WO1996036881A2

WO1996036881A2 - Screening for inhibitors of tcr-mhc interactions

Info

Publication number: WO1996036881A2
Application number: PCT/GB1996/001165
Authority: WO
Inventors: Dan Hung Barouch; Stephen John Rupert Vessey; Bent Karsten Jakobsen
Original assignee: Cancer Research Campaign Technology Limited
Priority date: 1995-05-16
Filing date: 1996-05-16
Publication date: 1996-11-21
Also published as: WO1996036881A3; GB9509844D0; AU5769096A

Abstract

This invention relates to the interactions between T-cell receptors and their specific MHC-peptide ligand. A screening method is provided to identify substances which inhibit the specific interaction between a T-cell receptor (TCR) and an MHC-peptide ligand involving incubating responder cells expressing a TCR with (i) an MHC-peptide ligand which stimulates said responder cells; and (ii) a candidate inhibitory substance; and monitoring a signal produced by said responder cells when the TCR engages the MHC-peptide ligand; and comparing said signal produced by said responder cells on the engagement of TCR with the MHC-peptide ligand with a control signal produced by said responder cells when incubated with (i) but not (ii); wherein a reduction in signal associated with the presence of (ii) in the incubation represents the efficacy of the inhibitor.

Description

Screening for inhibitors of TCR-MHC interactions

This invention relates to interactions between T-cell receptors and their specific MHC/peptide ligand and the potential therapeutic applications of inhibitors of this interaction.

According to one aspect of the present invention there is provided a method of screening for substances which inhibit the specific interaction between T-cell receptors (TCRs) and MHC- peptide ligands, involving incubating a candidate inhibitory substance with stimulatory MHC- peptide ligands and responder cells expressing TCRs. These cells produce a signal when the TCR engages the stimulatory MHC-peptide ligands, and the reduction of this signal by the candidate inhibitory substance represents the efficacy of the inhibitor.

The responder cell expressing the TCR preferably has the following properties: (i) it should be easy to culture; (ii) the correctly folded TCR should be stably expressed at high levels on the cell surface; (iii) the signal generated by the TCR-MHC interaction should be easily detectable by conventional means and simulates the functioning of the TCR in its native T- cell environment. An example of such a responder cell is a mast cell transfected with recombinant DNA encoding the TCR protein and which undergoes degranulation in response to the TCR-MHC interaction on the cell surface.

Stimulatory MHC-peptide complexes may be created by immobilising soluble recombinant MHC on a solid support, such as "Dynabeads" (a form of plastic beads). The MHC is typically obtained by recombinant DNA expression, and is a single specific MHC molecule or fragment thereof.

Another aspect of the invention provides a multimerized array of MHC, with or without an associated peptide, suitable for use in the foregoing process.

The present invention is based upon the following experimental data.

Summary of the experimental findings

The critical event in the generation of a T cell response is recognition of a specific MHC- peptide ligand by a T cell receptor (TCR)¹. It remains unclear whether the TCR discriminates between individual peptfdes on the basis of relative affinity, a specific conformational effect or both². The present work describes an assay for studying this interaction using purified MHC-single peptide complexes³ and an antigen specific TCR-zeta chimeric receptor (TCZR) expressed on the surface of the basophil cell line⁴. These transfectants respond with the same specificity as the parent T cell without the additional binding and signalling effects of accessory molecules on target cells. Stimulation through the TCZR occurs only with multivalent arrays of the specific ligand suggesting that receptor multimerisation is necessary for productive signalling. Competition studies show that the MHC complexed to a variety of different peptides can bind the TCZR but cannot elicit a response, implying that productive signalling requires an additional effect such as a conformational change. Explanation of the drawings

Figure la shows the cloning and expression of TCR genes encoding Vα2.2 and VjSl.l.

Figure lb shows the FACS analysis of stably transfected RBLs expressing the TCZR.

Figure lc shows, in the preparation and purification of MHC-peptide complexes, a sample HPLC gel filtration profile of purified, soluble, recombinant MHC protein.

Figure 2a-d shows specific response of RBL/TCZR transfectants to peptide-pulsed target cells and purified MHC proteins. The results are means from triplicate assays. A typical experiment is shown in each case.

Figure 3a-f shows competition with monomeric MHC-peptide complexes and controls.

Figure 4 shows diagrammatically a model for TCZR signalling.

Detailed description of the experiments

Referring to Figure la; TCR genes were amplified by PCR from cDNA prepared from a CTL clone specific for HLA A2.1 restricted HIV pol peptide. Primer sequences were: Vα2.2 forward (5' CAC CGC TCG AGC CGC CAT CAT GAT GAA ATC CTT GAG A 3'), Nc.2.2 back (5' CTG GTA CAC GGC CGG GTC AGG GTT CTG GAT ATT 3') VjSl.l forward (5'ATG CAA GTC AGT CGA CCC GCC ACC ATG GGC TTC AGG CTC CTC T 3'), and VjSl. l back (5' CAC AGC GAC CTC GGG TGG GAA CAC GTT CTT AAG GTC CTC 3'). Products were cloned into pBS(KS-) encoding Cα and Cβ sequences mutagenised to contain an Eag I and an Afl II site respectively. A Vβ/Cβ Blunt/Xma I fragment and a VOJ/CC. Xho I/Xmal fragment were subcloned into expression vectors encoding the CD3 zeta chain. RBL cells were transfected with lOμg of each expression vector (linearised with Hind III) by electroporation (500μF, 250mV) and grown for 18 hours in DMEM 10% FCS. Stable transfectants were selected by the addition of 700 g/ml G418 (Sigma), 15μg/ml hypoxanthine, lOμg/ml aminopterin, 2μg/ml thymidine, 250μg/ml xanthine and 5μg/ml mycophenolic acid.

Referring to Figure lb; for the FACS analysis of stably transfected RBLs expressing the TCZR, 2xl0⁵ cells were stained with monoclonal antibodies (αFl (Serotec), /3F1) to the constant domain of the α^* and β chains of the TCR.

Referring to Figure lc; all recombinant MHC-single peptide complexes were prepared essentially as described³. Briefly, heavy chain and /52M inclusion bodies were overexpressed separately in E. coli. The inclusion bodies were purified, denatured in urea, and refolded around a synthetic peptide by an in vitro peptide-dependent folding mediod. Peptides were synthesized using standard Fmoc chemistry, verified by mass spectrometry and reverse phase HPLC, and HPLC purified if necessary. The folded MHC-single peptide complexes were purified by HPLC gel filtration and concentrated to 5mg/ml. Protein concentrations were determined by a combination of spectrophotometry, scanning densitometry on SDS-PAGE gels, and HPLC peak quantitation. The concentrations of active protein in each preparation were confirmed by an ELISA detecting immobilized MHC protein with a panel of monoclonal antibodies (data not shown).

Referring to Figure 2; peptides complexed with the A2 proteins were: pol (ILKEPVHGV), gag (SLYNTVATL), a random library of A2-binding peptides, pol-8E (ILKEPVHEV), a self peptide TLW (TLWVDPYEV), and null (SLAAAAAAL).

RBLs were cultured in flat bottomed 96 well plates (Falcon) at a density of 5x10"* cells/well for 12 hours at 37°C in DMEM 10%FCS (Sigma) with ³H-hydroxytryptamine creatinine sulphate (DuPont) at a final concentration of 0.2_/xCi/ml. The RBLs were washed 3 times in warm DMEM 10%FCS, incubated or 20 mins and then washed once more.

C1R cells were resuspended at 5xl0⁶/100μl and pulsed with various concentrations of peptide for 2hrs at 37°C. After one wash these APCs were added to the RBLs and coincubated for 60mins at 37° C. Supernatants were collected and responses were measured as corrected counts per minute (ccpm) by liquid scintillation counting.

Figure 2a shows the response to HA-A2.1 C1R cells pulsed with a range of concentrations of the HIV pol peptide.

Figure 2b shows the response to HA-A2.1 or HA-B7 C1R cells pulsed with individual peptides.

Referring to Figure 2c; this shows the response to purified, recombinant, monomeric A2-pol and multimerised MHC-peptide complexes.

Degranulation assays were performed as above except either monomeric MHC protein or 10 million MHC-saturated dynabeads were added to the wells instead of peptide-pulsed cells.

In order to create multimerised MHC-peptide complexes, sheep anti-mouse dynabeads were first saturated with an affinity-purified monoclonal coupling antibody according to manufacturer's protocols (Dynal). Coupling antibodies used were W6/32 (monomorphic anti- MHC) and BB7.2 (anti-A2, directed against the alpha-2 helix of the MHC). Following three washes, the beads were saturated with MHC-peptide complexes for 2 hours at 4° and then washed again three times before addition to the assay.

Referring to Figure 2d; this shows the response to biotinylated A2-peptide complexes multimerised on streptavidin coated dynabeads.

Streptavidin dynabeads were incubated with various amounts of purified, biotinylated A2- peptide complexes for 30 minutes at 4°C and then washed three times to eliminate any free A2 monomers. Biotinylated A2-peptide complexes were prepared by surface biotinylation of refolded β2M protein for 1 hour at room temperature using a 3-fold molar excess of NHS- SS-biotin (Pierce) gel filtration purification to remove free biotin, and seeding a folding reaction essentially as before (Fig lc) using folded biotinylated β2M, denatured heavy chain, and a synthetic peptide. Degranulation assays were performed as above with 10 million MHC-coated dynabeads per well. Referring to Figures 3a/b; these show competition of RBL-TCZR response to multimerised A2-pol with non biotinylated monomeric MHC-peptide complexes.

Results shown are means of 3 to 8 independent experiments each performed in duplicate and are reported as a percent of the uncompeted response. Standard error of the mean is shown in Fig 3a. Degranulation assays were performed (as in Fig 2) using 10 million A2-pol coated streptavidin dynabeads (as in Fig 2d) per well. A sub-saturating concentration of MHC protein was used (lμg MHC per 10 million beads), and the response with no added competitor was approximately 1000 ccpm. Soluble, monomeric, non-biotinylated MHC protein was checked by gel filtration to ensure that there was no detectable aggregation of the protein. The protein was diluted in media and added to the assay together with the A2- pol coated dynabeads.

Figure 3c shows that the stimulatory capacity of streptavidin coated dynabeads bearing multivalent arrays of MHC-peptide is unaffected by preincubation with monomeric complexes. Biotinylated A2-pol or A2-gag complexes were linked to streptavidin coated dynabeads as described above. Beads were then incubated for 60 mins at 37°C with either medium alone or 10μM non-biotinylated monomeric complexes. Multivalent A2-pol was incubated with monomeric A2-gag and vice versa. After washing the beads 3 times in warm DMEM 10% FCS to remove free monomeric complexes they were used in degranulation assays as described above.

Figure 3d shows that the response to multivalent A2-pol is unaffected by the presence of free gag peptide. Assays were performed as described above in the presence of ascending concentrations of uncomplexed gag peptide.

Figure 3e shows that RBL/TCZR degranulation induced by a monoclonal antibody to the constant domain of the TCR (_/3F1) is not affected by the presence of monomeric A2-pol or A2-gag. Degranulation assays were performed as in Fig 2 but lμg of mAb jSFl was added in place of APCs in addition to monomeric A2-peptide complexes prepared as in Fig 3a/b.

Figure 3f shows that RBL/TCZR degranulation is unaffected by preincubation of transfectants with monomeric MHC-peptide complexes. RBL/TCZR transfectants were preincubated with monomeric MHC-peptide complexes for 40 mins at 37 °C and men washed with warm DMEM 10% FCS. Degranulation assays were then performed as in Fig 2 but with 10 million streptavidin dynabeads coated with A2-pol per well.

Figure 4 proposes a model for TCZR signalling based on the results presented here together with the findings in references 20-22 and possible similarities with the EGF receptor²³. Figure 4a shows TCZR engaged by a multivalent array of MHC-peptide complexes. Figure 4b shows how specific ligand can induce conformational change allowing stable receptor aggregation. Non-specific ligand induces no conformational change. Figure 4c shows TCZR engaged by soluble monomeric MHC-peptide complexes. Figure 4d shows conformational change induced, but insufficient cross-linking of receptors takes place. Results and discussion

Eukaryotic expression vectors encoding and β TCZR chimeric receptor chains⁴ were constructed using TCR genes from a CTL clone specific for an HLA -A2.1 restricted HIV pol peptide (residues 476-484; ILKEPVHGV)⁵ (Fig la). Stably transfected RBL 2H3 cells expressing the TCZR (Fig lb) showed peptide specific and MHC restricted degranulation using peptide pulsed target cells (Fig 2a, b). These results indicate that the chimeric receptor retains the specificity of the original TCR, implying that the antigen binding site of the receptor is correctly folded.

We next studied the interaction between the TCZR and a series of purified recombinant MHC-single peptide complexes³ (Fig lc). No stimulation was observed using monomeric A2-pol complexes (Fig 2c). Multivalent arrays of antigen were prepared by coupling MHC- peptide complexes to dynabeads. Specific degranulation was demonstrated in response to multivalent arrays of A2-pol complexes multimerised on sheep anti-mouse or streptavidin coated dynabeads using either mAb W6/32⁶ (Fig 2c) or biotinylated jS2M respectively (Fig 2d). This degranulation is dependent on the accessibility of the peptide binding groove since no response was observed when BB7.2⁷ a mAb specific to a polymorphic residue on the o;2 domain of HLA-A2 was used to multimerise the A2-pol complexes (Fig 2c). These results demonstrate that a specific response can be generated by purified MHC protein in the absence of other accessory molecules on target cells.

In order to test whether A2-pol monomers can bind to the TCZR we performed competition experiments between monomeric and multivalent A2-pol complexes. The results indicate that despite being unable to stimulate degranulation, A2-pol monomers are capable of engaging the TCZR since they inhibited the response to multivalent A2-pol (Fig 3a). Lack of inhibition by monomeric MHC/peptide complex B8-gag pl7 (residues 24-32) - ie a complex in which both the MHC and the associated peptide are presumed irrelevant to the TCZR - demonstrates that this effect is MHC-restricted (Fig 3a).

Remarkably, the response to multivalent A2-pol was also inhibited by monomeric soluble A2 protein complexed with some other peptides, including a variant of the index pol peptide, a random library of A2 binding peptides⁸, and a supposedly irrelevant HIV gag peptide pl7 (residues 77-85)⁹ (Fig 3b). This suggests that these complexes also bind the TCZR. The inhibition observed with non-cognate complexes cannot be explained by displacement of the A2-pol from the streptavidin coated beads since the monomeric competitors were not biotinylated. This point was confirmed by preincubation of A2-pol coated beads with monomeric A2-gag and vice versa, which did not alter the stimulatory capacity of the beads (Fig 3c). Exchange of heavy chain or peptide between the monomeric complexes and the multimerised A2-pol as a cause of inhibition was also excluded by this observation. Peptide exchange was further excluded by demonstrating tfiat a significant excess of free gag pl7-8 peptide had no inhibitory effect on stimulation by multivalent A2-pol (Fig 3d). In addition purified A2-peptide complexes did not exhibit a nonspecific inhibitory or toxic effect on the RBLs since they failed to inhibit degranulation induced by an anti-TCR mAb (Fig 3e). This conclusion is reinforced by the observation that preincubation of RBLs with monomeric A2 complexes followed by washing did not affect their subsequent responsiveness to multivalent A2-pol (Fig 3f). Moreover, as internal controls in competition experiments, A2-TLW¹⁰ (a self peptide TLWVDPYEV) and A2-null (SLAAAAAAL) showed little inhibition of degranulation in response to A2-pol coated beads (Fig 3b).

The finding that A2 complexes with some non-cognate peptides can compete for binding to the TCZR is consistent with a previous study in which T cell hybridoma responses were inhibited by a soluble Ig-TCR chimera which shared MHC-restriction but not peptide specificity¹¹. In addition, the process of positive selection, which is considered to arise from interaction between the TCR and non-cognate peptide complexes, might result in some inherent binding a variety of A2-peptide complexes by an A2-restricted TCR¹²"¹⁷.

Stimulation of degranulation by cross-linking antibodies and multimerised A2-pol complexes, but not by the same complex in monomeric form, suggests that multimerisation of the TCZR is required to induce a response. Ligand multivalency has also been shown to be of importance in signalling through both hapten-specific¹⁸ and allospecific TCRs¹⁹. Successful TCZR signalling cannot simply be a consequence of differential ligand affinity, since our competition data demonstrate that non-cognate ligands can bind to the TCZR with a similar profile to A2-pol yet are unable to induce degranulation even when presented in multivalent form at concentrations 100 fold greater than those required to observe a response with A2-pol (data not shown).

If ligand binding alone is insufficient for productive signalling of the TCZR then an additional effect is required. One possibility is that local TCZR aggregation is induced by exposure of a contact surface between correctly engaged receptors possibly through a conformational change (Fig 4). There is evidence to support the view that signalling through the native TCR requires a change in conformation as well as a multimerisation. Reports have shown that crosslinking of the TCR with a low potency antibody can lead to T cell activation only when combined with a non-cross linking Fab fragment from a second antibody that is thought to induce a conformational change²⁰. In addition no correlation was found between the affinity or avidity of anti-TCR mAbs and induction of a T cell response^21,22.

In summary these experiments indicate that engagement of the TCR by a multivalent array of MHC-peptide complexes is necessary but not sufficient for productive signalling. These data suggest that the affinity of the interaction between the TCR and specific MHC-peptide complexes is unlikely to account fully for the T cell signalling associated with antigen recognition. One possible model is that, as seen with the EGF receptor²³, a conformational change in the extracellular domain of the TCR is also required for productive multimerisation and subsequent signalling.

The present invention thus allows a very concise assay to be set up for the screening of substances, eg peptides or mimetics, that inhibit the interaction between TCRs and MHC- peptide ligands in vitro. The use of a RBL (a type of mast cell) expressing TCRs as repsonder cells is useful since degranulation of these cells provides a convenient signal of effective TCR-MHC-peptide interactions. The RBLs are also useful in that other surface proteins are not known to interact with MHC, such as would be the case with T-cells which normally express TCRs. However, other responder cells could be used, for example employing expression of a suitable reporter molecule such as jS-galactosidase as the detectable signal. By using this combination of multimerised MHC-peptide complexes and recombinant TCRs, one can readily screen for inhibitors of their binding. These inhibitors could typically be highly specific peptides, MHC-peptide complexes or MHC-peptide mimetics, antibodies, TCRs, or other immunospecific molecules, or fragments of any of the above. Other inhibitors could be more general in their action and inhibit many or all TCR-MHC interactions. These inhibitors could act by direct competition, allosteric changes, or other means.

Substances which inhibit TCR-MHC-peptide interactions could be used in a variety of therapeutic applications, for example in blocking T-cells which give rise to autoimmune disease such as diabetes, rheumatoid arthritis, groves disease etc, organ transplant rejection, or other T-cell mediated conditions.

References

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8. Flynn, G.C., Pohl, J., Flocco, MX, and Rothman, J.E. Nature 353, 726-730 (1991).

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796 (1992).

12. Schumacher, T.N.M. and Ploegh, H.L. Immunity 1, 721-723 (1994).

13. Janeway, C.A. Immunity 1, 3-6 (1994).

14. Hogquist, K.A., Gavin, M.A., and Bevan, M.J. J. Exp. Med. 177, 1469-1473 (1993).

15. Ashton-Rickardt, P.G., Van Kaer, L, Schumacher, T.N.M. , Ploegh, H.L, and Tonegawa,

S. Cell 73, 1041-1049 (1993).

16. Hogquist, K.A. etal. Cell 76, 17-27 (1994).

17. Ashton-Rickardt, P.G. et al. Cell 76, 651-663 (1994).

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1430 (1992).

19. Schneck, J., Maloy, W.L, Coligan, J.E., and Margulies, D.H. Cell 56, 47-55 (1989).

20. Rojo, J. and Janeway, C.A. J. Immunol. 140, 1081-1088 (1988).

21. Janeway, C.A. et al. Cold Spring Harbor Symp. Quant. Biol. 14, 657-666 (1989).

22. Yoon, S.T., Dianzani, U., Bottomly, K., and Janeway, C.A. Immunity 1, 563-569 (1994).

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Claims

CLAIMS :

1. A screening method to identify substances which inhibit the specific interaction between a T-Cell Receptor (TCR) and an MHC-peptide ligand, which comprises the steps of:

incubating responder cells expressing a TCR with (i) an MHC-peptide ligand which stimulates said responder cells; and (ii) a candidate inhibitory substance; and

monitoring a signal produced by said responder cells when the TCR engages the MHC-peptide ligand; and

comparing said signal produced by said responder cells on the engagement of TCR with the MHC-peptide ligand with a control signal produced by said responder cells when incubated with (i) but not (ii)

wherein a reduction in signal associated with the presence of (ii) in the incubation represents the efficacy of the inhibitor.

2. A method according to claim 1 wherein the responder cells comprise high levels of TCR stably expressed at the cell surface.

3. A method according to claim 1 or claim 2 wherein said signal comprises the generation of a detectable substance by the responder cell.

4. A method according to claim 3 wherein the detectable substance is radioactively labelled.

5. A method according to any one of the preceding claims wherein the responder cell is transfected with recombinant DNA encoding the TCR protein.

6. A method according to claim 5 wherein the responder cell is also transfected with recombinant DNA encoding a CD3 zeta chain.

7. A method according to any one of the preceding claims wherein said responder cell is a granulocyte.

8. A method according to claim 7 wherein said granulocyte is a mast cell.

9. A method according to claim 7 or claim 8 wherein the granulocyte degranulates in response to the engagement of TCR with the MHC-peptide ligand, said degranulation comprising the signal.

10. A method according to any one of the preceding claims wherein the MHC-peptide ligand is immobilised on a solid support.

11. A method according to any one of the preceding claims wherein the MHC is recombinant MHC.

12. A method according to any of the preceding claims wherein the MHC-peptide ligand comprises a multimerised multivalent array of MHC-peptide ligands.

13. A method according to any one of the preceding claims wherein the candidate inhibitory substance is selected from: highly specific peptides, MHC-peptide complexes or MHC-peptide mimetics, antibodies, TCRs, or other immunospecific molecules, or fragments of any one of the above.

14. A multimerised multivalent array of MHC molecules suitable for use in the method of claim 1.

15. A multimerised multivalent array of MHC molecules according to claim 14 wherein said MHC molecules are complexed with peptides.

16. A kit for a screening method as defined in claim 1 which comprises:

a responder cell expressing a TCR; and

an MHC molecule which when complexed to a ligand can function to stimulate said responder cell.

17. A kit according to claim 16 which comprises a multimerised multivalent array of MHC molecules.

18. A kit according to claim 16 or claim 17 wherein ligand is complexed to MHC.

19. An inhibitor of the specific interaction between a TCR and an MHC-peptide ligand obtainable by a method according to any one of claims 1 to 13.

20. A method which comprises the preparation of a pharmaceutical composition comprising an inhibitor of the specific interaction between a TCR and an MHC-peptide ligand identified following a method according to any one of claims 1 to 13.

21. A pharmaceutical composition comprising an inhibitor of the specific interaction between a TCR and an MHC- peptide ligand identified following a method according to any one of claims 1 to 13.