Mutant MHC class I molecules

Details Mutant MHC class I molecules Mutant MHC class I molecules

1997-08-29

2001-06-19

Saunders, David (Department: 1644)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C424S093700, C424S178100, C424S193100, C435S069600, C530S336000, C530S380000, C530S387300, C530S395000, C530S402000, C530S404000

Reexamination Certificate

active

06248564

ABSTRACT:

BACKGROUND OF THE INVENTION
The major histocompatibility complex (“MHC”) plays a central role in the immune system. Antigen-specific T cells recognize antigenic peptides in association with MHC class I or II molecules on the cell surface. Class I molecules consist of two noncovalently associated subunits: a highly polymorphic &agr; heavy chain and a conserved &bgr;2-microglobulin (“&bgr;2-M”) light chain. Two of the three extracellular domains of the heavy chain, i.e., domains &bgr;1 and &agr;2, are folded into a “groove” structure which anchors an antigenic peptide for presentation to T cells.
Human class I molecules (or “complexes”) have been refolded from
E. coli
-produced heavy chains and &bgr;2-M subunits in the presence of synthetic peptides (Garboczi et al.,
Proc. Natl. Acad. Sci. USA,
89:3429-3433, 1992). The three-dimensional structures of such recombinant complexes as determined by X-ray crystallography are virtually identical to the structure of the class I molecule as isolated from human cells (Madden et al.,
Cell,
75:693-708, 1993; Bjorkman et al.,
Nature,
329:506-512, 1987). Further, subtype A0201* of HLA-A2 produced in
E. coli
and assembled with synthetic HIV-1 nonapeptides has been shown to elicit cytolytic CD8
+
T cell responses (Walter et al.,
Int. Immunology,
9:451-459, 1997).
The classical class I gene family includes the highly polymorphic human class I molecules HLA-A, -B, and -C, and murine class I (i.e., H-2) molecules D, K, and L. A series of structural relatives (non-classical class I molecules) has been found in humans (e.g., HLA-E, -F, -G, -H, -I, and -J; and CD1) and mice (Q, T, M, and CD1) (Shawar et al.,
Annu. Rev. Immunol.,
12:839-880, 1994). These molecules have the typical structure of an antigen-presenting molecule, where a polymorphic heavy chain is noncovalently associated with the conserved &bgr;2-M subunit. The T cell repertoire reacting with these non-classical ligands has been characterized to only a limited extent.
SUMMARY OF THE INVENTION
The invention features a method of preparing a conjugate of an MHC class I molecule and a compound. In this method, one first obtains an MHC class I molecule, where a cysteine residue (i.e. a non-natural or new cysteine residue) has been engineered into its &bgr;2-microglobulin subunit. The compound (e.g., a protein, a carbohydrate, a lipid molecule, or any other organic compound) is then conjugated to the mutant class I molecule specifically via a linkage formed between the sulfhydryl group of the new cysteine residue in the &bgr;2-microglobulin subunit and a functional group of the compound. Alternatively, the compound can be first conjugated to the new &bgr;2-M subunit, and then the subunit is mixed with an &agr; heavy chain (from the same or different species as the &bgr;2-M subunit) in the presence of an appropriate peptide to form a compound-class I conjugate. The cysteine residue is preferably introduced into a region of &bgr;2-M that faces away from the interface between &bgr;2-M and the &agr; heavy chain. Exemplary regions are those corresponding to residues 15-23, 35-53, or 66-97 of SEQ ID NO: 1. SEQ ID NO: 1 shows the amino acid sequence of a human &bgr;2-M. Minor sequence variations can exist among &bgr;2-M molecules from different or the same species; and residues from two different &bgr;2-M sequences are said to be corresponding to each other when they are equivalent in function or relative position to the conserved residues in the two &bgr;2-M sequences, or both. The new residue, alone or together with one or more (e.g., two to five) amino acid residues, can be inserted into the &bgr;2-M region without any deletion of the region, or replace one or more (e.g., two to five) residues of the region. For instance, the new cysteine residue can replace a residue that corresponds to serine 52, tyrosine 67, or lysine 91 of SEQ ID NO: 1.
The compound can be, for example, a ligand for a multivalent binding molecule, an antibody (e.g., one that is specific for a tumor antigen), a molecule on the surface of a cell (e.g., an antigen-presenting cell or any other hematopoietic cell), or a ligand for a surface receptor of a cell.
The new compound-class I conjugates have several uses. For instance, when a multivalent binding molecule is supplied, conjugates of the ligands for the binding molecule with the new, monomeric class I molecules (e.g., ones that consist of an &agr; heavy chain, the new &bgr;2-M, and a peptide associated with the heavy chain) can be multimerized. Multimeric class I molecules can be used to, e.g., label, isolate and quantitate specific T cells. Exemplary multivalent binding molecules are avidin (or a derivative thereof, e.g., streptavidin), whose ligands are biotin and biotin derivatives.
A conjugate of the present invention can also be used to stimulate the immunity of an individual (e.g., a human or a mouse). To accomplish this, a conjugate of a new class I molecule and a cell (e.g., an antigen-presenting cell) is introduced into an individual, and the conjugate can stimulate the immune cells, particularly T cells specific for the peptide in the conjugate. In this method, the cell and the class I molecule in the conjugate are preferably syngeneic with this individual.
Another conjugate of the present invention can be used to eradicate a tumor (or any other undesired cell) in an individual. In this method, a conjugate of a new class I molecule and an antibody specific for an antigen (or a ligand for a receptor) expressed exclusively or primarily on the cell is introduced into the individual. The &agr; heavy chain in the conjugate can be allogeneic or xenogeneic) to the patient. If the heavy chain is syngeneic to the patient, the class I molecule would be associated with an antigenic peptide that can elicit a strong T cell response.
Use of the new recombinant &bgr;2-M, monomeric and multimeric class I molecules containing the new &bgr;2-M, and the new compound-class I conjugates are also within the scope of the invention.
The new &bgr;2-M eliminates the need for genetically engineering MHC heavy chains to create a chemically reactive site in each of the differing MHC class I molecules. This is a significant advantage in view of the enormous polymorphism of MHC heavy chains.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.


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