METHOD FOR THE DETECTION OF ANTIGEN PRESENTATION
20170254803 · 2017-09-07
Assignee
Inventors
- Michael Platten (Heidelberg, DE)
- Wolfgang Wick (Heidelberg, DE)
- Lukas BUNSE (Heidelberg, DE)
- Theresa BUNSE (Heidelberg, DE)
- Felix SAHM (Heidelberg, DE)
Cpc classification
G01N2800/56
PHYSICS
A61K39/00
HUMAN NECESSITIES
G01N33/542
PHYSICS
International classification
Abstract
The present invention pertains to a method for detecting antigen presentation via antigen presenting molecules such as major histocompatibility complex (MHC) class I or II. The invention deploys a first binding agent specific for the antigen epitope and a second binding agent specific for the antigen presenting molecule. The binding agents of the invention are coupled to proximity probes which upon antigen presentation elicit a detectable signal. The method of the invention allows detecting antigen presentation via MHC in-vitro and in a tissue sample in-situ. Thus the method of the present invention finds application as a new diagnostic tool, for example in the diagnosis of various diseases such as infectious diseases, immunological disorders, in particular autoimmune diseases, and proliferative disorders such as cancer.
Claims
1. A method for detecting antigen presentation of an epitope by an antigen presentation molecule, comprising (a) Providing a first binding agent and a second binding agent, wherein the first binding agent is capable of specifically binding the epitope, and the second binding agent is capable of specifically binding the antigen presentation molecule; wherein the first and the second binding agent are characterized in that spatial proximity of the first binding agent and the second binding agent induces a detectable signal, (b) Providing an antigen presentation molecule, wherein the antigen presentation molecule is suspected of presenting the epitope, (c) Providing the epitope, (d) Bringing into contact the epitope, the antigen presentation molecule, the first binding agent and the second binding agent, wherein the presence of the detectable signal is indicative for the antigen presentation of the epitope by the antigen presentation molecule.
2. The method according to claim 1, wherein the first binding agent comprises a first proximity probe and the second binding agent comprises a second proximity probe, wherein spatial proximity of the first proximity probe and the second proximity probe induces the detectable signal.
3. The method according to claim 1, further comprising providing a third binding agent capable of specifically binding to the first binding agent; providing a fourth binding agent capable of specifically binding to the second binding agent; and wherein step (d) comprises bringing into contact the epitope, the antigen presentation molecule, the first binding agent, the second binding agent, the third binding agent, and the fourth binding agent.
4. The method according to claim 3, wherein the third binding agent comprises a first proximity probe and the fourth binding agent comprises a second proximity probe, wherein spatial proximity of the first proximity probe and second proximity probe induces the detectable signal.
5. The method according to claim 1, wherein the binding agents are monoclonal- or polyclonal antibodies, preferably monoclonal antibodies.
6. The method according to claim 1, wherein in step (b) the antigen presentation molecule is provided in a biological cell, preferably on the surface on a biological cell such as an antigen presenting cell selected from a dendritic cell, a B lymphocyte or a tumor cell.
7. The method according to claim 1, wherein the epitope is a disease associated antigen, preferably an epitope associated with an immunological disorder, such as an autoimmune disorder, or a tumor associated antigen (TAA), or an epitope derived from a TAA, preferably wherein the TAA is selected from the group of cancer mutated antigens, cancer germ line expressed antigens, cancer viral antigens or cancer overexpression antigens.
8. The method according to claim 1, wherein the epitope is derived from IDH1 and comprises the IDH1R132H mutation, most preferably a peptide comprising an amino acid sequence according to SEQ ID NO: 30 (peptide IDH1R132H p125-137), or wherein the epitope is derived from NY-ESO-1.
9. The method according to claim 1, wherein the epitope is provided by providing a biological cell expressing the epitope or a precursor thereof; or alternatively ectopically expressing the epitope, or a precursor thereof, in a biological cell, preferably a cell which further comprises the antigen presentation molecule, most preferably an antigen presenting cell such as a dendritic cell, a B lymphocyte or a tumor cell.
10. A method for generating a personalized disease therapy plan for treating a subject suffering from a disease, the method comprising the steps of (a) Providing a biological sample obtained from the subject, (b) Detecting antigen presentation of at least one known epitope or antigen in the biological sample using the method according to claim 1, wherein the epitope and the antigen presentation molecule are provided in the biological sample, and (c) Generating a therapy plan for treating the subject by selecting a vaccine composition comprising vaccine-molecules corresponding to the epitope or antigen as detected in (a).
11. A method for producing a personalized vaccine composition, the method comprising the steps of (a) Providing a biological sample obtained from the subject, (b) Detecting antigen presentation of at least one known epitope or antigen in the biological sample using the method according to claim 1, wherein the epitope and the antigen presentation molecule are provided in the biological sample, and (c) Producing a personalized vaccine composition by admixing vaccine compounds into a composition which correspond to the epitopes/antigens as detected in (b).
12. The method according to claim 10, wherein the biological sample is a tissue sample, and the detecting antigen presentation is performed in the tissue sample in-situ.
13. The method according to claim 10, wherein the epitope is, or is derived of, a TAA, preferably a mutated tumor antigen.
14. A method for diagnosing, stratifying, monitoring or classifying a subject suffering from a disease, the method comprising the steps of: (e) Providing a biological sample of the subject suffering from a disease to be diagnosed, (f) Detecting antigen presentation of at least one known epitope or antigen in the biological sample, the epitope being characteristic for a candidate disease, using the method according to claim 1, wherein the epitope and the antigen presentation molecule are provided in the biological sample, wherein a diagnosis is provided based on the presence or absence of the presentation of the epitope/antigen in said cellular sample or tissue sample.
15.-17. (canceled)
18. A proximity ligation assay (PLA) kit for detecting antigen presentation of an epitope by an antigen presentation molecule, said PLA kit comprising: a first binding agent and a second binding agent, wherein the first binding agent is capable of specifically binding the epitope, and the second binding agent is capable of specifically binding the antigen presentation molecule; wherein the first and the second binding agent are characterized in that spatial proximity of the first binding agent and the second binding agent induces a detectable signal.
19. The PLA kit according to claim 18, wherein the first binding agent comprises a first proximity probe and the second binding agent comprises a second proximity probe, wherein spatial proximity of the first proximity probe and the second proximity probe induces the detectable signal.
20. The PLA kit according to claim 18, further comprising: a third binding agent capable of specifically binding to the first binding agent and a fourth binding agent capable of specifically binding to the second binding agent.
21. The PLA kit according to claim 20, wherein the third binding agent comprises a first proximity probe and the fourth binding agent comprises a second proximity probe, wherein spatial proximity of the first proximity probe and second proximity probe induces the detectable signal.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0102] The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:
[0103]
[0104]
[0105]
[0106]
[0107]
[0108]
EXAMPLES
Materials and Methods
Peptides
[0109] Human IDH1wt and IDH1R132H amino acid sequences IDH1 p118-146 PRLVSGWVKPIIIGRHAYGDQYRATDFVV (SEQ ID NO: 1) and PRLVSGWVKPIIIGHHAYGDQYRATDFVV (SEQ ID NO: 2), respectively, cover the amino acid exchange from Arg to His at position 132, including all possible 15-mer IDH1 peptides containing position 132, and are identical to mouse sequence except for position 122 (Thr in mouse). Peptide libraries for ELISA and in vitro stimulation of IDH1wt and IDH1R132H of 10 and 20-mers contained the following peptides:
TABLE-US-00001 (SEQ ID NO: 3) IDH1wt p118-132: PRLVSGWVKPIIIGR; (SEQ ID NO: 4) IDH1wt p120-134: LVSGWVKPIIIGRHA; (SEQ ID NO: 5) IDH1wt p122-136: SGWVKPIIIGRHAYG; (SEQ ID NO: 6) IDH1wt p124-138: WVKPIIIGRHAYGDQ; (SEQ ID NO: 7) IDH1wt p126-140: KPIIIGRHAYGDQYR; (SEQ ID NO: 8) IDH1wt p128-142: IIIGRHAYGDQYRAT; (SEQ ID NO: 9) IDH1wt p130-144: IGRHAYGDQYRATDF; (SEQ ID NO: 10) IDH1wt p132-146: RHAYGDQYRATDFVV; (SEQ ID NO: 11) IDH1R132H p118-132: PRLVSGWVKPIIIGH; (SEQ ID NO: 12) IDH1R132H p120-134: LVSGWVKPIIIGHHA; (SEQ ID NO: 13) IDH1R132H p122-136: SGWVKPIIIGHHAYG; (SEQ ID NO: 14) IDH1R132H p124-138: WVKPIIIGHHAYGDQ; (SEQ ID NO: 15) IDH1R132H p126-140: KPIIIGHHAYGDQYR; (SEQ ID NO: 16) IDH1R132H p128-142: IIIGHHAYGDQYRAT; (SEQ ID NO: 17) IDH1R132H p130-144: IGHHAYGDQYRATDF; (SEQ ID NO: 18) IDH1R132H p132-146: HHAYGDQYRATDFVV;
20-mers
TABLE-US-00002 (SEQ ID NO: 19) IDH1R132H p123-142 GWVKPIIIGHHAYGDQYRAT and (SEQ ID NO: 20) IDH1wt p123-142 GWVKPIIIGRHAYGDQYRAT.
[0110] Negative control peptide for ELISA and in vitro stimulation was mouse myelin oligodendrocyte glycoprotein (MOG) p35-55 MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 21) MOG peptide was synthesised by Genscript, IDH1 (wt, IDH1R132H) 15- and 20-mers were synthesised by Bachem Distribution Services GmbH. Peptides were diluted in PBS 10% DMSO at 2.5 mg/ml and stored at −80° C.
In Silico MHC Class II Peptide Binding Prediction
[0111] Human IDH1R132H 29-mer peptide PRLVSGWVKPIIIGHHAYGDQYRATDFVV includes all possible processed IDH1R132H 15-mers including the point mutation at codon 132 which leads to the amino acid change from arginin (R) to histidin (H). Binding of 15-mer IDH1R132H peptides to available HLA-DR types was predicted by the NetMHCII 2.2 algorithm.
IDH1R132H and IDH1 Wildtype Peptide Coated ELISA
[0112] ELISA plates (Costar) were coated with IDH1R132H and IDH1wt p118-132, p120-134, p122-136, p124-138, p126-140, p128-142, p130-144, p132-146, p123-142 (10 μg per well in PBS), washed with PBS 0.05% Tween 20, and blocked with 3% FBS in PBS 0.05% Tween 20. As negative controls, MOG p35-55 was used at equal concentrations and peptide diluent PBS 10% DMSO was used at equal volume. As primary antibody monoclonal mouse anti-IDH1R132H (1:1000, H09, Dianova) was used. HRP-conjugated secondary antibody was sheep anti-mouse IgG (1:5000, Amersham). HLA-DRB1*01:01 MHC class II tetramer bound to IDH1R132H p123-142 and HLA-DRB1*01:01 MHC class II tetramer control tetramer bound to CLIP were kindly provided by NIH tetramer core facility and used 1:200 during pre-incubation with H09 for competitive ELISA. Substrate was TMB (ebioscience) and reaction was stopped with 1M H2SO4. OD at 450 nm was measured with an ELISA reader (Thermo Fisher).
Glioma Tissue
[0113] Assays were performed with glioma tissue from patients diagnosed at the Department of Neuropathology at Heidelberg University. IDH1 mutation status was routinely diagnosed by IHC. Tissues were obtained from the archives of the Department of Neuropathology, University Hospital Heidelberg, according to the regulations of the Tissue Bank of the National Center for Tumour Diseases, University Hospital Heidelberg, and used after approval of the local regulatory authorities.
Cell Lines and Modification of Gene Expression
[0114] Glioma cell line LN229 endogenously expressing HLA-DR1 was lentivirally transduced with full-length cDNA of human IDH1R132H or IDH1wt (NCBI GenBank CR641695.1) in pLenti6.2/V5-DEST for stable expression. Virus was produced by transfection of HEK293T packaging cell line. Since IDH1R132H protein dimerizes and produces the oncometabolite R-2-HG which impacts proliferation and thereby impairs stable IDH1R132H expression, a second point mutation leading to the amino acid exchange D252G was introduced into IDH1R132H and wt-coding sequences by site-directed mutagenesis. This dimerization-deficient mutation results in an inert enzyme, thus to increased expression stability. Transduced cells were selected with 10 μg/ml blasticidin (Sigma-Aldrich) for stable overexpression. Stable overexpression of cancer testis antigen CTAG1A (NY-ESO-1) in LN229 was done by transfection with full length NY-ESO-1 cDNA (NCBI Genbank BC160040) provided by the DKFZ Genomics Facility, in the retroviral vector pMXs-IRES-BsdR (Cell Biolabs, Inc.) using FuGene HD transfection reagent (Promega). Cells were selected with 9 μg/ml blasticidin (Sigma-Aldrich) 72 h after transfection. Melanoma cell line SK-Mel-37 endogenously expressing NY-ESO-1 and HLA-DR1 and -DR3 was used for analysis of endogenous presentation of NY-ESO-1-derived peptides. For knockdown of HLA-DR in LN229 and HLA-DR and NY-ESO-1 in SK-Mel-37, ON-TARGET SMARTpool® siRNA (Dharmacon RNA Technologies, Lafayette, Colo., USA) were used. The siRNA target sequences were as follows:
TABLE-US-00003 for CTAG1B (NY-ESO-1): (SEQ ID NO: 22) CUGAAUGGAUGCUGCAGAU; (SEQ ID NO: 23) CCGGCAACAUACUGACUAU; (SEQ ID NO: 24) CGCCAUGCCUUUCGCGACA; (SEQ ID NO: 25) GCUGGAGGAGGACGGCUUA; for HLA-DRA: (SEQ ID NO: 26) UGACAAAGCGCUCCAACUA; (SEQ ID NO: 27) UGACCAAUCAGGCGAGUUU; (SEQ ID NO: 28) GGAAUCAUGGGCUAUCAAA; (SEQ ID NO: 29) CAACUGAGGACGUUUACGA;
[0115] As negative control ON-TARGETplus siCONTROL Non-targeting Pool (D-001810-10-05, Dharmacon) was used. The transfection was performed with lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol.
Proximity Ligation Assay
[0116] Tumour cell lines were seeded on glass coverslips, grown until 70-90% confluent and fixed and permeabilized with Cytofixx Pump Spray (Cell Path) for 30 min at −20° C. and subsequent 4% PFA in PBS for 30 min at room temperature. Glioma tissue was deparaffinized with HistoClearTMII (National Diagnostics) and rehydrated. Antigen retrieval was performed using Cell Conditioning Solution CC1 (Ventana Medical Systems, Inc.) for 30 min. PLA was performed using Detection Reagents Red, PLA Probe anti-mouse PLUS, PLA Probe anti-rabbit MINUS and Wash Buffers Flourescence (all Duolink, Olink Bioscience) according to manufacturer's instructions. Briefly, blocking was done with blocking solution for 30 min at 37° C. and primary antibodies monoclonal mouse anti-human IDH1R132H (1:100, H09, Dianova) or mouse anti-human monoclonal NY-ESO-1 (1:50, E978, Sigma Aldrich) with monoclonal rabbit anti-human HLA-DR (1:50, EPR3692, Abcam) in antibody diluent were incubated over night at 4° C. PLA Probe anti-mouse PLUS and PLA Probe anti-rabbit MINUS were incubated for 1 h at 37° C. Ligation and amplification were performed using the Detection Reagents Red. Immunofluorescent (IF) co-staining was performed as described below after amplification of PLA signal. Vectashield HardSet Mounting Medium with DAPI (Vector laboratories) was used for mounting and nuclear staining.
Immunofluorescent Staining
[0117] For IF staining cells were seeded on glass coverslips, grown until 70-90% confluent and fixed and permeabilized as described above. For blocking and staining blocking solution (Duolink, Olink Bioscience) and antibody diluent (Duolink, Olink Bioscience) were used as for PLA, respectively. As primary antibodies anti-IDH1R132H was used as described above. Additional IF stainings were performed using monoclonal rabbit anti-myc-tag (1:200, 71D10, Cell Signalling) and polyclonal rabbit anti-human IBA-1 (1:100, Wako) and secondary antibodies used were donkey anti-mouse AlexaFluor® 488 and goat anti-rabbit AlexaFluor 546® (all 1:300, Molecular Probes, Invitrogen). DAPI staining and mounting were performed as described above. For PLA co-staining secondary antibody anti-mouse AlexaFluor® 488 for IDH1R132H or myc-tag detection and anti-rabbit AlexaFluor® 488 (all 1:300, Molecular Probes, Invitrogen) for IBA-1 detection were used.
Immunohistochemistry
[0118] Glioma tissue was deparaffinized with HistoClearTMII (National Diagnostics) and rehydrated. Antigen retrieval was performed using Cell Conditioning Solution CC1 (Ventana Medical Systems, Inc.) for 30 min. Endogenous peroxidase was blocked with 3% hydrogen peroxide in PBS. Blocking was performed with 5 FBS for one hour. Primary antibodies (monoclonal mouse anti-human IDH1R132H (1:100, H09, Dianova) and monoclonal rabbit anti-human HLA-DR (1:50, EPR3692, Abcam) were incubated over night at 4° C. Colour reaction was performed using Liquid DAB+ Substrate Chromogen System (DAKO). Counterstaining was performed using hemalum (Carl Roth GmbH+Co. KG).
Western Blot
[0119] Total protein was isolated by cell lysis with ice cold TRIS-HCl, 50 mM, pH 8.0 (Carl Roth) containing 150 mM NaCl (J. T. Baker, Deventer, Netherlands), 1% Nondiet P-40 (Genaxxon Bioscience, Ulm, Germany), 10 mM EDTA (GerbuBiotechnik, Gaiberg, Germany), 200 mM dithiothreitol (Carl Roth), 100 μM PMSF and complete EDTA-free (1:50, Roche, Mannheim Germany) for 20 min and centrifuged to pellet debris. Protein concentrations were measured via the Bio-Rad protein assay (Bio-Rad, Hercules, Calif., USA) at 595 nm and 30 μg of protein diluted in Laemmli sample buffer were denatured at 95° C. for 5 min and electrophoretically separated on 12% acrylamide-polyacrylamide SDS-containing gels. Proteins were blotted onto nitrocellulose membranes by wet blot at 1.5 mA/cm2 for 1 h. After blocking with 5% milk powder in 0.5 M TBS, pH 7.4, 1.5 M NaCl, 0.05% Tween 20, membranes were incubated consecutively with primary monoclonal mouse anti-IDH1R132H (1:500, H09, Dianova), monoclonal rat anti-panIDH1 (1:500, W09, Dianova) for detection of wt and R132H IDH1, monoclonal rabbit anti-myc tag (71D10, 1:1000, Cell Signalling), anti-NY-ESO-1 (1:500, Sigma Aldrich) overnight at 4° C., and mouse anti-α-tubulin (1:5000, Sigma-Aldrich) as loading control for 1 h at room temperature. Staining with secondary HRP-conjugated anti-rat (1:(1000×F), Dako) or anti-mouse (1:5000, GE Healthcare, Buckinghamshire, UK) antibodies was performed at room temperature for 1 h and was followed by chemiluminescent development using ECL or ECL prime (both Amersham).
Flow Cytometry
[0120] Cells were harvested, washed once in PBS, 3% FBS, 2 mM EDTA, and blocking was done with human serum. Surface HLA-DR was stained using eFluor-450®-conjugated mouse anti-HLA-DR antibody (1:100, L243, ebioscience). Cells were acquired on a FACS Canto II (Beckton Dickinson) and analysed using FlowJo software.
HLA-Typing
[0121] Genomic DNA was isolated from patient blood or tumor samples using the FFPE LEV DNA Purification KIT AS1130, from cell lines using the QIAamp DNA Mini Kit (Qiagen). PCR-based typing was performed using HLA-A and HLA-DR type-specific primer pairs lyophilized in a 96 well plate (HLA-A* CTS-PCR-SSP Minitray Kit and HLA-DRB1* CTS-PCR-SSP Minitray Kit) and Mastermix 5.0% for HLA-DRB1* and Mastermix 7.5% for HLA-A* (all from Department of Transplantation Immunology, University Clinic Heidelberg) with Taq-polymerase (Fermentas) PCR was performed according to manufacturer's instructions. PCR products were separated on a 1.5 agarose gel containing GelRed® (1:10000, Genaxxon bioscience). Analysis was done according to manufacturer's instruction.
2-HG Measurement
[0122] 2-HG production in cells was analyzed as described previously [33]. As a control for 2-HG production enzymatically competent retrovirally transduced LN229 IDH1 R132H were used. Vectors were generated as described previously (Schumacher Bunse nature 2014 Referenz).
Image Analysis
[0123] IF images were taken on LEICA DM IRB microscope, using 63× objective for PLA, detecting PLA signal with N2.1 filter, signal of immunofluorescent co-staining with GFP filter, using 40× objective for immunofluorescent images. Immunohistochemical images were taken on Zeiss Axioplan microscope. Images were linearly optimised with Adobe Photoshop CS3®.
Statistical Analysis
[0124] Data are expressed as mean+s.e.m. and analysis of significance (
Example 1: MHC Class II-Restricted Immunogenicity of IDH1R132H
[0125] The IDH1R132H mutation is expressed in about 80% of gliomas, defining a distinct glioma subtype [15-17]. The high incidence of this point mutation led to the development of a mutation-specific monoclonal antibody (H09) [18, 19], which finds routine application for histological diagnostics. This mouse antibody has been generated by immunization with synthetic peptide IDH1R132H p125-137 CKPIIIGHHAYGD (SEQ ID NO: 30) coupled to keyhole limpet hemocyanin and allows specific staining of diffuse infiltrating single tumor cells in gliomas (
TABLE-US-00004 TABLE 1 IDH1R132H 15-mer peptides bind to human MHC class II in silico. NetMHCII algorithm was used to predict binding of IDH1R132H 15-mer peptides to available HLA-DR types. Peptides with IC50 below 500 nM are defined as weak binders, those with IC50 below 50 nM are defined as strong binders. Only 15-mers predicted to bind are shown. allel 15-mers position IC50 (nM) binding level HLA-DRB1*0101 PRLVSGWVKPIIIGH 118-132 39.1 s RLVSGWVKPIIIGHH 119-133 60.4 w HLA-DRB1*0701 PRLVSGWVKPIIIGH 118-132 331.5 w RLVSGWVKPIIIGHH 119-133 392.6 w HLA-DRB1*0802 SGWVKPIIIGHHAYG 122-136 192.5 w VSGWVKPIIIGHHAY 121-135 217.6 w HLA-DRB1*1101 VKPIIIGHHAYGDQY 125-139 222.7 w WVKPIIIGHHAYGDQ 124-138 250.7 w HLA-DRB1*1501 VKPIIIGHHAYGDQY 125-139 37.2 s KPIIIGHHAYGDQYR 126-140 38.6 s HLA-DR84*0101 KPIIIGHHAYGDQYR 126-140 145.3 w VKPIIIGHHAYGDQY 125-139 146.4 w HLA-DR85*0101 SGWVKPIIIGHHAYG 122-136 309.9 w VSGWVKPIIIGHHAY 121-135 311.0 w
[0126] The inventors then sought to address IDH1R132H epitope processing and presentation in vitro using PLA. PLA has been developed for protein-protein interaction analysis in unmodified cells and tissues by applying specific antibodies for proteins of interest coupled to PCR probes. Rolling circle PCR amplification then allows for the fluorescent visualization of co-localization of native proteins in situ. Thus, in principle, this technique is applicable for the analysis of natural peptide processing, as it does not require structural modifications of the system by exogenous expression or introduction of fluorescent labels. In order to employ the PLA for epitope presentation on MHC, a specific antibody detecting the epitope of interest is required. A peptide library was generated encompassing the IDH1R132H region (
Example 2: Establishment of an In Vitro System to Detect IDH1R132H-MHC Class II Co-Localization
[0127] As an in vitro system to evaluate the applicability and specificity of PLA for detection of IDH1R132H epitope presentation on HLA-DR (
Example 3: IDH1R132H-MHC Class II Co-Localization can be Detected In Vitro
[0128] In IDH1D252G/R132H transduced cells, but not cells expressing IDH1D252G, /WT an epitope-specific proximity of MHC class II HLA-DR and IDH1R132H peptide was detected using PLA (
Example 4: PLA Also Demonstrates Co-Localization of an Endogenously Expressed Tumor-Associated Antigen (TAA) In Vitro
[0129] To confirm the applicability of PLA as a tool to detect epitope presentation on HLA-DR, the inventors next aimed to extend this finding to an established TAA of known functional relevance. The cancer testis antigen NY-ESO-1 (CTAG1B) represents a suitable antigen, because it has been shown to be a potent immunogenic target in various studies, inducing not only specific CD8+ T cell-mediated, but also CD4+ T cell-mediated responses and containing several MHC class II HLA-DR-binding epitopes e.g. p119-143, p121-138, and p123-137 for HLA-DR1 [20, 21]. Therefore, the inventors overexpressed myc-tagged NY-ESO-1 in LN229 (
Example 5: IDH1R132H-MHC Class II PLA Demonstrates Co-Localization In Situ
[0130] The inventors next assessed the applicability of PLA to detect epitope presentation in tumor tissue in situ. To this aim, PLA was performed on paraffin-embedded glioma tissue.
[0131] With the increased interest in targeting true tumor antigens, which typically are mutated antigens, by active immunotherapy [22-24] there is an increasing requirement to evaluate whether these antigens are indeed presented in the tumor tissue. In contrast to TAA, mutated antigens have not undergone central tolerance, however, they are often minor antigens presented on MHC class II rather than MHC class I [25]. While the latter has been considered a disadvantage for a long time there is an increasing evidence that an antigen-specific CD4+ T cell response is capable of executing an effective antitumor immunity and not just provide help for CD8+ T cells [26, 27]. Potential mechanisms include direct cytotoxicity by antigen-specific CD4+ T cells towards tumor cells presenting the antigen on MHC class II and activation of innate immune cells by antigen-specific CD4+ T cells stimulated through intratumoral professional APC presenting tumor antigens. The inventors have previously demonstrated that IDH1, which is frequently mutated in gliomas and other types of tumors, represents a novel tumor neo-antigen. The IDH1R132H mutation, which represents the most common mutation in IDH1, is capable of inducing a mutation-specific class II-restricted CD4+ T cell response in patients with IDH1R132H-mutated gliomas and in MHC-humanized A2.DR1 mice after vaccination (Schumacher Bunse Nature 2014). Two lines of evidence in our previous study suggest that IDH1R32H is endogenously processed and presented on MHC class II: (i) In patients with IDH1R132H+ gliomas but not IDH1wt gliomas, mutation-specific CD4+ T cells can be detected after ex vivo stimulation with IDH1R132H (p123-142) in an MHC class II-restricted manner. (ii) Mutation-specific CD4+ T cells can be detected after vaccination of MHC-humanized mice with irradiated syngeneic sarcomas expressing human IDH1R132H. Here the inventors present a novel approach to detect presentation of antigenic epitopes in situ applying PLA, a combined immunofluorescence and PCR method.
TABLE-US-00005 TABLE 2 Mutational and genetic analysis and clinical information of glioma patients. patients gender age diagnosis IDH1 status (IHC) PLA signal HLA-type HLA-DR expression p001 f 25 A°III IDH1R132H + DRB1*07,10:01 DRB3* + p002 m 50 GBM IDH1R132H + DRB1*10:01,15 DRB5* + p003 m 71 GBM IDH1wt − n.d. + p004 m 41 A°III IDH1R132H − DRB1*03,07 DRB4* +++ p005 f 52 A°III IDH1R132H − DRB1*07,07 +* p006 m 35 A°III IDH1R132H + DRB1*03,11, DRB3* + p007 f 55 A°III IDH1R132H − DRB1*11,13 DRB3* − p008 m 42 A°III IDH1wt − n.d. ++* p009 f 58 A°III IDH1wt − n.d. + p010 m 42 A°III IDH1wt − n.d. ++++ p011 m 48 A°III IDH1wt − n.d. ++ p012 f 88 A°III IDH1wt − n.d. +* p013 f 60 A°III IDH1wt − n.d. − p014 m 55 A°III IDH1wt − n.d. + p015 m 45 A°III IDH1wt − n.d. − p016 m 40 A°III IDH1R132H + n.d. + p017 m 42 A°III IDH1R132H + DRB1*01,15 DRB5* + p018 f 71 A°III IDH1R132H − n.d. +++ F, female; m, male; A, astrocytoma, GBM, glioblastoma (WHO °IV); IHC, immunohistochemistry; semi-quantitative analysis of HLA-DR expression: −, negative; +, low; ++, moderate; +++, strong; ++++, very strong; *,mainly microglial HLA-DR expression; n.d., not determined.
[0132] While this approach offers the advantage of detecting presentation in situ on paraffin-embedded tissue slides it is restricted in its use by the requirements for the antibody targeting the antigen: (i) The antibody targeting the antigen must recognize the presented epitope bound to MHC (class II) in a native state and in situ. (ii) In case of mutated antigens, the antibody needs to be mutation-specific. For IDH1R132H both requirements are fulfilled. The mutation-specificity has been established in numerous studies [18, 19, 28]. In fact, IDH1R132H IHC is now implemented in routine diagnostics. In this study, the inventors demonstrate that the antibody recognizes the same epitope, which is presented on MHC class II, which is part of IDH1R132H p123-142 and the shorter peptides p122-136, p124-138 and p126-140 (
[0133] The inventors further extend this observation to a classic tumor-associated antigen, NY-ESO-1. PLA using an NY-ESO-1 specific and a HLA-DR-specific antibody detected co-localization of an NY-ESO-1 epitope with HLA-DR in SK-Mel-37 endogenously expressing NY-ESO-1 and HLA-DR1 and HLA-DR3 (
[0134] The inventors also present evidence that IDH1R132H is presented in human IDH1R132H-mutated gliomas. While larger prospective series are required to determine the biological relevance of PLA-positivity, it is conceivable that this method may find application in pre-selecting patients for a targeted immunotherapy. With respect to deciphering the cellular context of antigen-presentation in the tumor tissue, there is room for improvement with respect to multi-labelling. The attempts to do so in this study suggest that IDH1R132H is not only presented by tumor cells themselves, which can be MHC class II positive (
[0135] In summary, the inventors present a novel method to detect presentation of antigens in situ which may find application in but is not restricted to the identification of relevant tumor (associated) antigens.
REFERENCES
[0136] 1. Kantoff, P. W., et al., Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med, 2010. 363(5): p. 411-22. [0137] 2. Mocellin, S., et al., Part I: Vaccines for solid tumours. Lancet Oncol, 2004. 5(11): p. 681-9. [0138] 3. Mocellin, S., et al., Part II: Vaccines for haematological malignant disorders. Lancet Oncol, 2004. 5(12): p. 727-37. [0139] 4. Prins, R. M., et al., Gene expression profile correlates with T-cell infiltration and relative survival in glioblastoma patients vaccinated with dendritic cell immunotherapy. Clin Cancer Res, 2011. 17(6): p. 1603-15. [0140] 5. Sampson, J. H., et al., An epidermal growth factor receptor variant III-targeted vaccine is safe and immunogenic in patients with glioblastoma multiforme. Mol Cancer Ther, 2009. 8(10): p. 2773-9. [0141] 6. Sliwkowski, M. X. and I. Mellman, Antibody therapeutics in cancer. Science, 2013. 341(6151): p. 1192-8. [0142] 7. Zhao, L. L., et al., Pathological significance of epidermal growth factor receptor expression and amplification in human gliomas. Histopathology, 2012. 61(4): p. 726-736. [0143] 8. Jager, E., et al., Monitoring CD8 T cell responses to NY-ESO-1: correlation of humoral and cellular immune responses. Proc Natl Acad Sci USA, 2000. 97(9): p. 4760-5. [0144] 9. Lundegaard, C., et al., NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res, 2008. 36(Web Server issue): p. W509-12. [0145] 10. Nielsen, M. and O. Lund, NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction. BMC Bioinformatics, 2009. 10: p. 296. [0146] 11. Rammensee, H., et al., SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics, 1999. 50(3-4): p. 213-9. [0147] 12. Regner, M., et al., An improved method for the detection of peptide-induced upregulation of HLA-A2 molecules on TAP-deficient T2 cells. Exp Clin Immunogenet, 1996. 13(1): p. 30-5. [0148] 13. Dutoit, V., et al., Exploiting the glioblastoma peptidome to discover novel tumour-associated antigens for immunotherapy. Brain, 2012. 135(Pt 4): p. 1042-54. [0149] 14. Weiler, M., et al., mTOR target NDRG1 confers MGMT-dependent resistance to alkylating chemotherapy. Proc Natl Acad Sci USA, 2014. 111(1): p. 409-14. [0150] 15. Balss, J., et al., Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol, 2008. 116(6): p. 597-602. [0151] 16. Verhaak, R. G., et al., Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell, 2010. 17(1): p. 98-110. [0152] 17. Yan, H., et al., IDH1 and IDH2 mutations in gliomas. N Engl J Med, 2009. 360(8): p. 765-73. [0153] 18. Capper, D., et al., Characterization of R132H mutation-specific IDH1 antibody binding in brain tumors. Brain Pathol, 2010. 20(1): p. 245-54. [0154] 19. Capper, D., et al., Monoclonal antibody specific for IDH1 R132H mutation. Acta Neuropathol, 2009. 118(5): p. 599-601. [0155] 20. Ayyoub, M., et al., Assessment of vaccine-induced CD4 T cell responses to the 119-143 immunodominant region of the tumor-specific antigen NY-ESO-1 using DRB1*0101 tetramers. Clin Cancer Res, 2010. 16(18): p. 4607-15. [0156] 21. Zarour, H. M., et al., NY-ESO-1 119-143 is a promiscuous major histocompatibility complex class II T-helper epitope recognized by Th1- and Th2-type tumor-reactive CD4+ T cells. Cancer Res, 2002. 62(1): p. 213-8. [0157] 22. Choi, B. D., et al., EGFRvIII-targeted vaccination therapy of malignant glioma. Brain Pathol, 2009. 19(4): p. 713-23. [0158] 23. Sampson, J. H., et al., Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma. J Clin Oncol, 2010. 28(31): p. 4722-9. [0159] 24. Sensi, M. and A. Anichini, Unique tumor antigens: evidence for immune control of genome integrity and immunogenic targets for T cell-mediated patient-specific immunotherapy. Clin Cancer Res, 2006. 12(17): p. 5023-32. [0160] 25. Accolla, R. S. and G. Tosi, Optimal MHC-II-restricted tumor antigen presentation to CD4+ T helper cells: the key issue for development of anti-tumor vaccines. J Transl Med, 2012. 10: p. 154. [0161] 26. Hunder, N. N., et al., Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N Engl J Med, 2008. 358(25): p. 2698-703. [0162] 27. Quezada, S. A., et al., Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts. J Exp Med, 2010. 207(3): p. 637-50. [0163] 28. Sahm, F., et al., Addressing diffuse glioma as a systemic brain disease with single-cell analysis. Arch Neurol, 2012. 69(4): p. 523-6. [0164] 29. Lehtinen, M., et al., A T-helper cell epitope overlaps a major B-cell epitope in human papillomavirus type 18 E2 protein. APMIS, 1992. 100(11): p. 1022-6. [0165] 30. Rees, R. C. and S. Mian, Selective MHC expression in tumours modulates adaptive and innate antitumour responses. Cancer Immunol Immunother, 1999. 48(7): p. 374-81. [0166] 31. Takamura, Y., et al., Regulation of MHC class II expression in glioma cells by class II transactivator (CIITA). Glia, 2004. 45(4): p. 392-405. [0167] 32. Nielsen, M., C. Lundegaard, and O. Lund, Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method. BMC Bioinformatics, 2007. 8: p. 238. [0168] 33. Balss, J., et al., Enzymatic assay for quantitative analysis of (D)-2-hydroxyglutarate. Acta Neuropathol, 2012. 124(6): p. 883-91.