A Novel Her2/VEGFR2/CD3 trispecific antibody with an optimal structural design showed improved T-cell-redirecting antitumor efficacy

Abstract

Rationale: T-cell-redirecting bispecific antibodies (bsAbs) and trispecific antibodies (tsAbs) designed to recognize different epitopes or antigens have emerged as promising cancer therapies. Current approaches are all designed to include another antibody specific to the site of the primary antibody, and the molecular structures are generally established. However, the dimensions of target molecule and epitope location play a key role in the efficiency of the immunological synapse (IS) formation and subsequent T-cell-redirecting activities, therefore the connection flexibility of these antibodies determines the geometries of different formats of these molecules and will have a major impact on the efficacy. Methods: We describe a novel recombination strategy using various linker designs to site-specifically fuse anti-Her2 (2Rs15) or anti-VEGFR2 (3VGR19) nanobodies to different positions of the anti-CD3 antibody fragment (Fab, SP34). Based on the comparison among the various antigen-specific bsAbs, we could determine the desired fusion site of each nanobody to SP34, and further ensure the optimal structure of tsAbs with synergistic dual-antigen enhanced T-cell-redirecting activities. Results: This approach allows precise control of the formation of IS between Her2- and/or VEGFR2-expressing cancer cells and T cells, to obtain the optimal structure of the Her2/VEGFR2/CD3 tsAb without the need to map antibody-binding epitopes. Optimization of Her2/VEGFR2/CD3 tsAb results in enhanced T-cell-redirecting in vitro and in vivo antitumor efficacy compared with the corresponding bsAbs alone or in combination, and the potency to overcome tumor relapse due to antigen escape or resistance to Herceptin and Cyramza therapy. Conclusion: The novel design strategy for developing tsAbs using a site-specific recombination approach represents a promising platform for immuno-oncology and in applications other than cancer therapy.

When three is not a crowd: trispecific antibodies for enhanced cancer immunotherapy

“…Moreover, the TsAb-based treatment inhibited tumor growth in a PC3 tumor model resistant to the approved mAb trastuzumab (anti-HER2) and ramucirumab (anti-VEGFR2). These results further supported the rationale that TAA dual targeting can improve the therapeutic index of TsAb 35 .…”

The clinical regimens and cell membrane camouflaged nanodrug delivery systems in hematologic malignancies treatment

“…This additional moiety allows for the targeting of an extra tumor-associated antigen, enhancing specificity and preventing immune evasion. Alternatively, it can target additional costimulatory receptors on immune cells, thereby enhancing their effector functions ( Liu D. et al, 2022 ). Until now, around 100 bispecific T cell engagers are in clinical trials, and NK cell engagers and TsAbs are currently undergoing early-stage clinical studies because of late starting ( Tapia-Galisteo et al, 2023 ).…”

Facile Access to Branched Multispecific Proteins

“…An incorrect light-chain pairing leads to low yields. 26 Although the utilization of the 2 + 1 antibody technology 26 and nanobody−Fab fragment fusion protein recombination strategy 37 can be used to generate trispecific immune engagers, these technologies are beyond the technical expertise of many research groups. Further, the optimal display of each binding domain in a trispecific IgG antibody might not be readily achievable within the constraints imposed by protein biosynthesis.…”