Solution structure and synaptic analyses reveal determinants of bispecific T cell engager potency
Abstract
Bispecific T cell engagers (TcEs) link T cell receptors to tumor-associated antigens on cancer cells, forming cytotoxic immunological synapses (IS). Close membrane-to-membrane contact (≤13 nm) has been proposed as a key mechanism of TcE function. To investigate this and identify potential additional mechanisms, we compared four immunoglobulin G1-based (IgG1) TcE Formats (A–D) targeting CD3ε and Her2, designed to create varying intermembrane distances (A < B < C < D). Small-angle X-ray scattering (SAXS) and modeling of the conformational states of isolated TcEs and TcE–antigen complexes predicted close contacts (≤13 nm) for Formats A and B and far contacts (≥18 nm) for Formats C and D. In supported lipid bilayer (SLB) model interfaces, Formats A and B recruited, whereas Formats C and D repelled, CD2–CD58 interactions. Formats A and B also excluded bulky Quantum dots more effectively. SAXS also revealed that TcE–antigen complexes formed by Formats A and C were less flexible than complexes formed by Formats B and D. Functional data with Her2-expressing tumor cells showed cytotoxicity, surface marker expression, and cytokine release following the order A > B = C > D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A = B = C > D. Addition of close contact requiring CD58 costimulation revealed phospholipase C-γ activation matching cytotoxicity with A > B = C > D. Our findings suggest that when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. Both the close/far-contact formation axis and the low/high flexibility axis significantly impact TcE potency, explaining the similar potency of Format B (close contact/high flexibility) and C (far contact/low flexibility).
Article Details
Journal Info
Proceedings of the National Academy of Sciences
National Academy of Sciences
Authors (26)
Alexander Leithner
The Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), University of Oxford, Roosevelt Drive
Oskar Staufer
The Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), University of Oxford, Roosevelt Drive
Tanmay Mitra
The Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), University of Oxford, Roosevelt Drive
Falk Liberta
Boehringer Ingelheim Pharma GmbH & Co. Kg.
Salvatore Valvo
The Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), University of Oxford, Roosevelt Drive
Mikhail Kutuzov
Sir William Dunn School of Pathology, University of Oxford, South Parks Road
Hannah Dada
The Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), University of Oxford, Roosevelt Drive
Jacob Spaeth
The Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), University of Oxford, Roosevelt Drive
Weijie Zhou
Biotherapeutics Discovery, Boehringer Ingelheim
Felix Schiele
Boehringer Ingelheim Pharma GmbH & Co. Kg.
Sophia Reindl
Boehringer Ingelheim Pharma GmbH & Co. Kg.
Herbert Nar
Boehringer Ingelheim Pharma GmbH & Co. Kg.
Stefan Hoerer
Boehringer Ingelheim Pharma GmbH & Co. Kg.
Maureen Crames
Biotherapeutics Discovery, Boehringer Ingelheim
Stephen Comeau
Biotherapeutics Discovery, Boehringer Ingelheim
David Young
Biotherapeutics Discovery, Boehringer Ingelheim
Sarah Low
Biotherapeutics Discovery, Boehringer Ingelheim
Edward Jenkins
The Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), University of Oxford, Roosevelt Drive
Simon J. Davis
Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital
David Klenerman
Department of Chemistry, University of Cambridge
Andrew Nixon
Biotherapeutics Discovery, Boehringer Ingelheim
Noah Pefaur
Biotherapeutics Discovery, Boehringer Ingelheim
David Wyatt
Boehringer Ingelheim Pharma GmbH & Co. Kg.
Omer Dushek
Sir William Dunn School of Pathology, University of Oxford, South Parks Road
Srinath Kasturirangan
Biotherapeutics Discovery, Boehringer Ingelheim
Michael L. Dustin