Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1997-10-29
2001-05-15
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S350000, C530S395000, C435S069300, C424S185100, C424S193100, C424S192100, C424S133100
Reexamination Certificate
active
06232445
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel complexes of major histocompability complex (MHC) molecules and methods of expressing and use of such complexes. For example, in one aspect, the invention relates to MHC class II molecules that include a modified class II &bgr;2 chain. In another aspect, the invention relates to MHC class I and class II complexes that include a covalently linked immunoglobin constant region. In still other aspects, the invention relates to polyspecific MHC complexes, as well as methods of expressing and purifying MHC complexes. The MHC complexes of the invention are useful for a variety of applications including screening peptides for the capacity to modulate T-cell activity in vitro and in vivo.
2. Background
Antigen-specific T-cell responses are invoked by antigenic peptides. The peptides generally bind to the binding groove of MHCs as part of an immune system mechanism for identifying and responding to foreign antigens. The bound antigenic peptides interact with T-cell receptors and modulate an immune response. The antigenic peptides are bound by non-covalent means to particular “binding pockets” comprised of polymorphic amino acid residues.
Naturally-occurring 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. Crystallographic analysis of human class II HLA-DR1 complex bound to an influenza virus peptide indicates 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. See e.g., J. Brown et al.,
Nature
, 364:33 (1993); L. Stern et al.,
Nature
, 368:215 (1994)). MHC class I and class II molecules have different domain organizations. See e.g., A. Rudensky et al.,
Nature
, 353:622 (1991). See also U.S. Pat. Nos. 5,284,935; 5,260,422; 5,194,425; 5,130,297; WO 92/18150; WO 93/10220; and WO96/04314 for discussions of MHC molecules.
Particularly, J. Brown, et al. supra have reported that the MHC class II &bgr;2 chain performs a critical role in the proper folding of MHC class II complexes.
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. The MHC &agr; and &bgr; chain transmembrane and cytoplasmic domains have been disclosed. See P. Cosson et al.,
Science
, 258:659 (1992); W. Wade et al.,
Immunology
, 32:433 (1995); H. Kozono et al.,
Nature
, 369:151 (1994) and J. Brown et al., supra.
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 (1993)).
More specifically, it has been reported that peptide-MHC complexes on the surface of antigen presenting cells (APCs) 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 includes inhibiting T-cell development with MHC molecules that contain a bound peptide. The bound peptide can be 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 TCR-bound antigenic peptides 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 into those amino acid sequences which can potentially play roles as TCR agonists or antagonists. 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).
Previous studies have shown that MHC class II heterodimeric molecules can bind exogenous peptide. However, the MHC class II chains often dissociate. In a dispersed state, the MHC class II chains may not be suitable for binding presenting peptide. See Stern, L. J. and D. C. Wiley,
Cell
68: 465 (1992); Scheirle, A. B. et al.,
J. Immunol
. 149: 1994 (1992); Kozano H. et al.,
Nature
369:151 (1994).
There have been several attempts to obtain fully soluble and functional MHC complexes. For example, in one approach, MHC complexes have been isolated from cells using biochemical techniques that include exposure to harsh agents such as proteolytic enzymes, salts, and/or detergents. These agents must often be removed by dialysis or binding reactions. See e.g., J. M Turner et al.
J. Biol. Chem
. 252: 7555 (1977); T. A. Springer et al.
PNAS
(USA) 73: 2481(1976).
However, these methods are often not optimal for isolating fully soluble and functional MHC complexes in significant quantities.
Highly useful MHC class I and class II complexes capable of modulating the activity of T-cells and methods of making the complexes have been disclosed in a published PCT Application No. WO 96/04314, filed Jul. 31, 1995. The MHC complexes disclosed generally bind a specific peptide ligand.
SUMMARY OF THE INVENTION
The present invention relates to novel MHC class I and class II complexes that are fully soluble and functional, e.g., empty single chain MHC class II complexes, loaded single-chain MHC class II complexes, single chain MHC class II peptide fusion complexes, polyspecific single chain MHC class II complexes (empty, loaded, or including fused peptide) and uses of such complexes.
Generally stated, we have found that soluble expression of the MHC complexes can be facilitated by fusing an immunoglobin light chain constant region to the MHC complexes. We have also found that soluble expression of the MHC complex can be facilitated by modifying a class II &bgr;-chain of the MHC class II complex including deletion of the entire class II &bgr;-chain.
We previously disclosed highly useful single-chain (“sc-”) MHC class I and class II complexes in the published PCT Application No. WO 96/04314 and in a published PCT application No. WO 97/28191, filed Jan. 31, 1996, the disclosures of which are each fully incorporated herein by reference. The disclosed sc-MHC class I and class II complexes include sc-MHC molecules with recombinantly fused presenting peptides (sc-MHC peptide fusion molecules), empty sc-MHC molecules (no recombinantly fused presenting peptides), and loaded sc-MHC complexes (include non-covalently attached presenting peptides).
We have discovered that it is possible to facilitate soluble expression of the previously disclosed sc-MHC class I and class II molecules by fusing an immunoglobin light chain constant region (i.e. Ig-C
L
) and/or modifying the &bgr;2 chain in a class II sc-MHC molecule. The Ig-C
L
fusion includes adding a Ig-C
L
chain or suitable fragment thereof to the sc-MHC class I or class II complex. The class II &bgr;2 chain modification includes deleting, substituting or adding amino acids to the class II &bgr;2 chain, including deletion of the entire class II B2 chain. The Ig-C
L
fusions and class II &bgr;2 chain modifications enhance soluble expression of the sc-MHC molecules and do not significantly impact the specific binding activity of the sc-MHC molecules.
The invention further provides novel polyspecific MHC complexes. The complexes generally include one or more sc-MHC class I or class II mol
Acevedo Jorge
Burkhardt Martin
Jiao Jin-An
Rhode Peter R.
Wong Hing C.
Buchanan Robert L.
Corless Peter F.
DeCloux Amy
Saunders David
Sunol Molecular Corporation
LandOfFree
Soluble MHC complexes and methods of use thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Soluble MHC complexes and methods of use thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Soluble MHC complexes and methods of use thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2562111