Readout was determined with anti-human F(ab) HRP. For in vitro T cell-mediated cytotoxicity assays, unfractionated and NK-cell depleted PBMCs were prepared from freshly-thawed and PHA + IL-2 prestimulated PBMCs. Daudi (CD20+) B lymphoblast cells were engineered to stably express a beta-gal reporter fragment using KILR retroparticles (Eurofins). Target cells were treated with KPMW135, RTX, and controls. PBMCs were introduced at an effector:target ratio of 15:1 and incubated for 18h. Luminescence signal was obtained with luminometer to reflect target cell death. Cynomolgus monkeys were intravenously injected with RTX or KPMW135 (30 μg/kg). Endpoints included clinical observations, cytokine profile and flow cytometric immunophenotyping of T cells, monocytes, granulocytes, NK cells, and B cells (CD45, CD3, CD16, CD14, NKG2A, HLA DR) and cellular activation (CD44 and CD69). Results Binding affinity of CD20 to KPMW135 was 0.75nM. CD20 binding to RTX and KPMW135 was similar, thus conjugation of the CD3-binding ScFv did not negatively affect affinity to CD20. ELISA results show KPMW135 binds to CD3εδ