Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical inhibitory checkpoint molecule, and monoclonal antibodies (mAbs) targeting CTLA-4 that restore anti-tumor T cell immunity have achieved clinical success. Here, we report a humanized IgG1 mAb, namely JS007, with high binding affinity to CTLA-4. JS007 shows superior binding affinity and T-cell activating efficiency over ipilimumab. Moreover, it demonstrates substantial in vivo tumor suppression efficacy at low doses. The crystal structure of JS007/CTLA-4 complex (PDB: 8HIT) shows JS007 adopts a heavy-chain-dominant binding mode, and mainly contacts the BC loop, DE loop and FG loop of CTLA-4. Notably, two Tyr residues (VH-Y100 and VL-Y32) from the complementarity-determining region loops insert into the two cavities formed by the residues from the loops of CTLA-4, which may contribute to the stabilization of the binding. Comparative analysis with other anti-CTLA-4 mAbs indicates that the double "wedge-into-hole" binding mode is unique for JS007 and may be responsible for the high-affinity binding to CTLA-4. These findings have provided an important molecular understanding of the high-affinity CTLA-4 blockade mAbs and shed light on future development of agents targeting CTLA-4.

Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a co-stimulatory receptor and an important target for cancer immunotherapy. We herein present a potent FcγR-independent GITR agonist IBI37G5 that can effectively activate effector T cells and synergize with anti-programmed death 1 (PD1) antibody to eradicate established tumors. IBI37G5 depends on both antibody bivalency and GITR homo-dimerization for efficient receptor cross-linking. Functional analyses reveal bell-shaped dose responses due to the unique 2:2 antibody-receptor stoichiometry required for GITR activation. Antibody self-competition is observed after concentration exceeded that of 100% receptor occupancy (RO), which leads to antibody monovalent binding and loss of activity. Retrospective pharmacokinetics/pharmacodynamics analysis demonstrates that the maximal efficacy is achieved at medium doses with drug exposure near saturating GITR occupancy during the dosing cycle. Finally, we propose an alternative dose-finding strategy that does not rely on the traditional maximal tolerated dose (MTD)-based paradigm but instead on utilizing the RO-function relations as biomarker to guide the clinical translation of GITR and similar co-stimulatory agonists.
Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

B-cell maturation antigen (BCMA) is an ideal target for the treatment of multiple myeloma (MM), and a bispecific antibody (bsAb) of BCMA × CD3 has entered clinical trials with great potential. In this study, we characterized GR1803, a novel bsAb for MM treatment. We reported that GR1803 bound to recombinant BCMA, CD3, BCMA-positive (BCMA + ) MM cells, and human T cells; that it induced T cell activation and cytokine release dependent on the presence of BCMA + cells; and that it effectively killed MM cells with EC 50 values less than 10 ng/mL. We found that GR1803 induced MM cells to release various cytokines, including interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, interferon-g (IFN-γ), GM-CSF, and TNF-α. Finally, we found that GR1803 significantly and dose-dependently inhibited the growth of tumors specifically expressing human BCMA in vivo . Taken together, these results demonstrate that GR1803, a novel BCMA × CD3 bsAb, efficiently and selectively kills MM cells and represents a novel immunotherapy for treating MM.

Engineered recombinant antibody-based reagents are rapidly supplanting traditionally derived antibodies in many cell biological applications. A particularly powerful aspect of these engineered reagents is that other modules having myriad functions can be attached to them either chemically or through molecular fusions. However, these processes can be cumbersome and do not lend themselves to high throughput applications. Consequently, we have endeavored to develop a platform that can introduce multiple functionalities into a class of Fab-based affinity reagents in a "plug and play" fashion. This platform exploits the ultra-tight binding interaction between affinity matured variants of a Fab scaffold (FabS ) and a domain of an immunoglobulin binding protein, protein G (GA1). GA1 is easily genetically manipulatable facilitating the ability to link these modules together like beads on a string with adjustable spacing to produce multivalent and bi-specific entities. GA1 can also be fused to other proteins or be chemically modified to engage other types of functional components. To demonstrate the utility for the Fab-GA1 platform, we applied it to a detection proximity assay based on the β-lactamase (BL) split enzyme system. We also show the bi-specific capabilities of the module by using it in context of a Bi-specific T-cell engager (BiTE), which is a therapeutic assemblage that induces cell killing by crosslinking T-cells to cancer cells. We show that GA1-Fab modules are easily engineered into potent cell-killing BiTE-like assemblages and have the advantage of interchanging Fabs directed against different cell surface cancer-related targets in a plug and play fashion.
© 2019 The Protein Society.

Programmed cell death 1 (PD-1) is an immuno-checkpoint receptor which is primarily expressed on T cells, monocytes, natural killer cells and macrophages. Programmed death-ligand 1 (PD-L1) is the primary ligand of PD-1 and is constitutively expressed on antigen presenting cells, mesenchymal stem cells and bone marrow-derived mast cells. In addition, PD-L1 is also expressed on a wide range of tumor cells, including lung cancer, breast cancer and melanoma. PD-1 and PD-L1 are important members of the immunoglobulin super-family and participate in immune regulation. In the present study, the immune-suppressive effects of a number of tumor cell lines were determined. The breast tumor cell lines MCF-7 and MDA-MB-231 displayed the largest inhibitory effects on T-cell activation and cytokine secretion in a co-culture system. The HepG2, A549 and A375 cells displayed limited inhibitory effects. MCF-7 and MDA-MB-231 cells expressed the highest level of PD-L1 among the cells used, which may explain their higher immuno-suppressive effects. Compound A0-L, a small molecule inhibitor of the PD-1/PD-L1 interaction, restored T cell functions. Additionally, it was demonstrated that the tumor cells with higher levels of PD-L1 expression suppressed signaling pathways involved in T-cell activation, such as the T-cell receptor- zeta chain of T cell receptor associated protein kinase ZAP70-RAS-GTPase-extracellular-signal-regulated kinases and CD28-PI3K-Akt serine/threonine kinases pathways. These findings suggest that tumor cells with higher expression levels of PD-L1 may exhibit higher immuno-suppressive activity, and that drugs targeting the PD-1/PD-L1 interaction may have improved therapeutic effects on tumors expressing higher levels of PD-L1.
Copyright: © Zheng et al.