PEPTIDES DERIVED FROM MELANOMA-ASSOCIATED ANTIGEN C2 (MAGEC2) AND USES THEREOF

Abstract

Peptides derived from melanoma-associated antigen C2 (MAGEC2), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules are described. Also provided by the present invention are binding moieties that bind to the peptides and/or complexes of the invention. Such moieties are useful for the development of immunotherapeutic reagents for the treatment of diseases such as cancer.

Claims

1. A method of effecting an in vivo immune response in cells expressing melanoma-associated antigen C2 (MAGEC2), in a subject with non-small cell lung cancer, comprising: administering to the subject a therapeutically effective amount of a soluble T cell receptor (“TCR”) fused to an anti-CD3 specific antibody fragment, wherein the TCR is capable of specifically binding a polypeptide complexed with HLA-A*02, wherein the polypeptide is 8 to 16 amino acids in lengths and comprises the amino acid sequence of SEQ ID NO: 1.

2. The method of claim 1, wherein the polypeptide is 12 amino acids in length.

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

4. The method of claim 1, wherein the TCR is a heterodimeric TCR comprising an alpha chain, the alpha chain comprising a Vα variable region, and a beta chain comprising a Vβ variable region.

5. The method of claim 2, wherein the alpha chain variable region and the beta chain variable region of the heterodimeric TCR comprises the CDRs of the variable regions respectively selected from SEQ ID NO:26 and SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, and SEQ ID NO:30 and SEQ ID NO:31.

6. A method of adoptive cell therapy in a subject with non-small cell lung cancer, comprising: administering to the subject T cells transfected with a vector encoding a TCR capable of specifically binding a polypeptide complexed with HLA-A *02, wherein the polypeptide is 8 to 16 amino acids in length and comprises the amino acid sequence of SEQ ID NO: 1.

7. The method of claim 6, wherein the polypeptide is 12 amino acids in length.

8. The method of claim 7, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 1.

9. The method of claim 8, wherein the TCR is a heterodimeric TCR comprising an alpha chain, the alpha chain comprising a Vα variable region, and a beta chain comprising a Vβ variable region.

10. The method of claim 9, wherein the alpha chain variable region and the beta chain variable region of the heterodimeric TCR comprises the CDRs of the variable regions respectively selected from SEQ ID NO:26 and SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, and SEQ ID NO:30 and SEQ ID NO:31.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0076] FIGS. 1 to 25 show the respective fragmentation spectra for the peptides of SEQ ID NOS: 1 to 25, eluted from cells. A table highlighting the matching ions is shown below each spectrum. FIG. 1 discloses “ALIEVGPDHFCV” as SEQ ID NO: 1. FIG. 2 discloses “ALIEVGPDHFC” as SEQ ID NO: 2. FIG. 3 discloses “KVWVQGHYLEY” as SEQ ID NO: 3. FIG. 4 discloses “ATVMASESL” as SEQ ID NO: 4. FIG. 5 discloses “SIKKKVLEF” as SEQ ID NO: 5. FIG. 6 discloses “AEEPVTEAEML” as SEQ ID NO: 6. FIG. 7 discloses “TIDTADDATVM” as SEQ ID NO: 7. FIG. 8 discloses “YAGREHFV” as SEQ ID NO: 8. FIG. 9 discloses “ALIEVGPDHF” as SEQ ID NO: 9. FIG. 10 discloses “EEVPSGVIPNL” as SEQ ID NO: 10. FIG. 11 discloses “FVYGEPREL” as SEQ ID NO: 11. FIG. 12 discloses “SSNVSFSE” as SEQ ID NO: 12. FIG. 13 discloses “KLNNTVPSSF” as SEQ ID NO: 13. FIG. 14 discloses “ALIEVGPDH” as SEQ ID NO: 14. FIG. 15 discloses “YKDYFPVIL” as SEQ ID NO: 15. FIG. 16 discloses “FSTSSSLIL” as SEQ ID NO: 16. FIG. 17 discloses “REFMELLFGL” as SEQ ID NO: 17. FIG. 18 discloses “NAVGVYAGR” as SEQ ID NO: 18. FIG. 19 discloses “IEVGPDHFCVF” as SEQ ID NO: 19. FIG. 20 discloses “MPENSLLII” as SEQ ID NO: 20 and “FVYGEPREL” as SEQ ID NO: 11. FIG. 21 discloses “PSSFSTSSSLI” as SEQ ID NO: 21. FIG. 22 discloses “SEEVIWEVL” as SEQ ID NO: 22. FIG. 23 discloses “SVMSSNVSF” as SEQ ID NO: 23 and “SVM[Oxi]SSNVSF” as SEQ ID NO: 38. FIG. 24 discloses “VYGEPRELL” as SEQ ID NO: 24. FIG. 25 discloses “YAGREHFVY” as SEQ ID NO: 25.

[0077] FIGS. 26(A and B) shows ELISA plates demonstrating the specificity of TCRs for a complex of the peptide of SEQ ID NOs: 1 and 11 respectively and HLA-A*02, by comparing binding with other peptide-HLA-A*02 complexes.

[0078] FIG. 27 shows the amino acid sequences of the respective alpha chain and beta chain variable chains of the TCRs of FIGS. 26(A and B). FIG. 27 discloses SEQ ID NOS 1, 26-31, 11, and 32-37, respectively, in order of appearance.

EXAMPLES

Example 1—Identification of Target-Derived Peptides by Mass Spectrometry

[0079] Presentation of HLA-restricted peptides derived from MAGEC2 on the surface of tumour cell lines was investigated using mass spectrometry.

[0080] Method

[0081] Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.

[0082] Class I HLA complexes were purified by immunoaffinity using commercially available anti-HLA antibodies BB7.1 (anti-HLA-B*07), BB7.2 (anti-HLA-A*02) and W6/32 (anti-Class 1). 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Results

[0087] The polypeptides set out in table 1, corresponding to SEQ ID NOs: 1-25, were detected by mass spec following extraction from cancer cell lines. An example cell line from which the peptide was detected is indicated in the table along with the HLA antibody used for immunoaffinity purification.

TABLE-US-00003 SEQ Amino Example ID acid HLA cancer NO sequence antibody cell line 1 ALIEVGPDHFCV HLA-A*02 U266 2 ALIEVGPDHFC HLA-A*02 A375 3 KVWVQGHYLEY class I EJM 4 ATVMASESL class I EJM 5 SIKKKVLEF HLA-A*02 EJM 6 AEEPVTEAEML class I U266 7 TIDTADDATVM class I VMRC LCD 8 YAGREHFV HLA-A*02 U266 9 ALIEVGPDHF class I A375 10 EEVPSGVIPNL class I A375 11 FVYGEPREL HLA-A*02 U266 12 SSNVSFSE class I VMRC LCD 13 KLNNTVPSSF class I EJM 14 ALIEVGPDH HLA-A*02 IGR37 15 YKDYFPVIL HLA-B*07 IGR37 16 FSTSSSLIL class I U266 17 REFMELLFGL class I U266 18 NAVGVYAGR class I MEWO 19 IEVGPDHFCVF class I U266 20 MPENSLLII class I U266 21 PSSFSTSSSLI HLA-B*07 SW982 22 SEEVIWEVL class I U266 23 SVMSSNVSF HLA-A*02 SKMel37 24 VYGEPRELL class I HT 144 25 YAGREHFVY class I VMRC LCD

[0088] FIGS. 1-25 show representative fragmentation patterns for the peptides of SEQ ID NOS: 1-25 respectively. A table highlighting the matching ions is shown below each spectrum.

Example 2—Preparation of Recombinant Peptide-HLA Complexes

[0089] The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide.

[0090] Class I HLA molecules (HLA-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.

[0091] 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 and 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.

[0092] The refold mixture was then dialysed against 20 L of deionised water at 4° C. for 16 h, followed by 10 mM Tris pH 8.1 for a 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.

[0093] 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.

[0094] 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 loaded onto the column and was developed with PBS at 0.5 ml/min. 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.

[0095] 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 3—Identification of TCRs that Bind to a Peptide-MHC Complex of the Invention

[0096] Method

[0097] Antigen binding TCRs were obtained using peptides 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.

[0098] 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.

[0099] 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 resonance (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 2) 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.

[0100] Results

[0101] TCRs that specifically recognise peptide-HLA complexes of the invention were obtained from the library. FIGS. 26(A and B) shows ELISA data for three such TCRs, per peptide.

[0102] Amino acid sequences of the TCR alpha and beta variable regions of the TCRs identified in FIGS. 26(A and B) are provided in FIG. 27.

[0103] These data confirm that antigen specific TCRs can be isolated.