Engineering A Novel Asymmetric Head-To-Tail 2+1 T-Cell Bispecific (2+1 Tcb) Igg Antibody Platform With Superior T-Cell Killing Compared To 1+1 Asymmetric Tcbs

T cell bispecific antibodies that recruit and engage T cells for tumor cell killing through binding to the T cell receptor (TCR) upon binding to a tumor antigen (TA) and subsequent crosslinking have attracted broad interest. Here, we describe a novel asymmetric head-to-tail 2+1 T cell bispecific antibody (2+1 TCB) platform characterized by the fusion of a flexible Fab fragment to the N-terminus of the CD3e Fab of a heterodimeric asymmetric bispecific TA-CD3e IgG1 antibody in head-to-tail geometry via a flexible linker. The resulting TCB is monovalent for CD3e (K D 70-100 nM) and binds bivalently with avidity to the TA on the target cell. Correct heavy chain pairing is enabled by knob-into-holes technology, correct light chain pairing by CrossMAb technology or using a common light chain. This enables production with standard processes in CHO cells. To exclude FcgR-mediated unspecific TCR and FcgR co-activation resulting in unspecific cytokine release, Fc- effector functions (ADCC, ADCP, CDC) are abolished by introduction of P329G LALA mutations while FcRn binding and IgG-like pharmacokinetic properties are retained as shown in mouse and Cynomolgus. For comparative profiling, the following TCBs were generated with specificity for the tumor antigens MCSP/CSPG4, FOLR1/FRalpha, CD19 and CD20: 2+1 TCB CD3-inside , 2+1 TCB CD3-outside , one-armed 1+1 TCB CD3-inside and a classical asymmetric 1+1 IgG TCB. In vitro Jurkat-NFAT, T cell killing, activation and proliferation assays show that both 2+1 TCB formats mediate superior potency of killing (for CSPG4, FOLR1, CD19, CD20) and superior absolute killing (for CSPG4, CD19) compared to the respective classical asymmetric 1+1 IgG TCB. Surprisingly, the 2+1 TCB CD3-inside format was found to be superior in potency compared to the 2+1 TCB CD3-outside format, although its binding affinity for CD3e is reduced. These data confirm that TCBs mediate extremely potent T cell killing with fM-pM EC 50 values based on CD3e antibodies with affinities of only 70-100 nM. Notably, for CD19 both, 2+1 TCB CD3-inside and one-armed 1+1 TCB CD3-inside , mediate comparable potency and overall killing, and both were superior compared to the asymmetric 1+1 IgG TCB. These data underline the importance of the head-to-tail geometry with two Fabs on one arm attached to each other via a flexible G 4 S-linker. Finally, using 2+1 and 1+1 FOLR1 TCBs we demonstrate that bivalent binding allows better differentiation in killing of cells with high vs. low FOLR1 expression as compared to monovalent binding. Taken together, we demonstrate that the 2+1 TCB CD3-inside is the most potent, efficacious and versatile TCB design. Due to its orientation with the CD3e Fab inside, it allows the conversion of existing antibodies into potent TCBs without format restriction. Based on this platform, CEA CD3 TCB (RG7802, Phase I/Ib) and CD20 CD3 TCB (RG6026, Phase I) have entered clinical trials. Citation Format: Christian Klein, Christiane Neumann, Tanja Fauti, Tina Weinzierl, Anne Freimoser-Grundschober, Inja Waldhauer, Linda Fahrni, Sylvia Herter, Erwin van Puijenbroek, Sara Colombetti, Johannes Sam, Sabine Lang, Sherri Dudal, Wolfgang Schafer, Jorg T. Regula, Samuel Moser, Oliver Ast, Ralf Hosse, Ekkehard Mossner, Peter Brunker, Marina Bacac, Pablo Umana. Engineering a novel asymmetric head-to-tail 2+1 T-cell bispecific (2+1 TCB) IgG antibody platform with superior T-cell killing compared to 1+1 asymmetric TCBs [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 3629. doi:10.1158/1538-7445.AM2017-3629