PEPTIDE-MHC COMPLEXES

Abstract

The present invention provides a stabilised peptide-MHC (pMHC) complex, such as a peptide-HLA-E complex. The complex has a non-native linkage, such as a disulphide bond, between the C terminal anchor residue of the peptide, and an amino acid residue in the F pocket of the MHC binding groove.

Claims

1. A stabilized peptide-MHC (pMHC) complex, comprising a non-native linkage between the C terminal anchor residue of the peptide, and an amino acid residue in the F pocket of the MHC binding groove.

2. The complex of claim 1, wherein the non-native linkage is a covalent bond.

3. The complex of claim 2, wherein the covalent bond is formed between amino acids substituted for amino acid residues in the F pocket of the MHC binding groove and/or the C terminal anchor residue of the native peptide, preferably in both the F pocket of the MHC binding groove and the C terminal anchor residue of the native peptide.

4. The complex of claim 3, wherein the substituted amino acid residue in the F pocket of the MHC binding groove is at position 116 or 147.

5. The complex of claim 1, wherein the non-native linkage is a disulfide bond.

6. The complex of claim 5, wherein the amino acid residue at position 116 or 147 of the MHC heavy chain is substituted to cysteine.

7. The complex of claim 3, wherein the amino acid substituted for the C-terminal anchor residue of the peptide is a non-natural amino acid.

8. The complex of claim 7, wherein the C terminal amino acid anchor residue of the peptide is substituted to an analogue of homocysteine that has an extended carbon side chain.

9. The complex of claim 8, wherein the analogue of homocysteine is 2-amino-5-sulfanyl-pentanoic acid or 2-amino-6-sulfanylhexanoic acid.

10. The complex of claim 1, wherein the complex is soluble.

11. The complex of claim 1, wherein the MHC includes a biotin tag, optionally wherein the tag is C terminal.

12. The complex of claim 1, wherein the MHC is HLA-E.

13. A multimer of the complex of claim 1.

14. A method of making the peptide-MHC complex of claim 1, comprising forming a covalent bond between the MHC heavy chain and the C terminal amino acid anchor residue of the peptide.

15. A method of screening, comprising combining the complex of claim 1 with a population of T cell receptors (TCRs), TCR mimic antibodies or T cells; and identifying TCRs, TCR mimic antibodies or T cells that bind to the complex.

Description

EXAMPLES

[0041] The invention will now be described with reference to the following non-limiting examples and figures in which:

[0042] FIG. 1 shows the stability of the indicated pMHC complexes as determined by loss of ILT2 binding over time. Native pHLA-E complexes show limited stability.

[0043] FIG. 2 shows the difference in TCR recognition between a native pMHC complex and the equivalent pMHC complex stabilised using existing methodology.

[0044] FIG. 3 shows the difference in TCR recognition between a native pMHC complex and the equivalent stabilised pMHC complex of the invention.

EXAMPLE 1—ISOLATED PEPTIDE HLA-E COMPLEXES HAVE LIMITED STABILITY

[0045] This example demonstrates that isolated peptide HLA-E complexes have a short half-life, which means that they are not sufficiently stable to be used for the identification and characterisation of binding agents, such as TCRs and antibodies. A half-life of at least 4 h is typically preferred for such purposes and a half-life substantially in excess of this is desirable.

[0046] Stability was assessed using a number of peptides that are known to be presented by HLA-E, including MTB and HIV peptides described in Joosten et al., (PLoS Pathog. 2010 Feb. 26; 6(2):e1000782) and Hansen et al., (Science. 2016 Feb. 12; 351(6274):714-20), respectively, as well as two self peptides corresponding to leader peptides from HLA-A*02 and HLA-Cw3.

Methods

[0047] Peptides

[0048] Peptides were obtained by chemical synthesis from Peptide Protein Research Ltd and solubilised in DMSO to a concentration of 4 mg/ml prior to use.

[0049] Production of HLA-E*01:01 and HLA-E*01:03 Peptide Complex

[0050] HLA-E heavy chain and beta 2-microglobulin (β2m) were expressed separately in E. coli as inclusion bodies and subsequently refolded and purified using previously described methods (Garboczi et al., Proc Natl Acad Sci USA. 1992 Apr. 15; 89(8):3429-33). The HLA-E heavy chain contained a C-terminal biotinylation tag (AviTag™ GLNDIFEAQKIEWHE) and excluded the transmembrane and cytoplasmic domains. Briefly, HLA-E heavy chain and β2m were mixed and refolded together with the peptide of interest, at a ratio of 30:5:2. The soluble refolded pHLAs were then purified using a two-step protocol incorporating anion exchange, followed by size exclusion chromatography (SEC)). To produce biotinylated complexes, a biotinylation step was included following anion exchange and prior to SEC using Biotin-protein ligase (BirA) as described in O'Callaghan et al., Anal Biochem. 1999 Jan. 1; 266(1):9-15.

TABLE-US-00002 Sequence of HLA-E*01:03 Heavy chain native + AviTag™ and F116 highlighted MGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVP RAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTL QWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQIS EQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTH HPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDG TFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPGSGGGLNDIFE AQKIEWHE Sequence of HLA-E*01:03 Heavy chain native + AviTag™ and S147 highlighted MGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVP RAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTL QWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQIS EQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTH HPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDG TFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPGSGGGLNDIFE AQKIEWHE

[0051] The sequence of the AviTag™ and its GSGG linker is underlined and F116 and 5147 are shown in bold and underlined.

TABLE-US-00003 Sequence of Human beta-2 microglobulin MIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKV EHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0052] Assessment of Peptide-HLA Complex Stability

[0053] The stability of peptide-HLA-E complexes was assessed by Surface Plasmon Resonance (SPR) using a BIAcore T200 instrument. Approximately 500-1000 response units (RUs) of purified biotinylated peptide-HLA-E monomers were captured onto streptavidin-coupled CM-5 Series S sensor chips. A soluble, affinity enhanced, form of Ig-like transcript 2 receptor (ILT2) at a concentration of 1 μM was flowed over the surface of the chip at a flow rate of 10 μl min.sup.−1 for 60 seconds. ILT2 binds to class I HLA molecules in a conformationally dependent manner and is therefore used as an indicator of complex stability. ILT2 binding to pHLA-E complexes was measured at regular intervals over a time course of 5 hours and responses were then normalised for ILT2 binding by subtracting the bulk buffer response on a control flow cell containing no peptide-HLA. Binding half-life (T.sub.1/2) was calculated by plotting % activity against time, using the BIA T200 evaluation software and GraphPad Prism 8.

Results

[0054] Table 1 below provides the half-life (T.sub.1/2) for each of the indicated peptides in complex with HLA-E*01:03, as determined by ILT2 binding. All of the complexes have a half-life of under 5 h and several complexes have a half-life of less than 1 h. Representative examples of binding data are provided in FIG. 1 (left-hand panels).

TABLE-US-00004 TABLE l Peptide origin Peptide sequence T .sub.1/2 (h) (HLA-E*01:03) MTB (Rv1484) RLPAKAPLL 4.50 MTB (Rv3823c) ILPSDAPVL 0.70 MTB (Rv 1518) VMATRRNVL 0.57 HIV (Gag 275) RMYSPTSIL 0.50 HIV (Gag 275) RMYSPVSIL 0.27 Self (HLA-Cw3) VMAPRTLIL 3.20 Self (HLA-A*02) VMAPRTLVL 3.00

[0055] These data indicate that native peptide-HLA-E complexes have limited stability, which is unsuitable for identification and characterisation of binding agents.

EXAMPLE 2—PEPTIDE HLA-E COMPLEXES CAN BE STABILISED VIA CYS TRAP METHODOLOGY BUT DEMONSTRATE PERTURBED TCR BINDING

A)

[0056] In this example, the HLA-E heavy chain was modified to incorporate a cysteine mutation at position Y84; and the peptide was modified to include three additional amino acids (Gly-Cys-Gly) at the C terminus. This approach is commonly referred to as ‘Cys trap’ and has been used successfully to improve the stability of various HLA complexes by ‘trapping’ the peptide in the binding groove (as described in Truscott J Immunol. 2007 May 15; 178(10):6280-9; Mitaksov et al., Chem Biol. 2007 August; 14(8):909-22).

Methods

[0057] The same experimental methods as described in Example 1 were used.

Results

[0058] Table 2 below provides the half-life for each of the indicated cys trapped peptide HLA-E complexes, as determined by ILT2 binding. All of the complexes have a substantially extended half-life compared to the unmodified complexes shown in Example 1, with the majority in excess of 20 h. Representative examples of binding data are provided in FIG. 1 (right-hand panels).

TABLE-US-00005 TABLE 2 Peptide origin Peptide sequence T .sub.1/2 (h) (HLA-E*01:03) MTB (Rv 1484) RLPAKAPLL 23.8 MTB (Rv 3823c) ILPSDAPVL 55.3 MTB (Rv 1518) VMATRRNVL 34.0 HIV (Gag 275) RMYSPTSIL 14.9 HIV (Gag 275) RMYSPVSIL 16.6 Self (HLA-Cw3) VMAPRTLIL 27.2 Self (HLA-A*02) VMAPRTLVL 48.3

[0059] These data indicate that modified peptide-HLA-E complexes incorporating a cys trap have improved stability.

B)

[0060] Cys-trap stabilised peptide-HLA-E complex comprising the MTB peptide RLPAKAPLL+GCG was subsequently tested for recognition by antigen specific TCRs and compared to the unmodified complex. This peptide was chosen since the unmodified native peptide HLA-E complex has a relatively long half-life and is therefore amenable to assessment of TCR binding.

Methods

[0061] Assessment of TCR Binding to Peptide-HLA-E Complex

[0062] Four TCRs that recognise MTB peptide RLPAKAPLL HLA-E complex were isolated from naïve phage libraries and prepared as soluble alpha beta heterodimers as previously described (Boulter et al., Protein Eng. 2003 September; 16(9):707-11).

[0063] Binding Characterisation

[0064] Binding analysis of purified soluble TCRs to peptide-HLA complexes was carried out by surface plasmon resonance (SPR), using a BIAcore T200 instrument. Biotinylated pHLA-E molecules were refolded with the peptide of interest as described in Example 1 above. All measurements were performed at 25° C. in Dulbecco's PBS buffer, supplemented with 0.005% surfactant P20.

[0065] Biotinylated peptide-HLA-E monomers were immobilized onto streptavidin-coupled CM-5 Series S sensor chips. Equilibrium binding constants were determined using serial dilutions of soluble TCR injected at a constant flow rate of 10-30 μl min.sup.−1 over a flow cell coated with ˜1000 response units (RU) of peptide-HLA-E*01:03 complex. Equilibrium responses were normalised for each TCR concentration by subtracting the bulk buffer response on a control flow cell containing no peptide-HLA. The K.sub.D value was obtained by non-linear curve fitting using GraphPad Prism 8 software and the Langmuir binding isotherm, bound=C*Max/(C+KD), where “bound” is the equilibrium binding in RU at injected TCR concentration C and Max is the maximum binding.

[0066] For high affinity interactions, binding parameters were determined by single cycle kinetics analysis. Five different concentrations of soluble TCR were injected over a flow cell coated with ˜50-200 RU of peptide-HLA complex using a flow rate of 50-60 μl min.sup.−1. Typically, 60-200 μl of soluble TCR or was injected at a top concentration of between 100-1000 nM with successive 2 fold dilutions used for the other four injections. The lowest concentration was injected first. To measure the dissociation phase, buffer was injected until 10% dissociation occurred, typically after 1-3 hours. Kinetic parameters were calculated using the BIAevaluation® or BIAcore T200 Evaluation software. The dissociation phase was fitted to a single exponential decay equation enabling calculation of half-life. The equilibrium constant K.sub.D was calculated from k.sub.off/k.sub.on.

Results

[0067] The binding affinities given in Table 3 below, along with the binding curves shown in FIG. 2, show that while the antigen specific TCRs are able to recognise the native, non-stabilised peptide HLA-E complex, recognition of the cys trap stabilised complex is substantially reduced and in some cases below the level of detection.

TABLE-US-00006 TABLE 3 K.sub.D (μM) TCR001 TCR002 TCR003 TCROO4 Native (non-stabilised) 3.97 53.69 52.07 17.78 Cys trap stabilised 38.02 ND ND 159.9 ND - low levels of binding, kinetic parameters could not be determined.

[0068] These data indicate that, although the cys trap approach produces stabilised complex, it also perturbs TCR recognition. This may be due to structural alterations of the peptide or MHC resulting from incorporation of the additional residues and formation of the disulphide bond. This approach is therefore not suitable for the production of stabilised peptide HLA-E complexes for the identification and characterisation of binding agents such as TCRs.

EXAMPLE 3—PRODUCTION OF STABLE PEPTIDE HLA-E COMPLEXES WITH MINIMAL ALTERNATION OF TCR RECOGNITION

A)

[0069] In this example, the peptide HLA-E complex was modified to incorporate a novel engineered disulphide bond between the peptide binding groove of the HLA-E heavy chain and the C terminal anchor residue of the peptide.

[0070] To create the novel disulphide, the P9 anchor residue of the MTB peptide RLPAKAPLL peptide was modified to the non-natural amino acid L-3-C homocysteine (2-amino-5-sulfanyl-pentanoic acid) (RLPAKAPL-h3C), and the HLA-E heavy chain was mutated to cysteine at either position F116 or position S147.

Methods

[0071] Peptide HLA-E complexes were prepared and assessed for stability as described in Example 1. TCR binding was assessed as described in Example 2.

Results

[0072] Table 4 below demonstrates that the novel disulphide resulted in a substantial improvement in stability as indicated by the longer half-life of the complex relative to the native complex

TABLE-US-00007 TABLE 4 T.sub.1/2 (h) peptide Stabilisation method HLA-E complex Native (non-stabilised) 4.5 Non-native disulphide (F116) 12.3 Non-native disulphide (S147) 16.6

[0073] To demonstrate that novel stabilised peptide-HLA-E complexes retain native-like TCR recognition, binding was assessed for 9 different TCRs that had been isolated from a phage library for improved recognition of peptide (RLPAKAPLL)-HLA-E complex. In each case, the kinetics of TCR binding to the stabilised complex was compared to the native complex.

[0074] Table 5 and 6 show that TCR binding to the stabilised complex (with a cysteine mutation at F116 or S147 respectively) is preserved in all cases. For each TCR only a small difference in binding is observed between the stabilised and native complex, indicating that the peptide is adopting a near native-like conformation.

[0075] FIG. 3 shows the binding curves for four of the TCRs in Table 5.

TABLE-US-00008 TABLE 5 Native Non-native complex disulphide (F116) Fold TCR K.sub.D (nM) K.sub.D (nM) difference 005 198 299 1.51 006 294 466 1.58 007 195 325 1.66 009 31.1 69.7 2.24 010 0.822 1.6 1.95 011 1877 2537 1.35 012 472 820 1.73 013 1372 1887 1.37 014 163 258 1.58

TABLE-US-00009 TABLE 6 Native Non-native complex disulphide (S147) Fold TCR K.sub.D (nM) K.sub.D (nM) difference S2a23bwt 1753 1346 1.30 S2a15bwt 462 343 1.35 S2a25bwt 97.6 81.2 1.20 S2a24b07 6280 5360 1.17 S2a22b07 765 681 1.12

B)

[0076] In a further example, the P9 anchor residue of the MTB peptide RLPAKAPLL peptide was modified to the non-natural amino acid L-4-C homocysteine (2-amino-6-sulfanylhexanoic acid) (RLPAKAPL-h4C), and the HLA-E heavy chain was mutated to cysteine at position F116 or S147. Complex stability and TCR binding were assessed as described in part A.

Results

[0077] The binding half life of the resulting complex was 24.47h, demonstrating that the novel disulphide resulted in a substantial improvement in stability relative to the native complex (as shown in Table 4). TCR binding was assessed for 6 TCRs. In all 6 cases, TCR binding to the stabilised complex was preserved. The difference in binding relative to the native complex ranged from 1.53 to 3.24 fold for the disulphide with F116 and from 1.11 to 2.83 fold for the disulphide with S147.

TABLE-US-00010 TABLE 7 Native Non-native Non-native complex disulphide disulphide Fold Fold K.sub.D (F116) (S147) difference difference TCR (nM) K.sub.D (nM) K.sub.D (nM) (F116) (S147) 1 3.88 12.57 10.97 3.24 2.83 2 53.69 124.00 95.86 2.31 1.79 3 3.01 8.13 7.38 2.70 2.45 4 0.48 1.34 0.99 2.80 2.08 5 1.36 2.42 1.57 1.78 1.15 6 0.67 1.03 0.75 1.53 1.11