Soluble single chain T cell receptors

Details Soluble single chain T cell receptors Soluble single chain T cell receptors

1991-05-08

2002-07-09

Ulm, John (Department: 1646)

Chemistry: molecular biology and microbiology

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

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

Reexamination Certificate

active

06416971

ABSTRACT:

BACKGROUND OF THE INVENTION
Antigen binding receptors are of two basic types: immunoglobulin molecules (i.e., antibodies) expressed on the surface of B lymphocytes and secreted by plasma cells and T cell receptors on the surface of T lymphocytes.
The T cell receptor (TCR) is a molecular complex consisting of multiple subunits that mediate the recognition of antigen in the context of a particular major histocompatibility complex (MHC) product. Meuer, S. C., et al., Ann. Rev. Immunol. 2, 23-50 (1984); Clevers, H., et al., Ann. Rev. Immunol. 6, 629-662 (1988); Davis, M. M. and P. J. Bjorkman, Nature 334, 395-402 (1988). The antigen/MHC binding moiety, termed Ti, is a disulfide-linked heterodimer of 90 kD consisting of one ∝ and one &bgr; subunit on the majority of peripheral T lymphocytes. Both subunits are immunoglobulin-like, being composed of variable and constant domains, the former encoding the unique specificity of a given T cell clone. Ti, in turn, is non-covalently associated with a set of four invariant monomorphic subunits (&ggr;, &dgr;, &egr; and &zgr;), collectively termed CD3. All six receptor subunits are trans-membrane proteins and all but the &egr; and &zgr; subunits possess N-linked glycan moieties. The Ti ∝ and &bgr; subunits likely form a binding site for antigen and major histocompatability complex (MHC) through interaction of their variable domains whereas the CD3 subunits are thought to subserve signal transduction functions. In addition, it is known that a subpopulation of T cells (≦5% of peripheral T lymphocytes) exist that contain T cell receptors which contain Ti &ggr; and Ti &dgr; subunits that form heterodimers which form a binding site for antigen and MHC through interaction of their variable domains. Furthermore, there is now direct evidence to show that at least in the case of one nominal antigen which is a hapten, there is a subsite on the Ti molecule which directly binds hapten in the absence of MHC with an affinity constant of ~10−
5
[Siliciano, R. F. et al., Cell 47: 161-171 (1996)].
Each Ti &agr; and &bgr; subunit contains two extracellular domains, created by intrachain disulfide bonding of cysteine residues and a carboxy terminal hydrophobic transmembrane region followed by 5-6 amino acid cytoplasmic tails. The genes encoding the T cell receptor are assembled from separate gene segments, one of which encodes an invariant carboxy terminal constant region, while two or three other segments (V, D and J) encode the variable region of the molecule which recognizes antigen and MHC. Within the variable region are three regions of hypervariability that form the antigen binding pocket.
The organization of the gene locus which encodes the Ti &bgr; subunit consists of two tandemly arrayed sets of segments termed D&bgr;1-J&bgr;1-C&bgr;1 and D&bgr;2-J&bgr;2-C&bgr;2 and a set of 5′ V genes. The two constant regions of the Ti &bgr; protein differ from each other by only six amino acids in the translated region. Located 5′ to each C&bgr; region is a cluster of six functional J segments. Approximately 50 V&bgr; genes are known to exist in humans within the Ti &bgr; locus on chromosome 7 at 7q35. The V&agr; gene pool may be somewhat larger than V&bgr;, ~100 separated V genes. Furthermore, the organization of the Ti &agr; locus is distinct from Ti &bgr; as it contains only a single constant region gene and multiple J&agr; segments (>25) dispersed over more than 60 Kb [Wilson, R. K. et al., Immunol. Rev. 101, 149 (1988)]. The Ti &ggr; and Ti &dgr; subunits are similar in structure to the Ti ∝ and Ti &bgr; subunits. Brenner, M. B. et al., Nature 322; 145-149 (1986).
Because of the obligatory association of Ti subunits with CD3 subunits in the endoplasmic reticulum prior to surface T cell receptor expression, genetic analysis and engineering of T cell receptors in secreted form has, to the present time, been impractical. Furthermore, the present inventors have observed that truncated forms of Ti &agr; and Ti &bgr; subunits lacking transmembrane and intracytoplasmic segments have failed to coassociate and/or be secreted when expressed in eukaryotic systems, including CHO, baculovirus-SF9 and yeast.
SUMMARY OF THE INVENTION
The present invention circumvents these and other problems in the art.
The present invention concerns a soluble, single chain T cell receptor. Preferably, the soluble, single chain T cell receptor is a Ti &bgr; subunit fragment joined to a Ti &agr; subunit fragment or a Ti &ggr; subunit fragment joined to a Ti &dgr; subunit fragment by an amino acid linker. Additionally preferred is a soluble, single chain T cell receptor that is biologically active.
The present invention further concerns a nucleic acid molecule comprising a nucleic acid sequence coding for a soluble, single chain T cell receptor. Preferably, the soluble, single chain T cell receptor is a Ti &bgr; subunit fragment joined to a Ti &agr; subunit fragment or a Ti &ggr; subunit fragment joined to a Ti &dgr; subunit fragment by an amino acid linker. It is also preferred that the nucleic acid molecule is a DNA molecule, and the nucleic acid sequence is a DNA Sequence.
The present invention additionally concerns an expression vector containing a DNA sequence coding for a soluble, single chain T cell receptor. Preferably, the soluble, single chain T cell receptor is a Ti &bgr; subunit fragment joined to a Ti &agr; subunit fragment or a Ti &ggr; subunit fragment joined to a Ti &dgr; subunit fragment by an amino acid linker.
The present invention also concerns prokaryotic or eukaryotic host cells containing an expression vector which contains a DNA sequence coding for a soluble, single chain T cell receptor. Preferably, the soluble, single chain T cell receptor is a Ti &bgr; subunit fragment joined to a Ti &agr; subunit fragment or a Ti &ggr; subunit fragment joined to a Ti &dgr; subunit fragment by an amino acid linker.


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