Production of Subunits of Soluble T Cell Receptors by Co-Transfection

1. Field of the Invention

The present invention relates to soluble T receptors and more particularly to secreted forms of soluble T receptors (sTR) V.alpha.C/.alpha./V.beta.C/.beta., V.gamma.C.gamma./V.delta.C.delta. or V.alpha.C.delta./V.beta.C/.gamma. and to their diagnostic and therapeutic applications.

2. Description of the Related Art

T lymphocytes are capable of recognizing, in a highly specific manner, myriads of antigens (Ag); this is by means of extremely diverse surface structures belonging to the superfamily of immunoglobulins (Ig), the T receptors (TR).

In man and in mice, most T lymphocytes in adults express sTR consisting of 2 variable glycoprotein sub-units called .alpha. and .beta.. Like the Ig heavy and light chains, these subunits contain an amino-terminal variable (V) domain and a carboxy-terminal constant (C) domain and are, in addition, very generally covalently associated with each other via an interchain disulphide bridge. The nature of the antigens recognized by the .alpha..beta. T receptor is relatively well established: they are complexes formed by an oligopeptide antigen (derived from the intracellular degradation of endogenous or exogenous proteins) closely associated with the polymorphic gene products situated in the so-called class I or II major histocompatibility complex (MHC). The interaction between the .alpha..beta. T receptor and the MHC/Ag complexes is conventionally reinforced by so-called coreceptor or accessory molecules (CD4 and CD8), which recognize conserved portions of the class II and I MHC molecules respectively.

Another subpopulation of T lymphocytes which can be distinguished by the nature of the genes (.gamma. and .delta.) encoding these T receptors has more recently been described. Contrary to the .alpha..beta. T lymphocytes, the antigenic specificity of the .gamma..delta. T cells still remains unclear. Based on the relative homology of the primary sequences of the .alpha..beta. and .gamma..delta. chains of the T receptor, some have predicted a structural similarity of the ligands for these receptors. In agreement with this hypothesis, a fraction of the .gamma..delta. T lymphocytes was found to be directed against molecules structurally similar or identical to the products of the MHC conventionally recognized by the .alpha..beta. T lymphocytes. However, there are also several examples of recognition by this T subpopulation of molecules of more distant structure, such as stress proteins or certain activating molecules such as CD48.

The present inventors have sought to generate "soluble" (secreted) forms of the .gamma..delta. T receptor, which could be used (like the Ig's) as probes permitting the isolation, localization and possibly the purification of specific ligands.

Moreover, such soluble T receptors also have a number of clinical applications. Traunecker et al. (1989. Inununol. Today 10:29) have reported attempts to produce soluble T receptors which consisted in removing the transmembrane (TM) portion of the .alpha. chains or .beta. chains by introducing a translational termination codon upstream of the sequences encoding the TM region which proved unsuccessful, no secretion having been detected.

Following these initial failures, other strategies were then adopted. In most cases, the principle consisted in constructing chimetic proteins comprising the V, or V and C regions of the .alpha. and .beta. subunits, joined to the C regions of immunoglobulins or to anchors of the glycosyl phosphatidylinositol (GPI) type. In the case of the TR/Ig fusion proteins, the main problem proved to be the sometimes predominant secretion of monomeric or homodimeric forms. In addition, the .alpha..beta. sTR heterodimeric forms sometimes exhibited significant structural differences with the membrane forms; in particular, the 2 .alpha. and .beta. chains were very generally non-covalently associated. This could consequently have effects on the overall structure and the fine antigenic specificity of such chimeric molecules. In the case of "lipidated" T receptors (anchored to the membrane by a GPI sequence), a sometimes quite high proportion of covalently associated .alpha..beta. heterodimers could be obtained. However, the main disadvantage of this technique was the need for an enzymatic treatment (with phospholipase C), in order to liberate the T receptors in the medium, and therefore a production which is costly and of low yield. A procedure for producing so-called monochain T receptors, consisting in joining a V.alpha. domain to a V.beta. domain via a peptide bridge, has been proposed more recently. However, the use of this technique proved to be delicate. In particular, it assumed the introduction of a large number of mutations in certain hydrophobic zones of the V regions normally masked on the native protein, in order to render these monochain T receptors hydrosoluble.

All the examples of the production of soluble forms of T receptors described in the literature, in all cases in hybrid form, have shown an extreme variability of efficiency from one chain combination to another.

The present inventors have discovered that soluble T receptors could be easily obtained and with a high yield, regardless of the combination of chains used, by means of a process consisting in producing DNA molecules encoding each of the constituent T receptor submits from which the transmembrane portion has been deleted, and in co-transfecting these DNAs into a host cell.

The subject of the present invention is also a process for producing soluble T receptors, wherein the DNA sequences encoding each of the constituent T receptor submits, from which the transmembrane portion of the T receptor has been deleted, are co-transfected into a host cell.

According to the invention, V.alpha.C/.alpha./V.beta.C.beta. soluble T receptors are produced by co-transfecting, into a host cell, DNA sequences encoding the .alpha. and .beta. submits of the T.alpha..beta. receptor from which the transmembrane portion of the T.alpha..beta. receptor has been deleted.

V.gamma.C.gamma./V.delta.C.delta. soluble T receptors are also produced by co-transfecting, into a host cell, DNA sequences encoding the .gamma. and .delta. submits of the T.gamma..delta. receptor from which the transmembrane portion of the T.gamma..delta. receptor has been deleted.

V.alpha.C.gamma./V.beta.C.delta. and V.alpha.C.delta./V.beta.C/.gamma. heterodimeric soluble T receptors are further produced, in which the constituent subunits are associated via a covalent bond, by co-transfecting, into a host cell, DNA sequences encoding the C.gamma. and C.delta. domains of the .gamma. and .delta. subunits of the T.gamma..delta. receptor from which their transmembrane portion has been deleted, fused respectively to the DNA sequences encoding the V.alpha. and V.beta. domains of the .alpha. and .beta. subunits of the T.alpha..beta. receptor in order to obtain V.alpha.C.gamma./V.beta.C.delta. receptors, or fused respectively to DNA sequences encoding the V.beta. and V.alpha. domains of the .beta. and .alpha. subunits of the T.alpha..beta. receptor in order to obtain V.alpha.C.delta./V.beta.C.delta. receptors.

V.gamma.C.gamma./V.alpha.C.delta. hybrid soluble T receptors are also produced by co-transfecting, into a host cell, DNA sequences encoding the .gamma. subunit of the T.gamma..delta. receptor from which its transmembrane portion has been deleted, with the DNA sequences encoding the C.delta. domain of the .delta. subunit fused to the DNA sequences encoding the V.alpha. domain of the .alpha. subunit of the T.alpha..beta. receptor. This construction is particularly advantageous and is based on the fact that certain V.alpha. genes can be used either by .alpha..beta. clones, or by .gamma..delta. clones.

Advantageously, the DNA sequences of the V.delta.2 and V.gamma.9 genes are used, for the constructions of the soluble T receptors of the invention, for the variable parts.

It may however be advantageous to produce V.gamma.C/65 /V.delta.C.delta. receptors using a V.gamma.9 DNA sequence on the one hand, and by replacing the V.delta.2 DNA sequence by other V.delta. DNA sequences for the same reasons as those mentioned above for the construction of the V.gamma.C.gamma./V.alpha.C.delta. hybrid receptor. This construction makes it possible to obtain anti-.alpha. antibodies, or antibodies directed against V.delta.'s distinct from V.delta.2.

Conversely, it is also possible to conserve the V.delta.2 DNA and to replace the V.gamma.9 DNA sequence with other V.gamma. DNA sequences, in order to obtain anti-V.gamma. antibodies.

The invention also encompasses these embodiments of V.gamma.C.gamma./V.delta.C.delta. soluble T receptors.

It should be noted that several V.delta. segments (especially V.delta.1) can be considered as V.alpha.'s, in the sense that they can be equally used by the .alpha. or .delta. chains of the T receptor. Thus, it can be considered that the examples of receptors produced in soluble form, which are provided here, demonstrate especially the usefulness of the process within the framework of the generation of monoclonal antibodies directed not only against the .gamma. and .delta., but also .alpha., variable regions.

Advantageously, the deletion of the transmembrane portion of the constituent T receptor submits is carried out by introducing a translational termination codon upstream of the sequences encoding the transmembrane portion of these submits, especially by P.C.R. (Polymerase Chain Reaction) directed mutagenesis.

The DNA sequences are genomic DNA or cDNA sequences.

Preferably, the co-transfection is carried out into eukaryotic cells, especially hamster ovary cells (CEO).

The subject of the invention is also a fusion protein formed between a soluble T receptor and a peptide sequence, the peptide sequence being constitutive of a peptide or of a protein, the fusion protein being obtained by fusing the DNA sequence encoding the peptide or the protein to one of the chains or to the two chains of DNA encoding the submits of a T receptor from which their transmembrane portion has been deleted, followed by a co-transfection of the DNA sequences thus fused into a host cell.

Advantageously in this case, the peptide sequence is that of interleukin-2 (IL-2).

The subject of the invention is also human or animal polyclonal or monoclonal antibodies directed against a soluble T receptor obtained by the process of the invention or an sTR-IL2 fusion protein as defined above.

The monoclonal antibodies according to the invention can be prepared according to a conventional technique. To this effect, the soluble T receptors, optionally fused with interleukin-2 or another protein, can be coupled if necessary to an immunogenic agent, such as tetanus toxoid, via a coupling agent such as a his diazotized benzidine.

The present invention also encompasses the fragments and the derivatives of monoclonal antibodies according to the invention. These fragments are especially F(ab').sub.2 fragments which can be obtained by enzymatic cleavage of the antibody molecules with pepsin, the Fab' fragments which can be obtained by reducing the disulphide bridges of the F(ab').sub.2 fragments and the Fab fragments which can be obtained by enzymatic cleavage of the antibody molecules with papain in the presence of a reducing agent. These fragments, as well as the Fc fragments, can also be obtained by genetic engineering.

The derivatives of monoclonal antibodies are for example antibodies or fragments of these antibodies to which markers such as a radioisotope are linked. The derivatives of monoclonal antibodies are also antibodies or fragments of these antibodies to which therapeutically active molecules are linked.

The subject of the invention is also hybridomas producing monoclonal antibodies specific for the peptide sequence described above. These hybridomas can be obtained by the conventional techniques of cell fusion between spleen cells activated in vitro by the antigen or obtained from an animal immunized against the peptide sequence of the invention, and cells from a myelomatous line.

The subject of the invention is also a diagnostic composition comprising a soluble T receptor obtained by a process according to the invention or an sTR-peptide sequence, especially sTR-IL2, fusion protein as defined above, or alternatively a monoclonal antibody according to the invention.

The diagnostic composition according to the invention can be used for the typing of cellular specificities linked to the T receptor. Indeed, a soluble T receptor can be used as such. However, because of the probably weak affinity of the latter for its specific ligand, it is advantageous to couple the soluble T receptors to a support, in order to increase their avidity by increasing their valency.

The support may consist of any support traditionally used, such as organic or magnetic beads.

Such supports are for example plastic plates used for the ELISA tests on which the soluble T receptor is attached in the same manner as immunoglobulins, tosyl-activated magnetic beads, for example those marketed by Dynal, Oslo, Norway, or alternatively AFFIGEL type activated gels such as those marketed by BIORAD.

The coupling techniques are those conventionally used and indicated by the distributor for the supports commercially available.

These methods may consist in a chemical coupling or by means of monoclonal antibodies directed against the soluble T receptors in question, the latter being themselves coupled to the support by chemical coupling.

Advantageously, the diagnostic compositions comprise a fused protein as described above, consisting of a soluble T receptor and an antigenic determinant against which specific antibodies are available.

Such diagnostic compositions can be used for the typing of cellular specificities not detected by conventional serological techniques.

The diagnostic composition according to the invention may also comprise monoclonal antibodies as defined above, and preferably a panel of monoclonal antibodies directed against the V and C portions of the chains of the T receptors obtained by immunizing animals against the soluble T receptors obtained according to the invention, previously purified.

In order to improve the efficacy of the immunizations, it is also possible to inject sTR-IL2 fusion proteins as defined above.

Such a diagnostic composition can be used especially for the detection of mono- or oligoclonal proliferations, such as those encountered in T leukaemias for example.

According to the invention, the diagnostic composition is brought into contact with a biological sample, for example a blood sample containing pathological T lymphocytes, and the complex formed with the ligand specific for the T receptor and the soluble T receptor or the fusion protein comprising the soluble T receptor and an antigenie determinant or the complex formed by the monoclonal antibodies according to the invention and the soluble T receptor or the soluble T receptor-IL2 fusion protein against which they are specifically directed, is detected.

These processes can be based on an RIA, RIPA or IRMA type radioimmunological method, or an immuno-enzymatic method of the WESTERN-BLOT type on strips or of the ELISA type.

For the implementation of these processes of detection, unlabelled cold molecules or molecules labelled by means of a suitable marker which may be biotin or its derivatives, an enzyme such as peroxidase, a fluorescent marker such as fluorescein, a radioactive marker and the like, are used.

These in vitro diagnostic processes comprise for example the following steps:

depositing a determined quantity of a composition containing a soluble T receptor, a soluble receptor fused with an antigenie determinant or a monoclonal antibody according to the invention directed against the soluble T receptor or the soluble T receptor-Interleukin 2 fusion protein according to the invention, in the wells of a microtitre plate or on another support such as beads or a nitrocellulose membrane,

depositing, in the wells, the biological sample to be tested, or incubating the latter with the beads or the membrane, in the presence of saturating agents or after prior saturation of the activated supports,

after incubating and rinsing the microplates or the beads, depositing in the wells or incubating with the beads a system for revealing the soluble T receptor-ligand complex which may have formed.

The kits for implementing the diagnostic process of the invention comprise:

at least one diagnostic composition according to the invention,

reagents for preparing a medium suitable for producing a complex between the ligand(s) which may be present in a biological sample,

one or more optionally labelled reagents capable of reacting with the complex formed.

The subject of the invention is also a therapeutic composition characterized in that it comprises a soluble T receptor obtained according to the process of the invention or a fusion protein as defined above, especially an sTR-IL2 according to the invention.

Such a therapeutic composition is useful especially in the treatment of pathological processes in which a pauciclonal proliferation of T lymphocytes is observed, such as T leukaemias or lymphomas and certain autoimmune diseases.

It is preferably administered by injection in an appropriate vehicle.

The administration of this therapeutic composition has a double purpose. It permits, on the one hand, the induction of an anti-idiotypic immuno response, resulting, in this case, in the active and selective removal of the cells carrying these idiotypes, and, on the other hand, the blocking, by competition, of the recognition of autologous antigens in the case of auto-immune proliferations.

Advantageously, the therapeutic composition according to the invention comprises a heterodimeric soluble T receptor as defined above, optionally carried by a fusion protein.

The therapeutic composition according to the invention may also comprise a monoclonal antibody according to the invention, optionally coupled to a therapeutically active molecule, for example a cytotoxic molecule, or a monoclonal antibody fragment or derivative as defined above.

Such a composition permits the direct removal of mono- or oligoclonal cells encountered in certain types of T leukaemias.

The production of soluble T receptors in the case of TR.gamma..delta. will be described in detail below with reference to the accompanying figures in which:

FIGS. 1 and 1B represent products of assembly of the .gamma. and .delta. genes. The sequences of the 5' and 3' primers used to amplify the cDNAs permitting the production of the soluble T.gamma..delta. receptors (.gamma.s and .delta.s cDNA) are represented above and below the .gamma. and .delta. cDNAs respectively. The positions of the termination codons are represented in bold characters. The grey parts in 3' of the .gamma. and .delta. cDNAs correspond to the hydrophobic transmembrane (TM) regions. FIG. 1 shows the sequences SEQ ID NO:1-SEQ ID NO:15 as depicted on the attached sequence listing.

FIGS. 2A and 2B represent the corresponding nucleotide and peptide sequences of the soluble .delta. and .gamma. chains of the clone used for the construction of the soluble T receptor described above. In particular, FIG. 2A shows the sequences SEQ ID NO:16 and SEQ ID NO:17 and FIG. 2B shows the sequences SEQ ID NO: 18 and SEQ ID NO:19 as depicted on the attached sequence listing.

FIG. 3 represents the results of the tests for detection of sTR.gamma..delta. by the IRMA technique in medium packaged from CHO cells transfected with .gamma.s/.delta.s.

SN represents the supernatant from the culture of the CHO cells, transfected with a non-pertinent cDNA (C) or with the cDNAs of the soluble .gamma. and .delta. subunits according to the invention (s.gamma..delta.).

The monoclonal antibodies giving a significant radioimmunological signal are represented as bold rectangles.

FIG. 4 represents the titration in soluble TR activity expressed in .mu.g/ml, as attested by the IRMA test (sandwich 7B6/TiV.delta.2), of the fractions eluted from an affinity column coupled with the anti-V.gamma. 7B6 antibody (marketed by Immunotech), onto which have been applied about 500 ml of supernatant from the culture of .gamma..delta.sFS-CHO cells.

FIG. 5 represents the SDS-PAGE analysis of fractions positive for the soluble TR activity, as attested by the IRMA test (sandwich 7B6/TiV.delta.2), of the fractions eluted from an affinity column coupled with the anti-V.gamma.9B6 antibody.

Two independent preparations (#1 and #2) were analysed under non-reducing (on the left) and reducing (on the right) conditions. MW=molecular weight markers.

The .gamma..delta.s G 115 human lymphocyte clone (whose nucleotide and peptide sequences corresponding to the soluble .delta. and .gamma. chains are represented in FIG. 2, in A and B respectively) expressing T V9JPCI.gamma./V2D3J1C.delta. receptors was used for the construction of the .gamma..delta.s genes and the expression of the soluble T receptors.

This clone was used for several reasons of which the main ones are:

the great majority of the .gamma..delta. T receptors of peripheral blood human leucocytes comprise similar V(D)J regions such that the structural and functional results obtained with the soluble form of the specific TR used can be easily applied to the TR expressed by a large proportion of .gamma..delta. cells,

monoclonal antibodies specific for the C.gamma., C.delta., V.gamma.9 and V.delta.2 regions are easily available and can be used to monitor the production and the purification of the soluble TR molecules,

unlike most .gamma..delta. V.delta.1-positive human T lymphocytes, the .gamma. and .delta. chains of the T receptors of the G 115 clone are covalently linked by a disulphide bridge which highly stabilizes the molecule after its secretion into the medium,

the antigenic specificity of the G 115 clone is fairly well known. In particular, this clone kills the cells of a Burkitt's lymphoma (called Dadi) and also recognizes an antigen present in water-soluble extracts of Mycobacterium tuberculosis.

The G 115 clone, obtained from T.gamma..delta. lymphocytes derived from human peripheral blood leucocytes, was maintained in an RPMI 1640 medium containing 8% human serum, 2 mM L-glutamin and 150 BRMP (Biological Response Modifier Program) units of IL2 and stimulated for one week out of two with 0.5 .mu.g/ml of leucoagglutinin (Pharmacia, France), irradiated peripheral blood leucocytes and irradiated and EBV-transformed B lymphoblasts.

After two washes in phosphate buffered saline solution, 5.times.10.sup.6 cells were lysed on ice in a Tris-HCl buffer (80 mM, pH 7.5) containing 100 mMNaCl, 5 mM EDTA and 0.5% by weight of Triton X100. After centrifuging, the supernatant was harvested and mixed with an equal volume of phenol at 65.degree. C. The RNA was extracted by a phenol/CHCl.sub.3 treatment, precipitated in 2.5 volumes of ethanol and solubilized in 40 .mu.l of 10 mM Tris/1 mM EDTA). 5 .mu.l of total RNA were reverse-transcribed for 1 hour at 37.degree. C. by means of a 3'-phosphated primer containing translational termination codons upstream of the hydrophobic transmembrane region of the .gamma. and .delta. genes, after the Lys.sup.247 and Gln.sup.274 codons, as represented in FIG. 1, at a concentration of 50 pM, the four dNTPs at a concentration of 1 mM each and 200 units of mouse mammary tumor virus reverse transcriptase (MMTV) (Boehringer Mannheim, Germany), in a final volume of 25 .mu.l. 1.75 .mu.l of a mixture for PCR (containing 13 mM Tris-HCl (pH 8.2), 66 mM KCl, 2 mM MgCl.sub.2, 2 U of Taq polymerase (Boehringer) and 50 pM of 5'-phosphated primer represented in FIG. 1 were added to the material obtained by reverse transcription and 30 amplification cycles (94.degree. C.--1 min, 45.degree. C.--1 min, 72.degree. C.--1 min) were carried out. The amplified DNA was purified after electrophoresis on a low melting point agarose gel and cloned into a plasmid Bluescript SK+ (Stratagene, La Jolla, Calif.) digested with SmaI. The sequencing was carried out using a system of double-stranded template according to the procedure provided by the supplier of the USB Sequenase kit. The fragments were cloned into an expression vector pKCR6 (Matrisian et al., Proc. Natl. Acad. Sci. USA. 83:9413) digested with EcoRI.

The plasmid DNA was then introduced into DHFR (dihydrofolate reductase)-negative hamster ovary cells DUKX-B11, cultured in RPMI 1640 medium, supplemented with 8% foetal calf serum, 2 mM L-glutamin, thymidin, adenosin and deoxyadenosin at 10 .mu.g/ml each, by the calciumphosphate precipitation technique (Wiglet et al., 1979 Cell. 16:777). The DHFR-positive cells were selected by culturing the transfected cells for three weeks in RPMI medium, supplemented with foetal calf serum and n-glutamin (2 mM) without nucleosides. The stable transfectants were then cloned by the limiting dilution technique.

a) Detection of the soluble T receptors

The monoclonal antibodies used for the detection of the soluble TRs were labelled with .sup.125 I by the Iodogen method (Fraker et al., 1978, Biochem.-Biophys. Res. Commun. 80:849). The T receptors were detected by a sandwich immunoradiometric assay (IRMA) by means of pairs of monoclonal antibodies specific for the .gamma. and .delta. chains.

Immulon-1 microtitre plates (Dynatech, Marnes, France) were coated for 90 min at 37.degree. C. with 50 .mu.l of Y102 (or 7B6) monoclonal antibody at 40 .mu.g/ml in a phosphate buffered saline solution. After removal of the antibody, the unbound sites were saturated with a phosphate buffered saline solution containing 0.5% bovine serum albumin for 1 hour at room temperature. The samples to be analysed were then added in an amount of 40 .mu.l at the same time as 10 .mu.l of labelled TiV.delta.2 monoclonal antibody. After incubating for 90 min at 37.degree. C., the wells were rinsed four times with 100 .mu.l of a phosphate buffered saline solution supplemented with bovine serum albumin.

The bound radioactivity was measured in a .gamma. scintillation counter. The following set of antibodies was used to measure the secretion of soluble TR.gamma..delta. by the IRMA technique: anti-V.gamma.9 (Y102, B37, 7B6), anti-C.gamma. (B121) and anti-V.delta.2 (TiV.delta.2) antibodies (Miossec et al., 1989, J. Exp. Med. 171:1171). A monoclonal antibody specific for IL2 was also used as negative control.

With the various combinations of antibodies, no signal was observed with the supernatants of non-transfected hamster ovary cells (CHO), of cells transletted with a non-pertinent cDNA or of cells transfected either with a truncated .gamma.s cDNA or a truncated .delta.s cDNA (FIG. 3). But the soluble .gamma..delta. hetero-dimers were clearly detected by IRMA (radioimmunological assay) in the supernatants of CHO cells co-transfected with soluble .gamma. and soluble .delta. assembly products (.gamma..delta.sFS-CHO) when pairs of antibodies specific for V.delta.2/C.gamma. or V.delta.2/V.gamma.9 were used (FIG. 3), which suggests that the soluble TR molecules secreted by the .gamma..delta.F5-CHO cells were predominantly heterodimers.

b) Purification of the soluble T receptors

10 mg of Y102 or 7B6 monoclonal antibody (anti-V.gamma.9) were covalently linked to a matrix of activated agarose beads (Affigel, Biorad, Richmond, Calif.) according to the instructions of the supplier.

The culture supernatants were applied to an affinity column at a rate of 30 ml/h at 4.degree. C. After washing with a phosphate buffered saline solution, the bound material was eluted with a 0.2M glycine buffer (pH 2.5). The eluted fractions were neutralized all at once with 1M Na.sub.2 HPO.sub.4.

The fractions positive for the soluble TR activity as attested by the IRMA test were combined, dialysed overnight against distilled water and concentrated by evaporation.

Soluble TR samples were prepared in a buffer for gel electrophoresis with or without reducing agent, separated by SDS-PAGE and transferred onto a nitro-cellulose membrane in accordance with the recommendations of the supplier. After saturating the unbound sites with a blocking buffer (dried skimmed milk and Tween 20), the fingerprints obtained were incubated in the presence of primary antibody (hybridoma supernatant diluted one-third with the blocking buffer) for 2 hours at room temperature. After washing, an anti-Ig-horseradish peroxidase conjugate was added, and the incubation continued for another 2 hours. The bound antibodies were revealed with diaminobenzidine (1 mg/ml), H.sub.2 O.sub.2 and CoCl.sub.2.

In a typical preparation, 3.3 mg (calculated using a coefficient for 1% extinction of 1.5, as calculated for the immunoglobulins) of affinity-purified .gamma..delta. TRs were treated with Vibrio cholerae neuraminidase (Boehringer Mannheim) in 1 ml of buffer containing 50 mM sodium acetate, 150 mM NaCl and 4 mM CaCl.sub.2 at pH 5.5 for 1 hour at 37.degree. C.

Under these conditions, the reaction was estimated to be complete by determining control assays for digested samples by isoelectric focusing in IEF 3-9 PhastGel medium (Pharmacia).

After dilution with a 0.1M sodium phosphate buffer, pH 7.3, the sample was concentrated by means of a centripep column at 30,000 revolutions (Amicon) before protelysis.

The neuraminidase-treated .gamma..delta. receptors were digested at 37.degree. C. for 30 minutes with papain (Worthington) at an enzyme/substrate ratio of 1/500 in the presence of 1.5 mM 2-mercaptoethanol and 1.25 mM EDTA. The reaction was completed by addition of N-ethylmaleimide.

These conditions were sufficient to completely eliminate the interchain disulphide bridge as attested by SDS-PAGE analysis under non-reducing conditions. Higher enzyme/substrate ratios and/or longer incubation times provided no proof of an additional protein cleavage. The reaction medium was then applied to a Zorbax CF-250 size-exclusion chromatography column (DuPont - New England Nuclear) which made it possible to obtain, after elution, the T receptor treated with papain and neuraminidase in the form of a single peak at about 65 kDa compared with 75 kDa for the native protein. No sign of chain dissociation was apparent.

After concentrating on a centripep, the material described above was incubated overnight at 37.degree. C. in the presence of endoglycosidase F and N-glycosidase F (Boehringer Mannhein) under non-denaturing conditions (0.1M sodium phosphate buffer, pH 7.3), as recommended by the manufacturer. A final purification was carried out by means of a Mono Q high-performance anion-exchange chromatography column (Pharmacia).

The total yield from 3.3 mg of affinity-purified T receptor was 1.1 mg or about 34%.

The material eluted from the anti-V.gamma.9 column consisted essentially of .gamma..delta. heterodimers since it was precipitated by monoclonal antibodies specific for V.delta.2. In addition, an SDS-PAGE analysis under reducing and non-reducing conditions showed that these heterodimers were linked by a covalent bond.

Indeed, under non-reducing conditions, a diffuse principal band having an apparent molecular weight of 75-80 kD was observed, which separated under reducing conditions into two predominant components of 42 and 44 kD and two minor components of 50 and 39 kD. Identical patterns were obtained with material precipitated in stages with anti-V.gamma.9 and anti-V.delta.2 monoclonal antibodies. By means of monoclonal antibodies generated against this soluble receptor (monoclonal antibodies 360 and 389, cf. below), it was possible to show by the Western-blot technique that the 50 kD and 44 kD bands corresponded to the .gamma. chain, and that the 42 and 39 kD band corresponded to the .delta. chain. The differences in the sizes of the soluble .gamma. and .delta. species were due to the different degrees of N-glycosylation, as subsequently specified.

a) Generation of monoclonal antibodies directed against soluble forms of .gamma..delta. TR after immunization of mice against soluble .gamma..delta. TRs:

BALB/c mice were immunized with soluble .gamma..delta. T receptors, in accordance with the following procedure: on day 1, 50 .mu.g of protein in 500 .mu.l of emulsified complete Freund's adjuvant at 50% in 0.9% NaCl were subcutaneously injected at four different points. On day 25, the same procedure was repeated in incomplete Freund's adjuvant. A booster was made by 3 intraperitoneal injections on days 50, 51 and 52, by means of 15 .mu.g of protein each in 250 .mu.l of 0.9% NaCl. Splenocytes harvested on day 53 were fused with X63 Ag 8653 myeloma. Hypoxanthine/aminopterin/thymidin-resistant colonies were screened by a radioimmunological assay (RIA) by means of an iodine-labelled soluble T receptor, in accordance with the IODOGEN method.

To this effect, 96-well microtitre plates coated with avidin (Immunotech) were incubated with biotinylated anti-mouse goat immunoglobulins (GAMIG, Immunotech) in PBS, BSA, NaN.sub.3 overnight at 4.degree. C., and then washed 3 times in Tween PBS. 100 .mu.l (10.sup.5 cpm) of radiolabelled soluble T receptors were incubated for 2 hours at room temperature and washed 3 times in PBS-Tween. The bound radio-labelled soluble T receptors were assayed by .gamma. counting.

Nine monoclonal antibodies recognizing all or part of the human .gamma..delta. T lymphocytes were obtained from an immunized mouse spleen, 2 anti-V.gamma.9 antibodies (292 and 360), 2 anti-V.delta.2 antibodies (1 and 389), 1 .gamma..delta. pan anti-body (510) and 4 antibodies directed against .gamma..delta. sub-populations (49, 60, 103 and 515).

b) Reactivity of anti-soluble TR monoclonal antibodies towards mono- and polyclonal human lymphold lines:

Monoclonal antibodies having produced an RIA signal were then tested by immunofluorescence to determine their ability to recognize T receptors linked to the membranes of the G9 clone. The fine specificity of these monoclonal antibodies was finally studied by screening their reactivity towards T lymphocyte clones and lines whose T receptor phenotype was known.

From a single fusion experiment, the supernatants of 16 colonies (3% of the inoculated wells) gave a positive RIA signal and among them, eleven contained monoclonal antibodies recognizing the G9 clone in an indirect immunofluorescence assay. The specificity of 7 monoclonal antibodies was measured by flow cytometric analysis.

Three monoclonal antibodies (52, 106 and 510) were directed against a determinant which was common to all the T.gamma..delta. receptors but not to the T.alpha..beta. receptors. Two monoclonal antibodies (292 and 360) were specific for T receptors comprising the V.gamma.9 region and two monoclonal antibodies (1 and 389) for T receptors comprising the .gamma..delta.2 region. No precise specificity could be attributed to the remaining monoclonal antibodies (49, 60, 103 and 515) which recognized subpopulations of .gamma..delta. lymphocytes but whose reactivity could not be correlated with the presence of a particular V region of T receptor (Table I below).

It should be noted that all the monoclonal antibodies were capable of recognizing non-reduced soluble T receptors in Western-blot analyses, and several also reacted with .gamma. or .delta. species isolated after reduction (Table II), unlike most V-specific monoclonal antibodies generated against native T receptors (linked to membranes). In agreement with attributions of specificity deduced from flow cytometric experiments, the monoclonal antibodies 389 and 360 recognized various species (molecular mass 39-42 kDa and 44-50 kDa, respectively), which could correspond to the .delta. and .gamma. chains respectively. In addition, since the .gamma..delta.52 and 510 pan monoclonal antibodies, and the antibody 389 specific for V.delta.2 reacting with the same species of 39-42 kDa, this indicating that the monoclonal antibodies 52 and 510 were directed against the C.delta. region (Table I).

Other .gamma..delta. soluble receptors were prepared as described below, after modification of the multiple cloning site of the expression vector pKCR6.

a) Modification of the multiple cloning site of the expression vector pKCR6

In order to facilitate and to permit the oriented integration of the complementary DNAs encoding the soluble gamma and delta chains in the eukaryotic system expression vector pKCR6, a DNA fragment previously cloned between the XbaI and SalI sites of the vector pKCSR.alpha. was introduced between the KpnI sites of this vector.

The digestion of the vector pKCR6 thus modified by the XhoI and XbaI enzymes liberated these two sites and permitted an oriented cloning, the XhoI site being situated between 5' of the coding sequence and the XbaI site in 3'.

b) Generation of a complementary DNA encoding a soluble V.gamma.8 chain

b1) PCR cloning of a soluble V.gamma.8 chain

The RNA used for this cloning is obtained from a T.gamma..delta. clone.

The oligonucleotide primer used for the synthesis of the first complementary DNA strand is the following:

The amplification of this cDNA was carried out by means of the oligonucleotide described above used as antisense primer and a sense primer containing a site for the XhoI restriction enzyme upstream of the translational initiation codon. The sequence of this oligonucleotide is the following:

The DNA fragment obtained at the end of this amplification was cloned into the vector pBS-SK opened by the SmaI restriction enzyme and then sequenced. The sequence obtained is in conformity with that described in the literature (Cell. (1986) 45:237-246) with the exception of the joining sequence involving the J.gamma.1 segment:

b2) Integration into the expression vector and transfection into eukaryotic cells

The cDNA fragment encoding a soluble V.gamma.8 chain was extracted from the vector pBS-SK after digestion with the restriction enzymes XhoI and XbaI and integrated into the modified expression vector pKCR6 described in a) digested with the same enzymes.

The vector thus obtained was co-transfected in combination with the expression vector containing the cDNA encoding the soluble V.delta.2 chain.

The procedure for transfection, screening of the producing clones and purification of the soluble TCRs produced is analogous to that described above for the production of soluble V.gamma.9 V.delta.2 TCR.

c) Generation of a complementary DNA encoding a soluble V.delta.3 chain

cl) PCR cloning of a soluble V.delta.3 chain

The RNA used for this cloning is obtained from a T.gamma..delta. clone.

The nucleotide primer used for the synthesis of the first complementary DNA strand is the following:

The amplification of this cDNA was carried out by means of the oligonucleotide described above used as antisense primer and a sense primer containing a site for the XhoI restriction enzyme upstream of the translational initiation codon. The sequence of this oligonucleotide is the following:

The DNA fragment obtained at the end of this amplification was cloned into the vector pBS-SK opened by the SmaI restriction enzyme and then sequenced. The sequence obtained is in conformity with that described in the literature (j. Exp. Med. (1989) 169:393-405) with the exception of the joining sequence involving the D.delta.2, D.delta.3 and J.delta.1 segments:

c2) Integration into the expression vector and transfection into eukaryotic cells

The cDNA fragment encoding a soluble V.delta.3 chain was extracted from the vector pBS-SK after digestion with the restriction enzymes XhoI and XbaI and integrated into the modified expression vector pKCR6 described in a) digested with the same enzymes.

The vector thus obtained was co-transfected in combination with the expression vector containing the cDNA encoding the soluble V.gamma.9 chain.

The procedure for transfection, screening of the producing clones and purification of the soluble TCRs produced is analogous to that described above for the production of soluble TCR V.gamma.9 V.delta.2.

d) Generation of a complementary DNA encoding a soluble V.delta.1 chain

The DNA complementary to a total V.delta.1 C.delta. chain cloned into the vector pBS-SK between the SalI and BamHI restriction sites was used.

This fragment was sequenced completely and exhibits no variation compared with the sequence described in the literature (Eur. J. Immunol. (1989) 19:1545-1549) with the exception of the joining sequence involving the D.delta.2 and J.delta.1 segments:

e) Integration into the expression vector and transfection into eukaryotic cells

The digestion, with the XhoI and EcoRI restriction enzymes, of the vector pBS-SK containing the V.delta.1 C.delta. cDNA liberates a DNA fragment encoding the entire variable part V.delta.1 D.delta.2 J.delta.1 and the portion of the first exon of the constant part C.delta. between the joining region and the unique EcoRI site.

This DNA fragment was purified and integrated into the expression vector pKCR6 containing the soluble V.delta.3 chain after it had been digested with the XhoI and EcoRI restriction enzymes. This strategy therefore made it possible to replace the variable part V.delta.3 with the variable part V.delta.1 and thus to construct a cDNA encoding a soluble V.delta.1 chain.

The vector thus obtained was co-transfected in combination with the expression vector containing the cDNA encoding the soluble V.gamma.9 chain.

The procedure for transfection, screening of the producing clones and purification of the soluble TCRs produced is analogous to that described for the production of soluble TCR V.gamma.9 V.delta.2.

a) Detection of various soluble receptors, control of specificity

In the same manner as described above, 2 IRMAs were developed with the antibody 510 as phase antibody and with the antibodies 360 and 389 as tracers. These 2 IRMAs were tested on the supernatants of CHOs transfected with the genes V.gamma.9/V.delta.2, V.gamma.9/V.delta.3, V.gamma.8/V.delta.2. Only the tracers corresponding to the transfected V give a signal, thus providing a good control of specificity.

b) Development of a general method of purification

The purification described previously for isolating the V.gamma.9V.delta.2 receptor consisted of an immunopurification with an anti-V.gamma.9 antibody (Y102 or 7B6). An affinity column of the same type but using the antibody 510 described above and which recognizes a determinant of the delta constant chain was used. The advantage of this new purification is the possibility of purifying any soluble receptor of the invention regardless of the .gamma., .delta. and even .alpha., .beta. variable chains which they contain. This method was first tested in order to purify the soluble receptor containing V.gamma.9/V.delta.3.

5 mg of antibody 510 were covalently linked to 1 g of a matrix of cyanogen bromide-activated sepharose 4B beads (PHARMACIA, Upsalla, Sweden) according to the instructions of the supplier.

The supernatant from a culture of the transfectant .gamma.9.delta.3 was applied to the affinity column thus formed at the rate of 10 ml/hour at room temperature. After washing with a phosphate buffered saline solution PBS (0.01M phosphate, 0.14M NaCl, pH 7.2, same flow rate), the bound material was eluted with a 0.05M titrate solution at pH 3.0. The eluted fractions were neutralized immediately with a 0.2M Tris buffer pH 9 (100 .mu.l for 1 ml of eluate).

The fractions positive for the soluble TR activity as attested by the IRMA test were combined and concentrated to 1 .mu.g/ml of proteins on a CENTRICON cell (30 KD barrier) (AMICON, Beverly, Mass., USA) according to the instructions of the manufacturer. This cell also made it possible to change the buffer for PBS.

The analysis of the eluted proteins was carried out by SDS-PAGE and by Western-blotting. The analysis gave slightly different results compared with .gamma.9.delta.2. Indeed, under non-reducing conditions, three highly predominant bands of molecular weights 65, 68, 70 kD, which separated into four predominant bands 32.5, 34, 36 and 40 kD ›sic!. Western-blot analysis with the anti-bodies 510 (anti-C.delta.) and 360 (anti-V.gamma.9) showed that all the predominant bands previously observed under non-reducing conditions reacted with both antibodies. Under reducing conditions, the bands reacted either with the antibody 360 or with the antibody 510.

From this analysis, it can be concluded that the material eluted from the affinity column consisted essentially of covalently linked .gamma..delta. heterodimers possibly present in the form of several glycosylation isomers.