Solution structure and synaptic analyses reveal determinants of bispecific T cell engager potency

  1. Alexander Leithner
  2. Oskar Staufer
  3. Tanmay Mitra
  4. Falk Liberta
  5. Salvatore Valvo
  6. Mikhail Kutuzov
  7. Hannah Dada
  8. Jacob Spaeth
  9. Sally Zhou
  10. Felix Schiele
  11. Sophia Reindl
  12. Herbert Nar
  13. Stefan Hoerer
  14. Maureen Crames
  15. Stephen Comeau
  16. David Young
  17. Sarah Low
  18. Edward Jenkins
  19. Simon J. Davis
  20. David Klenerman
  21. Andrew Nixon
  22. Noah Pefaur
  23. David Wyatt
  24. Omer Dushek
  25. Srinath Kasturirangan
  26. Michael L. Dustin
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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 modelling 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 co-stimulation 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).

Significance statement

Bispecific T-cell engagers (TcEs) are immunotherapeutic drugs that trigger the destruction of cancer cells by linking T cells to cancer cell through specific surface molecules (antigens). We designed a series of TcEs with varying distances between their binding sites and flexibilities of the TcE-antigen complexes. By combining structural and functional analyses, we confirmed close-contact formation between T cells and cancer cells as a critical determinant, mediated by co-activating receptors. Furthermore, we also identified molecular flexibility of the TcE-antigen complex as a further critical parameter for TcE potency. These findings provide novel insights into TcE function and highlight the importance of both parameters for future research and the design of improved immunotherapies.

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