Abstract 3637: Co-targeting of PD-1 and CTLA-4 inhibitory pathways with bispecific DART® and TRIDENT™ molecules
Authors: Chichili Gurunadh R. , Shah Kalpana , Motte-Mohs Ross La , Berezhnoy Alex , Liu Daorong , Hill Jessica , He Leilei , Shoemaker Christine , Brown Jennifer , Stahl Kurt , Li Hua , Li Jonathan , Gorlatov Sergey , Tamura Jim , Ciccarone Valentina , Alderson Ralph , Bonvini Ezio , Moore Paul A. , Johnson Syd 
Published: July 2017

Abstract
Introduction: Immunotherapy with the combination of monoclonal antibodies (mAbs) that block PD-1 (nivolumab) and CTLA-4 (ipilimumab) has shown clinical benefit beyond that observed with either mAb alone. We have developed PD-1 x CTLA-4 bispecific proteins aimed at inducing antitumor immunity through simultaneous blockade of both checkpoint molecules. Two proteins, a tetravalent PD-1 x CTLA-4 bispecific DART® protein (bivalent for both PD-1 and CTLA-4) and a trivalent PD-1 x CTLA-4 TRIDENT™ protein (bivalent for PD-1 but monovalent for CTLA-4) were engineered; the TRIDENT protein was designed to promote CTLA-4 blockade through avidity when bound to PD-1 on cells co-expressing both receptors, thus favoring PD-1 over CTLA-4 blockade in cells that do not co-express both molecules.
Methods: Proteins were engineered from humanized PD-1 and CTLA-4 mAb sequences and demonstrated favorable biophysical properties. Binding assays were performed by ELISA or flow cytometry. Co-ligation of PD-1 and CTLA-4 was assessed in an enzyme-fragment complementation assay (DiscoverX). T-cell activation was tested in reporter cells, staphylococcus enterotoxin B-stimulated PBMCs or MLR assays.
Results: Both DART and TRIDENT proteins showed equivalent potency in binding immobilized recombinant PD-1 or PD-1-expressing cell lines, inhibition of PD-1 interaction with PD-L1 or PD-L2 as well as reversal of PD-1/PD-L1 mediated T-cell signal inhibition. In all assays, both formats showed activities that were comparable to those of the precursor PD-1 mAb. With respect to CTLA-4, the DART protein showed a minor potency loss in binding to CTLA-4 expressing cells, inhibition of CTLA-4/B7 interaction and reversal of T-cell signal inhibition compared to the precursor mAb. The TRIDENT protein, however, showed substantial lower potency than the DART protein in all CTLA-4 assays, consistent with the monovalent nature of the CTLA-4 arm. Importantly, in cells that co-express both receptors, DART and TRIDENT proteins show comparable co-engagement of PD-1 and CTLA-4, as shown by enzyme-fragment complementation, suggesting that anchoring through PD-1 compensates for the decreased CTLA-4 avidity of the TRIDENT molecule when both target receptors are expressed. Similarly, in T-cell co-activation assays, both DART and TRIDENT proteins showed a comparable increase in IFN-gamma response that recapitulated that observed with the combination of the individual parental mAbs.
Conclusion: Both PD-1 x CTLA-4 DART and TRIDENT bispecific molecules block PD-1 and CTLA-4 pathways, with the TRIDENT protein demonstrating a PD-1-biased binding preference, consistent with its design intended to reduce CTLA-4 blockade in the absence of PD-1 co-expression. Both molecules showed comparable T-cell activation activity. Further development of bispecific PD-1 x CTLA-4 molecules for cancer treatment is warranted.
Citation Format: Gurunadh R. Chichili, Kalpana Shah, Ross La Motte-Mohs, Alex Berezhnoy, Daorong Liu, Jessica Hill, Leilei He, Christine Shoemaker, Jennifer Brown, Kurt Stahl, Hua Li, Jonathan Li, Sergey Gorlatov, Jim Tamura, Valentina Ciccarone, Ralph Alderson, Ezio Bonvini, Paul A. Moore, Syd Johnson. Co-targeting of PD-1 and CTLA-4 inhibitory pathways with bispecific DART® and TRIDENT™ molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3637. doi:10.1158/1538-7445.AM2017-3637