Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2002-06-04
2003-09-23
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S006120, C435S070100, C435S070300, C435S071100, C435S091400, C536S024100
Reexamination Certificate
active
06623957
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to cloning vectors useful for the expression of T-cell variable domains, to bacterial cells transformed by the vectors and to methods of producing T-cell variable domains in a prokaryotic host cell, either as single domains or as single chain heterodimers.
2. Description of Related Art
The production of single or heterodimeric T-cell receptor variable domains is of interest for purposes of studying T-cell receptor interaction with antigens and possibly developing approaches to therapies for autoimmune diseases and cancer. An important goal of molecular biology is a detailed understanding at the molecular level of the binding of T-cell receptors to cognate peptide-major histocompatibility complexes. This will be a step in the development, for example, of immunotherapy for T-cell mediated autoimmune disease. Despite this interest and the potential applications arising from the study of T-cell receptor domains, no methods are available for the production of only single T-cell receptor domains, nor has expression and secretion in prokaryotic hosts been successful.
The majority of T cells recognize antigenic peptides bound to class I or II proteins of the major histocompatibility complex (MHC) and are thus “MHC restricted”. The recognition of peptide-MHC complexes is mediated by surface-bound T-cell receptors (TCRs). These receptors are comprised of various heterodimeric polypeptides, the majority of which are &agr; and &bgr; polypeptides. A minor population (1-10%) of mature T-cells bear T-cell receptors (TCRS) comprising &dgr; &ggr; heterodimers (Borst et al., 1987; Brenner et al., 1986).
Several composite dimeric species incorporating the &agr; and &bgr; polypeptides have been produced in various systems. TCR &agr;&bgr; heterodimers have been expressed as phosphatidyl-inositol linked polypeptides (Lin et al., 1990) or TCR-immunoglobulin chimeras (Gregoire et al., 1991) in mammalian transfectomas. The production of V
&agr;
C&kgr; homodimers (Mariuzza & Winter, 1989) and V
&bgr;
-C&bgr; monomers (Gascoigne, 1990) in mammalian cells has also been described. The expression and secretion of immunoglobulin VH domains (Ward et al., 1989), Fv fragments (Skerra and Pluckthum, 1988; Ward et al., 1989) and Fab fragments (Better et al., 1988) has been reported. Molecular modeling analyses indicate that there are structural similarities between immunoglobulin F
ab
fragments and the extracellular domains of TCRs (Novotny et al., 1986; Chothia et al., 1988). Several expression systems for the production of recombinant TCRs in mammalian cell transfectomas have been documented but successful expression and secretion of these proteins in a prokaryotic host has not been reported.
Despite apparent expression of a single chain anti-fluorescein TCR in
E. coli
(Novotny et al., 1991), the product could not be isolated from the periplasm even though the leader sequence had been cleaved from the N-terminus of the recombinant protein. The single chain TCR was relatively insoluble, requiring the use of genetic manipulation to replace five of the “exposed” hydrophobic residues with relatively hydrophilic residues.
No methods are presently available for the production of single or heterodimeric T-cell receptor variable domains as secreted proteins. If available, such species would have potential use in the induction of antibodies as protective vaccines, for the therapy of autoimmune disease, and antibodies for targeting idiotypes (T-cell) or T-cell leukemias. Additionally, secretion of T-cell receptor domains from bacterial cell hosts should provide a convenient, economically attractive and rapid route for production of recombinant T-cell receptors.
Advantages of the production of the TCR variable domains in
E. coli
compared with expression of phosphatidyl-inositol linked heterodimers and TCR-immunoglobulin chimeras in mammalian cells are the following: (1)
E. coli
(and other prokaryotic hosts) grow much faster; thus, results of genetic manipulation of the fragments can be analyzed more quickly, (2) use of
E. coli
is much cheaper than mammalian hosts, (3) production of only the TCR variable domains in mammalian hosts has not been reported. For raising anti-idiotypic antibodies (which recognize variable domains only), this is particularly significant.
TCR fragments have been produced in mammalian cells but they are relatively large. Smaller size TCR segments may allow more rapid structural resolution using such techniques as NMR and X-ray crystallography. Since the variable domains are the regions which interact with peptide-MHC complexes, these regions of TCRs are of considerable interest. Additionally, the use of variable domains alone in immunization should result in the production of anti-variable domain antibodies. Such antibodies are expected to be particularly desirable for use in therapy and diagnosis since they block the interaction of the TCR with antigen and, due to the variable nature of the V
&agr;
/V
&bgr;
domains or other domains such as V
62
and V
65
are specific for subsets of T-cells. Large TCR fragments, such as those that can be expressed from mammalian cells, result in production of antibodies not only against the variable domains, but also against the TCR constant domains, (if present in the construct) and/or the immunoglobulin domains (if present in the construct). There would therefore be distinct advantages in having smaller variable domain TCR fragments available, particularly for immunization since any immune response generated is likely to be directed to particular regions of interest, i.e., the V domains.
SUMMARY OF THE INVENTION
The present invention seeks to address one or more of the foregoing problems associated with expression and secretion of T-cell receptor variable domains in a prokaryotic host cell. Recombinant V
&agr;
, V
&bgr;
and single chain V
&agr;
V
&bgr;
heterodimers have been produced in gram-negative hosts transformed with vectors containing DNA encoding one or more T-cell receptor variable domains. The T-cell receptor domains are efficiently secreted in
E. coli
or
S. marcescens
. Only the TCR proteins expressed in
E. coli
have been characterized by CD. These products contain a high proportion of &bgr;-sheet structure indicative of a native structure. Murine T-cell V
&agr;
and V
&bgr;
domains have been expressed and isolated in yields up to milligram quantities per liter of bacterial culture. Single T-cell variable domains (V
&agr;
and V
&bgr;
) and single chain (sc) V
&agr;
V
&bgr;
heterodimers have been produced employing the disclosed vectors.
The recombinant plasmids or expression vectors of the invention are particularly adapted for expression of T-cell receptor domains in transformed prokaryotic host cells. The recombinant plasmids comprise a DNA segment coding for one or more T-cell receptor variable domains. Any of a number of variable domains may be included but preferred domains are the V
&agr;
and V
&bgr;
. Murine T-cell receptor domain V
&agr;
V
&bgr;
heterodimer, derived from the 1934.4 hybridoma (Wraith et al., 1989) is particularly preferred. Segments of the V
&agr;
or V
&bgr;
domains as well as other variable domains such as V
&dgr;
V
&ggr;
, constant domains, C
&agr;
, C
&bgr;1
, C
&bgr;2
, C
&dgr;
, C
&ggr;
immunoglobulin CH1, CH2 and CH3 domains, etc. may also be employed. It is also contemplated that variations of T-cell receptor variable domains also fall within the scope of the invention. Such variations may arise from mutations such as point mutations and other alterations affecting one or more amino acids or the addition of amino acids at the N or C termini. While the invention has been illustrated with murine T-cell receptors, similar strategies are applicable to the receptor domains from other species, including rat, man and other mammals.
Other DNA segments may also be included linked to the variable domains described, for example, one or more recombinant T-cell receptor variable domains of one or more specificities linked to TCR c
Board of Regents University of Texas System
Fulbright & Jaworski
Scheiner Laurie
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