Engineering a tumor-selective prodrug T-cell engager bispecific antibody for safer immunotherapy

ABSTRACT

T-cell engaging (TCE) bispecific antibodies are potent drugs that trigger the immune system to eliminate cancer cells, but administration can be accompanied by toxic side effects that limit dosing. TCEs function by binding to cell surface receptors on T cells, frequently CD3, with one arm of the bispecific antibody while the other arm binds to cell surface antigens on cancer cells. On-target, off-tumor toxicity can arise when the target antigen is also present on healthy cells. The toxicity of TCEs may be ameliorated through the use of pro-drug forms of the TCE, which are not fully functional until recruited to the tumor microenvironment. This can be accomplished by masking the anti-CD3 arm of the TCE with an autoinhibitory motif that is released by tumor-enriched proteases. Here, we solve the crystal structure of the antigen-binding fragment of a novel anti-CD3 antibody, E10, in complex with its epitope from CD3 and use this information to engineer a masked form of the antibody that can activate by the tumor-enriched protease matrix metalloproteinase 2 (MMP-2). We demonstrate with binding experiments and in vitro T-cell activation and killing assays that our designed prodrug TCE is capable of tumor-selective T-cell activity that is dependent upon MMP-2. Furthermore, we demonstrate that a similar masking strategy can be used to create a pro-drug form of the frequently used anti-CD3 antibody SP34. This study showcases an approach to developing immune-modulating therapeutics that prioritizes safety and has the potential to advance cancer immunotherapy treatment strategies.

KEYWORDS:

• Antibody engineering
• bispecific antibody
• cancer
• CD3
• cytokine release syndrome
• HER2
• T cell activation
• T cell retargeting
• x-ray structure

Acknowledgments

This research used resources of the Advanced Photon Source; a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly and Company, which operates the facility.

This research is based in part upon work conducted using the UNC Proteomics Core Facility, which is supported in part by NCI Center Core Support Grant (2P30CA016086-45) to the UNC Lineberger Comprehensive Cancer Center.

Disclosure statement

S.D., K.F., S.A., and M.H. are or were employees of Eli Lilly. S.D. is an employee of Tentarix Biotherapeutics.

Author contributions

Conceptualization, B.K., S.D., K.F., and A.C.M.; Methodology, B.K., S.D., and A.C.M.; Data Collection, A.C.M.; S.A., and M.H.; Data Analysis, B.K. and A.C.M.; Writing – draft preparation, B.K. and A.C.M.; Writing – review and editing, B.K., A.C.M., K.F., S.A., and M.H.

Data availability statement

Coordinates and structure factors for E10 fab complexed to the CD3ε-N7 peptide at pH 6.5 have been deposited in the Protein Data Bank under accession code 8VY4. All other data are available in the manuscript or supplementary materials.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2024.2373325

Additional information

Funding

This work was supported by the National Institutes of Health grants R35GM131923 (BK), T32GM008570 (ACM), and by a UNC Dissertation Completion Fellowship (ACM).