MHC molecules and uses thereof

Details MHC molecules and uses thereof MHC molecules and uses thereof

1998-04-28

2001-10-30

Saunders, David (Department: 1644)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Fusion protein or fusion polypeptide

C424S185100, C424S198100, C435S069700, C530S395000, C530S350000, C530S387100

Reexamination Certificate

active

06309645

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel complexes of major histocompability complex (MHC) molecules and uses of such complexes. For example, in one aspect, the invention relates to empty MHC complexes that contain a MHC molecule with a peptide-binding groove and a presenting peptide non-covalently linked to the MHC protein. In another aspect, the invention relates to MHC class II-peptide fusion complexes which include a single chain MHC class II molecule and a presenting peptide covalently linked to the peptide binding groove of the MHC protein. MHC complexes of the invention are useful for a variety of applications including in vitro screens for identification and isolation of peptides that modulate activity of T cells.
2. Background
Antigen-specific T cell responses are invoked by antigenic peptides bound to the binding groove or cleft of major histocompatibility complex (MHC) glycoproteins as part of the mechanism of the immune system to identify and respond to foreign antigens. The bound antigenic peptides interact with T cell receptors and thereby modulate an immune response. The antigenic peptides are bound by non-covalent means to particular “binding pockets” comprised of polymorphic residues of the MHC protein's binding groove.
MHC class II molecules are heterodimeric glycoproteins consisting of &agr; and &bgr; chains. The &agr;1 and &bgr;1 domains of these molecules fold together to form a peptide binding grove. Antigenic peptides bind the MHC molecule through interaction between anchor amino acids on the peptide and the &agr;1 and &bgr;1 domains. Crystal structure of human class II HLA-DR1 complex with an influenza virus peptide indicate that the N- and C-terminal ends of the bound peptide extend out of the binding groove such that the C-terminus of the peptide is proximal to the N-terminus of the &bgr; chain [J. Brown et al.,
Nature
, 364:33-39 (1993); L. Stem et al.,
Nature
, 368:215-221 (1994)]. MHC class I molecules have different domain organizations than MHC class II molecules, but generally similar structure with a peptide binding site or groove that is distal to membrane domains [see, e.g., A. Rudensky et al.,
Nature
, 353:622-626 (1991)]. See also U.S. Pat. No. 5,284,935; 5,260,422; 5,194,425; 5,130,297; and WO 92/18150 and WO 93/10220 for discussions of MHC molecules.
The &agr; and &bgr; chain transmembrane domains play an important role in the assembly and/or intracellular transport of MHC molecules. For example, amino acid changes in the TM domains can result in defective MHC molecules [P. Cosson et al.,
Science
, 258:659 (1992); W. Wade et al.,
Immunology
, 32:433 (1995); H. Kozono et al.,
Nature
, 369:151 (1994)]. The &agr; and &bgr; chain transmembrane and cytoplasmic domains have been disclosed [(see e.g., Brown, supra and references therein)].
MHC molecules complexed with antigenic peptides can induce selective immunosuppression by several different mechanisms [see, e.g., J. Guery et al.,
Critical Reviews in Immunology
, 13(3/4):195-206 (1993)].
More specifically, it has been reported that peptide-MHC complexes on the surface of antigen presenting cells will only induce clonal expansion of a T cell line specific for the MHC bound peptide if the antigen presenting cells also deliver co-stimulatory signals. One proposed approach takes advantage of this requirement for T cell activation and reports inhibition of T cell development by interaction with the antigenic peptide bound to the MHC molecule in the absence of co-stimulatory signals [see M. Nicolle et al.,
J. Clin. Invest
., 93:1361-1369 (1994); and S. Sharma et al.,
Proc. Natl. Acad. Sci. USA
, 88:11465-11469 (1991)].
Another proposed approach inhibits T cell development with MHC molecules that contain a bound peptide that is an antagonist or partial agonist to a T cell receptor (TcR) [see B. Evavold et al.,
Immunology Today
, 14(12):602-609 (1993)].
Modifications of the antigenic peptides bound to T cell receptors have been attempted to examine residues responsible for specific T cell responses. Determination of such “activating” amino acids of the antigenic peptides could provide insight of suitable sequence of a TcR partial agonist or antagonist. See Evavold, B. et al., supra.
It also has been speculated that new vaccines might be developed based on determination of the nature of various antigenic peptides bound to MHC molecules [see R. Chicz et al.,
Immunology Today
, 15(4): 155-160 (1994)].
SUMMARY OF THE INVENTION
The present invention relates to novel complexes of major histocompability complex (MHC) molecules (class I or II), e.g., empty single chain MHC class II complexes, loaded single chain MHC class II complexes, and single chain MHC class II peptide fusion complexes and the uses of such MHC complexes.
We have now discovered that single chain MHC class complexes without a covalently linked presenting peptide (i.e. an empty MHC complex) can be loaded by contacting a presenting peptide to the complex so that the presenting peptide non-covalently binds to the peptide binding groove of the complex. Generally, the presenting peptide will non-covalently bind to the peptide binding groove of the empty MHC complex via stable hydrogen bonding. The single chain MHC class complexes of the present invention, particularly single chain MHC class II complexes, are surprisingly stable and are useful in a variety of applications. For example, loaded single chain MHC class II complexes or single chain MHC class II peptide fusion complexes can be used to modulate various immune system responses in a mammal, e.g., T cell apoptosis, T cell anergy, T cell cytokine release, immunosuppression and induction of T cells. Empty MHC molecules, particularly empty single chain MHC class II molecules, are useful in, e.g., in vitro screens for detecting peptides that modulate the activity of T cells, including peptides which are T cell receptor antagonists and partial agonists.
We previously disclosed highly useful MHC class I and class II peptide fusion molecules in unpublished PCT Application No. PCT/US95/09816, filed Jul. 31, 1995 (sometimes referred to herein as said “PCT Application”) as well as pending U.S. application Ser. No. 08/382,454, U.S. Pat. No. 5,869,270, filed Feb. 1, 1995. The MHC fusion complexes comprise a presenting peptide covalently linked (i.e. fused) to the MHC molecule. The PCT Application No. PCT/US95/09816 and said U.S. application Ser. No. 08/382,454, U.S. Pat. No. 5,869,270, are herein incorporated by reference in its entirety.
As used herein, the term “presenting peptide” refers to a peptide that is capable of modulating the activity of a T cell receptor, either to induce T-cell proliferation, to inhibit or inactivate T cell development such as determined by the assays disclosed below, including the assay that includes sequential steps of culturing T cells to proliferate same, and contacting the T cells with a single chain MHC peptide fusion complex of the invention or a loaded single chain MHC complex of the invention and then evaluating whether the complex inhibits further development of the T cells.
The term “empty” (particularly “empty MHC molecule” or similar phrase), as used herein, refers to an MHC molecule (class I or II) of the invention which lacks a covalently or non-covalently bound presenting peptide. Preferably, the empty MHC molecule is class II and is comprised of a single polypeptide chain, rather than separate polypeptides.
The term “loaded” (particularly “loaded MHC molecule” or similar phrase), as used herein, refers to an empty MHC molecule (class I or II) which includes a presenting peptide non-covalently bound to the peptide binding groove or cleft of the MHC molecule, preferably so that the loaded MHC molecule can modulate the activity of T cells. The non-covalent binding is suitably via stable hydrogen bonding between the presenting peptide and the peptide binding groove or cleft of the empty MHC molecule. The non-co

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