Regulation of Encephalitogenic T Cells with Recombinant TCR Ligands

Abstract

We have previously described recombinant MHC class II β1 and α1 domains loaded with free antigenic peptides with potent inhibitory activity on encephalitogenic T cells. We have now produced single-chain constructs in which the peptide Ag is genetically encoded within the same exon as the linked β1 and α1 domains, overcoming the problem of displacement of peptide Ag from the peptide binding cleft. We here describe clinical effects of recombinant TCR ligands (RTLs) comprised of the rat RT1.B β1α1 domains covalently linked to the 72–89 peptide of guinea pig myelin basic protein (RTL-201), to the corresponding 72–89 peptide from rat myelin basic protein (RTL-200), or to cardiac myosin peptide CM-2 (RTL-203). Only RTL-201 possessed the ability to prevent and treat active or passive experimental autoimmune encephalomyelitis. Amelioration of experimental autoimmune encephalomyelitis was associated with a selective inhibition of proliferation response and cytokine production by Ag-stimulated lymph node T cells and a drastic reduction in the number of encephalitogenic and recruited inflammatory cells infiltrating the CNS. The exquisitely selective inhibition could be observed between molecules that differ by a single methyl group (the single amino acid residue difference between RTL-200 (threonine) and RTL-201 (serine) at position 80 of the myelin basic protein peptide). These novel RTLs provide a platform for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease.

Recombinant TCR Ligand Induces Early TCR Signaling and a Unique Pattern of Downstream Activation

“…RTL201 (encoding Gp-MBP 72-89 ) was shown to have potent suppressive and therapeutic activities for actively induced disease, substantially reducing the proliferative response of draining lymph node T cells (15). Even more striking, RTL201 completely suppressed passive EAE induced after transfer of Gp-MBP 72-89 -specific T cells, but had no effect on passive EAE induced with a different I-E (RT1.D)-restricted T cell line specific for a distinct encephalitogenic determinant, MBP 87-99 (15). Treatment with RTL201 prevented infiltration of both CSFE-labeled donor T cells FIGURE 5.…”

“…The design, expression, and purification of RTLs (U.S. Patent 6,270,772) have been described previously (14,15). There is a single amino acid difference between the Ag-peptide of RTL200 (rat-MBP 72-89 , PQKSQRTQDENPVVHF) and RTL201 (gp-MBP 72-89 , PQKSQRSQ-DENPVVHF).…”

“…Toward the long term goal of targeted Ag-driven immunosuppression of pathogenic T cells, we have developed and previously described a family of novel recombinant TCR ligands (RTLs) (14 -16) that consist of the ␣ 1 and ␤ 1 domains of MHC class II molecules expressed as single exons, with and without antigenic peptides of interest genetically encoded at the 5Ј end (17). We previously reported that a recombinant TCR ligand (RTL201) containing the 72-89 peptide of guinea pig myelin basic protein (Gp-MBP 72-89 ), successfully prevented and treated active and passive experimental autoimmune encephalomyelitis (EAE) (15). Amelioration of EAE was associated with a selective inhibition of the proliferation response and cytokine production by Ag-stimulated lymph node T cells and a drastic reduction in the number of encephalitogenic and recruited inflammatory cells infiltrating the CNS.…”

Recombinant TCR Ligand Induces Tolerance to Myelin Oligodendrocyte Glycoprotein 35-55 Peptide and Reverses Clinical and Histological Signs of Chronic Experimental Autoimmune Encephalomyelitis in HLA-DR2 Transgenic Mice

“…Our design includes only the minimal TCR interface, which involves only the ␣ 1 and ␤ 1 MHC domains without CD4 binding covalently linked to peptide (28). These constructs can prevent and treat MBP-induced EAE in Lewis rats (29,30) and inhibit activation and induce IL-10 secretion in human DR2-restricted T cell clones specific for MBP-85-99 or BCR-ABL b3a2 peptide (CABL) peptides (31,32). To further evaluate the inhibitory activity and mechanism of the effects of recombinant TCR ligand (RTL) on encephalitogenic T cells in vivo, we designed a MOG-35-55/DR2 construct (VG312) for testing in DR2 mice undergoing MOG peptide-induced EAE.…”

Production, Characterization, and Immunogenicity of a Soluble Rat Single Chain T Cell Receptor Specific for an Encephalitogenic Peptide

“…Rat RTL201, consisting of the ␣ 1 and ␤ 1 domains of RT1.B with MBP-(69 -89) peptide covalently attached (37,38), was immobilized on the chip by the OX-6 mAb at an R max of 100 -400 resonance units for binding experiments. Kinetic measurements were performed on a Biacore 3000 in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20) at a flow rate of 50 l/min to minimize the effects of mass transfer limitation.…”