PEPTIDES

Abstract

The present invention provides a peptide comprising, consisting essentially of or consisting of (i) the amino acid sequence KMPEAGEEQPQV (SEQ ID NO: 1); (ii) the amino acid sequence ISQTPGINL (SEQ ID NO: 2); or (iii) the amino acid sequence of SEQ ID NO: 1 or 2 with the exception of 1, 2 or 3 amino acid substitutions and/or 1, 2 or 3 amino acid insertions, and/or 1, 2 or 3 amino acid deletions, wherein the peptide forms a complex with a Major Histocompatibility Complex (MHC) molecule. Also provided are a complex of the peptide a Major Histocompatibility Complex (MHC) molecule, a nucleic acid molecule comprising a nucleic acid sequence encoding the peptide, a vector comprising such a nucleic acid sequence, a cell comprising such a vector and a binding moiety that binds the peptide.

Claims

1. A polypeptide comprising: (i) the amino acid sequence KMPEAGEEQPQV (SEQ ID NO: 1); (ii) the amino acid sequence ISQTPGINL (SEQ ID NO: 2); or (iii) the amino acid sequence of SEQ ID NO: 1 or 2 with the exception of 1, 2 or 3 amino acid substitutions and/or 1, 2 or 3 amino acid insertions, and/or 1, 2 or 3 amino acid deletions, wherein the polypeptide is capable of forming a complex with a Major Histocompatibility Complex (MHC) molecule.

2. The polypeptide of claim 1, wherein the polypeptide consists of from 8 to 16 amino acids.

3. The polypeptide of claim 1, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

4. A complex of the polypeptide of claim 1 and a Major Histocompatibility Complex (MHC) molecule.

5. The complex of claim 4, wherein the MHC molecule is HLA-A*02 or HLA-CW*03.

6. A nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide as defined in claim 1.

7. A vector comprising the nucleic acid molecule as defined in claim 6.

8. A cell comprising the vector as claimed in claim 7.

9. A binding moiety capable of specifically binding the polypeptide of claim 1.

10. The binding moiety of claim 9, capable of specifically binding the polypeptide when it is in complex with WIC.

11. The binding moiety of claim 10, wherein the binding moiety is a T cell receptor (TCR) or an antibody.

12. The binding moiety of claim 11, wherein the binding moiety is a TCR.

13. A method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a binding moiety as defined in claim 9.

14. The method of claim 13, wherein the disease is cancer.

15. A pharmaceutical composition comprising a binding moiety as defined in claim 9 and a pharmaceutically acceptable carrier.

16. A method of identifying a binding moiety that binds the complex as defined in claim 4, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.

17. The polypeptide of claim 2, wherein the polypeptide consists of from 9 to 12 amino acids.

18. The complex of claim 4, wherein the complex further comprises a biotin tag.

19. The polypeptide of claim 1, consisting of the amino acid sequence of any one of SEQ ID NOS: 1-2.

20. The binding moiety of claim 12, wherein the TCR is on the surface of a cell.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0069] FIG. 1 shows RT-PCR analysis of XAGE1 expression in lung tumour samples and normal tissue samples.

[0070] FIG. 2 shows the fragmentation spectrum for the peptide corresponding to SEQ ID NO: 1, eluted from cells. A table highlighting the matching ions is shown below the spectrum.

[0071] FIG. 3 shows the fragmentation spectrum for the peptide corresponding to SEQ ID NO: 2, eluted from cells. A table highlighting the matching ions is shown with the spectrum.

[0072] FIG. 4(a) shows an ELISA plate demonstrating the specificity of a TCR for a complex of the peptide of SEQ ID NO: 1 and HLA by comparing binding with other peptide-HLA complexes, and

[0073] FIG. 4(b) shows an ELISA plate demonstrating the specificity of three TCRs for a complex of the peptide of SEQ ID NO: 2 and HLA by comparing binding with other peptide-HLA complexes.

[0074] FIG. 5 shows the amino acid sequences of the respective alpha chain and beta chain variable chains of the TCRs of FIG. 4.

EXAMPLES

Example 1 XAGE1 Expression in Tumour Tissue

[0075] Method

[0076] XAGE1 expression was analysed by Quantitative real-time PCR using a lung cancer array panel (Origene TissueScan HLRT503). The PCR assay was performed with an internal fluorescent probe 5′-CAGCAGCTGAAAGTCGGGATCCTACACC-3′ (SEQ ID NO: 3) synthesized by IDT Integrated DNA Technologies. Primers were designed in-house (forward 5′-AACACAGAACCACACAGCCAGTC-3′ (SEQ ID NO: 4) and reverse 5′-CAGCTGTATCCTGATCTTCTTCTGTC-3′ (SEQ ID NO: 5)) and synthesized by Eurofins MWG Operon. The assay spans over introns to avoid any genomic DNA amplification, and its specificity was validated by resolution on agarose gel and sequencing.

[0077] PCR reactions were performed on the lyophilised cDNA for the cancer panel with 500 nM of each primer, the fluorescent probe, and 2× Quantitect Probe Mastermix (Qiagen). PCR cycling conditions consisted of: 15 min at 95° C.; then 40 cycles of 15s at 95° C., 60s at 60° C.; and was performed using a QuantStudio 6 instrument (Life Technologies). Purified PCR products were previously cloned into a pCR®4-TOPO plasmid to produce a standard template of a known copy number. Serial 1:10 dilutions were used to generate a standard curve from 10.sup.1 to 10.sup.6 transcripts/reaction and run in parallel, thus allowing the calculation of absolute transcript number in the cancer samples.

[0078] Results

[0079] FIG. 1 shows mRNA transcript levels of XAGE1 are elevated in lung tissue compared to normal tissues, indicating that XAGE1 is a valid TAA.

Example 2 Identification of XAGE1-Derived Peptides by Mass Spectrometry

[0080] Presentation of HLA restricted peptides derived from XAGE1 on the surface of various tumour cell lines was investigated using mass spectrometry.

[0081] Method

[0082] Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.

[0083] Class I HLA complexes were purified by immunoaffinity using a commercially available anti-HLA-antibodies BB7.2 (anti-HLA-A*02) and W6/32 (anti-Class 1) conjugated to FITC (Abcam). Briefly, cells were lysed in buffer containing non-ionic detergent NP-40 (0.5% v/v) at 5×10.sup.7 cells per ml and incubated at 4° C. for 1 h with agitation/mixing. Cell debris was removed by centrifugation and supernatant pre-cleared using proteinA-Sepharose. Supernatant was passed over 5 ml of resin containing 8 mg of anti-HLA antibody immobilised on a proteinA-Sepharose scaffold. Columns were washed with low salt and high salt buffers and complexes eluted in acid. Eluted peptides were separated from HLA complexes by reversed phase chromatography using a solid phase extraction cartridge (Phenomenex). Bound material was eluted from the column and reduced in volume using a vacuum centrifuge.

[0084] Peptides were separated by high pressure liquid chromatography (HPLC) on a Dionex Ultimate 3000 system using a C18 column (Phenomenex). Peptides were loaded in 98% buffer A (0.1% aqueous trifluoroacetic acid (TFA)) and 2% buffer B (0.1% TFA in acetonitrile). Peptides were eluted using a stepped gradient of B (2-60%) over 20 min. Fractions were collected at one minute intervals and lyophilised.

[0085] Peptides were analysed by nanoLCMS/MS using a Dionex Ultimate 3000 nanoLC coupled to either AB Sciex Triple TOF 5600 or Thermo Orbitrap Fusion mass spectrometers. Both machines were equipped with nanoelectrospray ion sources. Peptides were loaded onto an Acclaim PepMap 100 trap column (Dionex) and separated using an Acclaim PepMap RSLC column (Dionex). Peptides were loaded in mobile phase A (0.5% formic acid: water) and eluted using a gradient of buffer B (acetonitrile:0.5% formic acid) directly into the nanospray ionisation source.

[0086] For peptide identification the mass spectrometer was operated using an information dependent acquisition (IDA) workflow. Information acquired in these experiments was used to search the Uniprot database of human proteins for peptides consistent with the fragmentation patterns seen, using Protein pilot software (Ab Sciex) and PEAKS software (Bioinformatics solutions). Peptides identified are assigned a score by the software, based on the match between the observed and expected fragmentation patterns.

[0087] Results

[0088] Peptides corresponding to SEQ ID NOs: 1 and 2 were detected on the following cell lines

TABLE-US-00001 Peptide Cell type SEQ ID NO: 1 NCI-H1975 (lung  (KMPEAGEEQPQV) adenocarcinoma) SEQ ID NO: 2 HT144 (malignant melanoma) (ISQTPGINL) U266 (multiple myeloma) MKN1 (gastric adenocarcinoma HGC 27 (gastric carcinoma) NCI H358 (non-small  cell lung cancer)

[0089] These data confirm that a peptide corresponding to SEQ ID NOs: 1 and 2 are presented on the cell surface in complex with HLA FIGS. 2 and 3 depict a fragmentation pattern for the peptide corresponding to SEQ ID NOs: 1 and 2 respectively. A table highlighting the matching ions is shown with the spectrum.

[0090] No other MHC class 1 restricted peptides from XAGE1 b were detected using the above methodology, including the following XAGE1 b nonamer and decamer peptides which are strongly predicted to bind to HLA-A*02 based on their algorithm score from SYFPEITHI (www.svfpeithi.de) (Rammensee, et al., Immunogenetics. 1999 November; 50(3-4):213-9.

TABLE-US-00002 Sequence SYFPEITHI Score QLKVGILHL 26 LDLGSGVKV 20 NLDLGSGVKV 24 GINLDLGSGV 21 DLGSGVKVKI 21

Example 3 Preparation of Recombinant Peptide—HLA Complexes

[0091] The following describes the method used for the preparation of soluble recombinant HLA loaded with TAA peptide.

[0092] Method

[0093] Class I HLA molecules (HLA-A*02 or HLA-Cw03-heavy chain and HLA light-chain (β2m)) were expressed separately in E. coli as inclusion bodies, using appropriate constructs. HLA-heavy chain additionally contained a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15). E. coli cells were lysed and inclusion bodies processed to approximately 80% purity.

[0094] Inclusion bodies of β2m and heavy chain were denatured separately in denaturation buffer (6 M guanidine, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mM EDTA) for 30 mins at 37° C. Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1, 2 mM EDTA, 3.1 mM cystamine dihydrochloride, 7.2 mM cysteamine hydrochloride. Synthetic peptide was dissolved in DMSO to a final concentration of 4 mg/ml is added to the refold buffer at 4 mg/litre (final concentration). Then 30 mg/litre β2m followed by 60 mg/litre heavy chain (final concentrations) are added. Refolding was allowed to reach completion at room temperature for at least 1 hour.

[0095] The refold mixture was then dialysised against 20 L of deionised water at 4° C. for 16 h, followed by 10 mM Tris pH 8.1 fora further 16 h. The protein solution was then filtered through a 0.45 μm cellulose acetate filter and loaded onto a POROS HQ anion exchange column (8 ml bed volume) equilibrated with 20 mM Tris pH 8.1. Protein was eluted with a linear 0-500 mM NaCl gradient using an AKTA purifier (GE Healthcare). HLA-peptide complex eluted at approximately 250 mM NaCl, and peak fractions were collected. a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.

[0096] Biotinylation tagged pHLA molecules were buffer exchanged into 10 mM Tris pH 8.1, 5 mM NaCl using a GE Healthcare fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCl2, and 5 μg/ml BirA enzyme (purified according to O'Callaghan et al., (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.

[0097] The biotinylated pHLA molecules were further purified by gel filtration chromatography using an AKTA purifier with a GE Healthcare Superdex 75 HR 10/30 column pre-equilibrated with filtered PBS. The biotinylated pHLA mixture was concentrated to a final volume of 1 ml and loaded and the column. was developed with PBS at 0.5 ml/min using an Akta purifier (GE Healthcare). Biotinylated pHLA molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA molecules were stored frozen at 20° C.

[0098] Such peptide-MHC complexes may be used in soluble form or may be immobilised through their C terminal biotin moiety on to a solid support, to be used for the detection of T cells and T cell receptors which bind said complex. For example, such complexes can be used in panning phage libraries, performing ELISA assays and preparing sensor chips for Biacore measurements.

Example 4 Identification of TCRs that Bind to a Peptide-MHC Complex of the Invention

[0099] Method

[0100] Antigen binding TCRs were obtained using peptide-HLA complexes of the invention to pan a TCR phage library. The library was constructed using alpha and beta chain sequences obtained from a natural repertoire (as described in WO2015/136072, PCT/EP2016/071757, PCT/EP2016/071761, PCT/EP2016/071762, PCT/EP2016/071765, PCT/EP2016/071767, PCT/EP2016/071768, PCT/EP2016/071771 or PCT/EP2016/071772). The random combination of these alpha and beta chain sequences, which occurs during library creation, produces a non-natural repertoire of alpha beta chain combinations.

[0101] TCRs obtained from the library were assessed by ELISA to confirm specific antigen recognition. ELISA assays were performed as described in WO2015/136072. Briefly, 96 well MaxiSorp ELISA plates were coated with streptavidin and incubated with the biotinylated peptide-HLA complex of the invention. TCR bearing phage clones were added to each well and detection carried out using an anti-M13-HRP antibody conjugate. Bound antibody was detected using the KPL labs TMB Microwell peroxidase Substrate System. The appearance of a blue colour in the well indicated binding of the TCR to the antigen. An absence of binding to alternative peptide-HLA complexes indicated the TCR is not highly cross reactive.

[0102] The amino acid sequences of the variable domains of TCRs from ELISA positive phage clones was determined.

[0103] Further confirmation that TCRs are able to bind a complex of comprising a peptide HLA complex of the invention can be obtained by surface plasmon reasonance (SPR) using isolated TCRs. In this case alpha and beta chain sequences are expressed in E. coli as soluble TCRs, (WO2003020763; Boulter, et al., Protein Eng, 2003. 16: 707-711). Binding of the soluble TCRs to the complexes is analysed by surface plasmon resonance using a BiaCore 3000 instrument. Biotinylated peptide-HLA monomers are prepared as previously described (Example 3) and immobilized on to a streptavidin-coupled CM-5 sensor chip. All measurements are performed at 25° C. in PBS buffer supplemented with 0.005% Tween at a constant flow rate. To measure affinity, serial dilutions of the soluble TCRs are flowed over the immobilized peptide-MHCs and the response values at equilibrium determined for each concentration. Data are analysed by plotting the specific equilibrium binding against protein concentration followed by a least squares fit to the Langmuir binding equation, assuming a 1:1 interaction.

[0104] Results

[0105] TCRs that specifically recognise peptide-HLA complexes of the invention were obtained from the library. FIG. 4 shows ELISA data for four such TCRs.

[0106] Amino acid sequences of the TCR alpha and beta variable regions of the TCRs identified in FIG. 4 are provided in FIG. 5