Conversion of CTLA-4 from Inhibitor to Activator of T Cells with a Bispecific Tandem Single-Chain Fv Ligand

Abstract

Abs or their recombinant fragments against surface receptors of the Ig superfamily can induce or block the receptors’ native function depending on whether they induce or prevent the assembly of signalosomes on their cytoplasmic tails. In this study, we introduce a novel paradigm based on the observation that a bispecific tandem single-chain variable region fragment ligand of CTLA-4 by itself converts this inhibitory receptor into an activating receptor for primary human T lymphocytes. This reversal of function results from increased recruitment of the serine/threonine phosphatase 2A to the cytoplasmic tail of CTLA-4, consistent with a role of this phosphatase in the regulation of CTLA-4 function, and assembly of a distinct signalosome that activates an lck-dependent signaling cascade and induces IL-2 production. Our data demonstrate that the cytoplasmic domain of CTLA-4 has an inherent plasticity for signaling that can be exploited therapeutically with recombinant ligands for this receptor.

Molecular Determinants of Inverse Agonist Activity of Biologicals Targeting CTLA-4

“…The doxycycline-inducible, CTLA-4 stably transfected Jurkat T cell panel used for these studies has been extensively described (9,(13)(14)(15)(16)(17). The experiments reported here were done with wild-type CTLA-4 and with Y165F CTLA-4, a fully functional molecule that is expressed at high levels on the cell surface or with the indicated mutants.…”

“…The mouse mAbs 11 and 24, the ScFv molecules 24, 26, and 24:26 against human CTLA-4, mouse anti-human CTLA-4 -26 F(abЈ) 2 and B7.2 Ig were obtained from Wyeth Research (9,(13)(14)(15)(16)(17). The following commercially available Abs were used in these studies: a mouse mAb against CD45 (Transduction Laboratories and BD Biosciences), a goat polyclonal antiserum against actin (Santa Cruz Biotechnology), an anti-human CD45 F(abЈ) 2 (Caltag Laboratories), and an anti-human CD28 Ab (BD Biosciences).…”

“…For CD28, this has been shown with a subset of Abs known as superagonist Abs (2)(3)(4). For CTLA-4, in contrast, historical data had claimed the ability of some Abs to have inverse agonist properties (5)(6)(7)(8), but this has only recently been demonstrated using a bispecific, in-tandem single-chain Fv (ScFv) 3 fragment against CTLA-4 known as 24:26 (9).…”

A bispecific antibody against two different epitopes on hepatitis B surface antigen has potent hepatitis B virus neutralizing activity

“…unattainable by a combination of two antibodies, including increased functional affinity through avidity, 24 receptor activation, 25 delivery of toxins, 26 tissue-specific targeting, 27 and immunodiagnostics. 28 Therefore, we constructed the bispecific antibody C4D2-BsAb from C4G4 and D2H2.…”

CTLA4 aptamer delivers STAT3 siRNA to tumor-associated and malignant T cells

“…Thus, our studies demonstrate the ability to target specific subsets of T cells -tumor-associated CD8 + T cells and Tregs. While its expression is associated with CD8 + T cell exhaustion, CTLA4 intracellular signaling has been reported to possess a broad plasticity of cellular responses ranging from inhibition of cytokine production and blunting clonal expansion to T cell survival (28,35,(41)(42)(43). In our prior investigations, we validated that STAT3 critically contributes to the inhibition of adaptive antitumor immune responses (6,9).…”