PROGESTERONE-ASSOCIATED ENDOMETRIAL PROTEIN (PAEP) AND USES THEREOF

Abstract

Peptides derived from Progesterone-associated endometrial protein (PAEP), complexes comprising such peptides bound to recombinant MHC molecules, and cells presenting said peptide in complex with MHC molecules as 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 to cells expressing progesterone-associated endometrial protein (PAEP), in a subject with lung adenocarcinoma, 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: 5.

2. The method of claim 1, wherein the polypeptide is 10 amino acids in length.

3. The method of claim 2, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 5.

4. The method of claim 3, 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 4, 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:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, and SEQ ID NO:19 and SEQ ID NO:20.

6. A method of adoptive cell therapy in a subject with lung adenocarcinoma 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: 5.

7. The method of claim 6, wherein the polypeptide is 10 amino acids in length.

8. The method of claim 7, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 5.

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:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, and SEQ ID NO:19 and SEQ ID NO:20.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0074] FIG. 1 shows the fragmentation spectrum for the peptide corresponding to SEQ ID NO: 1. A table highlighting the matching ions is shown below each spectrum.

[0075] FIG. 2 shows the fragmentation spectrum for the peptide corresponding to SEQ ID NO: 2, eluted from cells. A table highlighting the matching ions is shown below the spectrum.

[0076] FIG. 3 shows the fragmentation spectrum for the peptide corresponding to SEQ ID NO: 3, eluted from cells. A table highlighting the matching ions is shown below the spectrum.

[0077] FIG. 4 shows the fragmentation spectrum for the peptide corresponding to SEQ ID NO: 4, eluted from cells. A table highlighting the matching ions is shown below the spectrum. FIG. 4 discloses “KQMEEPCRF” as SEQ ID NO: 4 and “KQM[Oxi]EEPC[CAM]RF” as SEQ ID NO: 33.

[0078] FIG. 5 shows the fragmentation spectrum for the peptide corresponding to SEQ ID NO: 5, eluted from cells. A table highlighting the matching ions is shown below the spectrum.

[0079] FIG. 6 shows the fragmentation spectrum for the peptide corresponding to SEQ ID NO: 6, eluted from cells. A table highlighting the matching ions is shown below the spectrum.

[0080] FIG. 7 shows the fragmentation spectrum for the peptide corresponding to SEQ ID NO: 8, eluted from cells. A table highlighting the matching ions is shown below the spectrum.

[0081] FIG. 8 shows the fragmentation spectrum for the peptide corresponding to SEQ ID NO: 7, eluted from cells. A table highlighting the matching ions is shown below the spectrum.

[0082] FIG. 9 shows the ELISA plate demonstrating specificity of TCRs for a complex of the peptide of SEQ ID NO: 1 and HLA-A*02, by comparing binding with other peptide HLA-A*02 complexes.

[0083] FIG. 10 shows the ELISA plate demonstrating specificity of TCRs for a complex of the peptide of SEQ ID NO: 5 and HLA-A*02, by comparing binding with other peptide HLA-A*02 complexes.

[0084] FIG. 11 shows the ELISA plate demonstrating specificity of TCRs for a complex of the peptide of SEQ ID NO: 6 and HLA-A*02, by comparing binding with other peptide HLA-A*02 complexes.

[0085] FIG. 12 shows the ELISA plate demonstrating specificity of TCRs for a complex of the peptide of SEQ ID NO: 8 and HLA-A*02, by comparing binding with other peptide HLA-A*02 complexes.

[0086] FIG. 13 shows the amino acid sequences of the respective alpha chain and beta chain variable chains of the TCR of FIG. 9. FIG. 13 discloses SEQ ID NOS 1 and 9-14, respectively, in order of appearance.

[0087] FIG. 14 shows the amino acid sequences of the respective alpha chain and beta chain variable chains of the TCR of FIG. 10. FIG. 14 discloses SEQ ID NOS 5 and 15-20, respectively, in order of appearance.

[0088] FIG. 15 shows the amino acid sequences of the respective alpha chain and beta chain variable chains of the TCR of FIG. 11. FIG. 15 discloses SEQ ID NOS 6 and 21-26, respectively, in order of appearance.

[0089] FIG. 16 shows the amino acid sequences of the respective alpha chain and beta chain variable chains of the TCR of FIG. 12. FIG. 16 discloses SEQ ID NOS 8 and 27-32, respectively, in order of appearance.

EXAMPLES

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

[0090] Presentation of HLA-restricted peptides derived from PAEP on the surface of tumour cell lines was investigated using mass spectrometry.

[0091] Method

[0092] Immortalised cell lines obtained from commercial sources were maintained and expanded under standard conditions.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] Results

[0098] The polypeptides set out in table 1, corresponding to SEQ ID NOs: 1-8, were detected by mass spec following extraction from human cancer cell lines. An example human 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 ID Amino acid HLA Example cancer NO sequence antibody cell line 1 AMATNNISL HLA-A*02 G361 2 DTDYDNFLF HLA-A*02 G361 3 DTDYDNFLFL HLA-A*02 G361 4 KQMEEPCRF HLA-A*02 G361 5 LLDTDYDNFL HLA-A*02 G361 6 MMCQYLARV HLA-A*02 G361 7 TLLDTDYDNF HLA-A*02 G361 8 TLLDTDYDNFL HLA-A*02 G361

[0099] FIGS. 1-8 show representative fragmentation pattern for the peptide corresponding to SEQ ID NO: 1-8 respectively. A table highlighting the matching ions is shown below each spectrum. Note that SEQ ID NO: 1 was observed with the methionine residue at position 2 in both oxidised and non-oxidised forms.

Example 2—Preparation of Recombinant Peptide-HLA Complexes

[0100] The following describes a suitable method for the preparation of soluble recombinant HLA loaded with TAA peptide.

[0101] Class I HLA-A*02 molecules (HLA-A*02-heavy chain and HLA light-chain (β2m)) were expressed separately in E. coli as inclusion bodies, using appropriate constructs. HLA-A*02-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.

[0102] Inclusion bodies of β2m and heavy chain were denatured separately in 6 M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mM EDTA. Refolding buffer was prepared containing 0.4 M L-Arginine, 100 mM Tris pH 8.1, 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride and chilled to <5° C. Synthetic peptide 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 30 mg/litre heavy chain (final concentrations) are added. Refolding was allowed to reach completion at 4° C. for at least 1 hour.

[0103] Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently. The protein solution was then filtered through a 1.5 μm cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCl gradient in 10 mM Tris pH 8.1 using an Akta purifier (GE Healthcare). HLA-A*02-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.

[0104] 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.

[0105] The biotinylated pHLA-A*02 molecules were purified using gel filtration chromatography. A GE Healthcare Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min using an Akta purifier (GE Healthcare). Biotinylated pHLA-A*02 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-A*02 molecules were stored frozen at −20° C.

[0106] Such peptide-MHC complexes may be used in soluble form or may be immobilised through 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.

Example 3—Identification of TCRs that Bind to a Peptide-MHC Complex of the Invention

[0107] Method

[0108] 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.

[0109] 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.

[0110] 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.

[0111] Results

[0112] TCRs that specifically recognise peptide-HLA complexes of the invention were obtained from the library. FIGS. 9-12 show ELISA data for four such TCRs.

[0113] Amino acid sequences of the TCR alpha and beta variable regions of the TCRs identified in FIGS. 9-12 are provided in FIGS. 13-16.

[0114] These data confirm that antigen specific TCRs can be isolated.