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1196 Streamlining T cell engager development with a diverse panel of fully human CD3-binding antibodies, bispecific engineering technology, and an integrated discovery engine
  1. Lindsay DeVorkin,
  2. Juntao (Matt) Mai,
  3. Kate Caldwell,
  4. Tim Jacobs,
  5. Raffi Tonikian,
  6. Karine Herve,
  7. Yuri Hwang,
  8. Cristina Faralla,
  9. Wei Wei,
  10. Emma Lathouwers,
  11. Rhys Chappell,
  12. Stefan Hannie,
  13. Katherine Lam,
  14. Harveer Dhupar,
  15. Tran Tran,
  16. Melissa Cid,
  17. Lena Bolten,
  18. Tova Pinsky,
  19. Ping Xiang,
  20. Courtenay Lai,
  21. Ahn Lee,
  22. Patrick Chan,
  23. Jasmine Chin,
  24. Aaron Yamniuk,
  25. Kush Dalal and
  26. Bryan Barnhart
  1. AbCellera, Vancouver, Canada


Background CD3 T cell engagers have the potential to be a cornerstone of immuno-oncology. However, a limited pool of CD3-binding antibodies and technological challenges in engineering bispecifics have hindered development. Discovering effective T cell engagers requires two target-binding arms — a CD3 arm that fine-tunes T cell activation and a tumor arm with high specificity for cancer cells — optimized as a whole to work in concert with each other. Beginning with diverse panels of antibodies increases the probability of finding appropriately potent and developable T cell engagers and reduces the need for downstream engineering.

Methods We used microfluidic technology to screen more than 3.5 million single cells from humanized mice and identified >200 CD3-specific antibodies. Using high-throughput assays, we determined affinity for CD3εδ and CD3εγ, cross-reactivity to human and cyno primary T cells, CD3 binding kinetics, and epitope bins. We assessed T cell activation by measuring CD25 and CD69 expression by flow cytometry. We then used our bispecific engineering platform, OrthoMabTM, to generate a proof-of-concept panel of CD3 x EGFR bispecific antibodies. Developability properties were assessed, including hydrophobicity (aHIC), self-association (AC-SINS), polyspecificity (BVP-ELISA), stability (nanoDSF), and aggregation (aSEC). CD3 T cell engager potencies were measured using an NFAT reporter T cell activation assay and an xCELLigence tumor cell killing assay, and cytokine release was assessed by FLEXMAP CD.

Results We identified hundreds of fully human CD3-specific antibodies that are diverse, developable, and validated. The antibodies displayed a wide range of CD3 binding affinities (KD ~1 nM to 1 μM), binding kinetics, and T cell activation potencies (EC50 ~6 to 190 nM). Data on this novel panel includes epitope binning, which revealed human and cyno CD3-binders that are distinct from previously described cross-reactive antibodies. The antibodies were assessed using a range of biophysical assays and have favorable developability properties. In an expanded proof-of-concept study, we used OrthoMabTM to generate a panel of CD3 x EGFR bispecific antibodies. The resulting bispecifics had favorable developability properties, and displayed a wide range of antigen-dependent T cell activation (EC50 ~2 pM to 2 nM) and tumor cell killing potencies (EC50 ~0.01 to 1 nM). From this panel, we identified potent T cell engagers that achieved >90% tumor cell killing with low levels of cytokine release.

Conclusions By integrating our panel of CD3-binding antibodies with our bispecific engineering and high-throughput antibody assessment capabilities, we identified developable CD3 T cell engagers with potent tumor cell-killing activity and minimal cytokine release.

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