Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70539—MHC-molecules, e.g. HLA-molecules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers

Definitions

• beta2-microglobulin as a prognostic marker for tumour immune escape and resistance to cancer immunotherapy and a diagnostic biomarker for patient selection for specific gene therapy.
• the invention relates to the medical field, more specifically to the field of cancer immunotherapy and gene therapy.
• tumour-specific T-cells are frequently unable to reject the tumour, leading to cancer progression. This is attributable to the immune escape and expansion of cells with low immunogenicity and high metastatic capacity.
• HLA Human Leukocyte Antigen
• One of the central mechanisms of immune evasion is associated with an immune selection that favours the outgrowth of Human Leukocyte Antigen (HLA) class I-negative tumour cells.
• HLA Human Leukocyte Antigen
• Cell surface expression of HLA class I molecules in tumour cells is required for the recognition of the class I heavy chain/beta2 -microglobulin ⁇ 2 ⁇ .) /tumour peptide complex by cytotoxic T cells (CTLs).
• CTLs cytotoxic T cells
• HLA class 1 expression has been reported to be a predictive factor for the response to chemotherapy in early breast cancer patients. Moreover, in patients undergoing immunotherapy, the lack of response and generation of progressing metastases appears to be associated, with immune selection of HLA-negative tumour cells variants with irreversible defects.
• HLA Human Leukocyte Antigen
• the present invention provides the first experimental evidence of the fate of p2m gene mutations in successive metastatic lesions in a melanoma patient undergoing immunotherapy, demonstrating that the immune escape of HLA class I-negative tumour cells is directly correlated to these genetic events, namely to a mutation of one copy of the ⁇ 2 ⁇ gene and the loss of the other copy, i.e., loss of heterozygosity (LOH).
• LHO heterozygosity
• a first aspect of the present invention refers to a method of predicting or prognosticating the response of a human subject to immunotherapy (from hereinafter method of the invention), wherein the subject is suffering from a cancer disease, and wherein the method comprises using, as an indicator, expression levels of p2-micro globulin from the tumour cells of a biological sample of the subject; wherein the result is indicative of a negative response if the expression levels of ⁇ -microglobulin are under-expressed in comparison to a reference sample and/or a positive control.
• the expression levels are determined after said subject has been, treated with immunotherapy
• under-expressed is defined as a level of expression, lower than 2/3 of the maximum score achieved in the reference sample and/or positive control.
• under-expressed is defined, as a level of expression lower or equal to 1 ⁇ 2 of the maximum, score achieved in in the reference sample and/or positive control. in yet another preferred embodiment of the first aspect of the invention, under-expressed is defined as a level of expression lower than. 1/10 of the maximum score achieved in in the reference sample and/or positive control.
• the protein P2-microglobulin is used as an indicator. Furthermore, it is also particularly preferred that the result is obtained by using. immunohistochernistry. Moreover, it is also particularly preferred that the biological sample is fresh tissue or paraffin embedded tissue.
• the reference samples used in the above method can be selected from autologous normal cells from the subject.
• cancer disease subjected to prognosis in the above method can be selected from any type of cancer disease, particularly from the list consisting of carcinomas or adenocarcinomas, more particularly the cancer disease is melanoma or bladder carcinoma,
• a second aspect of the invention refers to method for allocating a human subject suffering from cancer in one of two groups, wherein group 1 comprises subjects identifiable by the method of the invention; and wherein . group 2 represents the remaining subjects.
• a third aspect of the invention refers to a composition comprising a polynucleotide sequence coding for p2-microglobulin protein, for the treatment of a human subject of group 1 as identifiable by the method of the second aspect of the invention.
• the composition is administered sequentially or simultaneously to a composition suitable for cancer immunotherapy.
• said composition suitable for cancer immunotherapy comprises dendritic cells, obtained from the subject suffering the cancer disease, transfected with mRNA from the tumour cells of the biological sample obtained from the subject.
• said composition suitable for cancer immunotherapy comprises Bacillus Calmette-Guerin (BCG), more particularly said treatment comprises the use of the BCG vaccine.
• the viral vector from, the expression system is an adenoviral vector.
• a fourth aspect of the invention refers to a composition suitable for cancer immunotherapy for the treatment of a human, subject of group 2 as identifiable by the method of the second aspect the invention.
• said composition suitable for cancer immunotherapy comprises dendritic cells, obtained from the subject suffering the cancer disease, transfected with mRNA from the tumour cells of the biological sample obtained, from the subject.
• said composition suitable for cancer immunotherapy comprises dendritic cells, obtained from the subject suffering the cancer disease, transfected with mRNA from the tumour cells of the biological sample obtained from the subject.
• said composition suitable for cancer immunotherapy comprises Bacillus Calmette-Guerin (BCG) 5 more particularly said treatment comprises the use of the BCG vaccine.
• BCG Bacillus Calmette-Guerin
• the subject of any of the previous aspects of the invention suffers from a cancer disease, wherein said cancer disease is melanoma.
• the subject of any of previous aspects one to four suffers from a cancer disease, wherein said cancer disease is bladder carcinoma.
• the treatment of the subject further comprises chemo- or radiotherapy.
• FIG. 1 Immunohistochemical analysis of HLA class I expression in paraffin-embedded tumour samples with antibodies that recognize free HLA-A, B, C heavy chain (EMR 8-5) and [Mm (L-368). Samples 13872, 8755, and 9168 are positive for HLA expression.
• Tumour 17130 has a heterogeneous (H) pattern of HLA- ABC and p2m expression. The last sample obtained during metastatic progression is positive for HLA-A 5 B,C and negative for p2m.
• Negative HLA class I surface expression on DNR-DC-M010 cells analysed by flow cytometry using the anti-[52m antibody L-368 (A, upper FACS image) and the anti-HLA[3 ⁇ 42m complex antibody W6/32 (A, lower FACS image). Immunofluorescence was measured either under baseline conditions (black lines) or after 48 h incubation with IFN-gamma (grey lines). Immunocytochemistry of the cells (B) confirmed the loss of both HLA class I complex (W6/32) and ⁇ 2 ⁇ (L-368) labeling; however, immunostaining with antibody HC-10 recognizing free HLA heavy chains was positive.
• FIG. 4 P2m, HLA-A,HLA-B, and HLA-C mRNA expression was analyzed by RT-PCR using specific primers.
• Figure 4 The ⁇ 2 ⁇ gene was sequenced in the cell line and in P2m-negative tumour nests and a mutation was found. The sequence from, codon 64 to 69 of the ⁇ 2 ⁇ gene in exon 2 is shown in the schematic diagram.. Asterisk indicates the location of the G-to-T transversion mutation (codon 67) creating a premature stop codon.
• C Heterogeneous pattern of HLA heavy chain. (EMR 8-5 and HC-10 antibody) and p2m (L-368 antibody) immunolabeling in tumor sample 17130. Microdissection was performed to isolate p2m -negative nodules.
• FIG. 1 Analysis of the heterozygosity of chromosomes 15 and 6 in melanoma cell line DMR-DC-MOIO and in tumour samples. icrosatellite analysis was performed on DNA from DNR-DC-MOIO cells and compared to DNA obtained from the patient's autologous PBMC, (A) Scans from the results of PCR analysis of five microsatellite markers on chromosome 15 reveal a pattern of allelic loss. Arrows indicate loss of heterozygosity at five markers.
• FIG. 6 (A) Restoration of surface HLA class I expression and function on DNR-DC-MOIO cell line 48 hours after infection with AdCMVp2m virus, as demonstrated by flow cytometry using L-368 (black filled histograms) and W6/32 antibodies (grey histograms). (B) Increase in IF y production (ELISPOT) by HLA-A2+ donor T-cells primed with influenza A peptide after co-culture with MO 10 cells infected with AdCMVp2m vims. HLA-A2+ melanoma cells with normal HLA class I expression were used as a positive control. (C) The percentage of specific T-cell mediated lysis (calculated by radioactive 51Cr release) of M010 cells transduced with ⁇ 2 ⁇ gene was higher than the lysis of luciferase-transfected control cells.
• FIG. 7 Clinical evolution of metastatic melanoma showing the early appearance of ⁇ 2 ⁇ mutation (sample 17130). The same mutation was detected in a late metastatic lesion (sample 17790) and in the cell line derived, from a lymph node fine needle biopsy. LOH in chromosome 6 is present in all samples; LOH in chromosome 15 was detected in all lesions except for the first tumor specimen. The intensity of HLA class I positive immunolabeling (+++, ++) was determined by using antibodies that recognize HLA-A.B,C heavy chain specificity.
• Figure 8 Positive expression of NK ligands (MICA/B, ULBP1 ,2 5 and 3) (A) and melanoma antigen peptide MelanMart- 1 (B) in DNR-DC-MOI O cells.
• Figure 9 Positive transduction of human bladder cells with a control AdCMVGFP- vector. Bladder cell lines were incubated with AdCMVGFP for 48 hours and positive GFP expression was confirmed by flow cytometry.
• Figure 10 Infection with AdCMVp2m -vector increases HLA class I expression on human bladder carcinoma cells.
• FIG. 11 In vivo experiments on immunodeficient mice by using an intratumoral injection of AdCMV luciferase (control) and AdCMVP2m adenoviral vectors. Human tumour cells were transplanted into nude/ nude mice. Viral vectors were injected into tumours of 5-8 mm in diameters.
• A In vivo imaging of luciferase expression in human xenograft tumours 5 days after injection of adenoviral vector AdCMVluc in a dose of 109 PFU.
• B Time- and dose- dependent intensity of bioluminescence of the luciferase in human tumour xenografts.
• C and D Positive re-expression of b2m protein in the tumours transfected with AdCMVb2m vector demonstrated by immunohistochemistry (C) and western blotting (D) using anti-P2m antibody.
• FIG. 12 Treatment protocol with AdCMVp2m -vector + BCG in patients with T1G3 urothelial cancer.
• patients will receive a combined treatment consisting of gene therapy and BCG.
• Six weekly BCG instillations will be combined with three bi-weekly instillations of adenovirus carrying human b2m gene ( 101 1-1012 pfu) diluted in 50ml of PBS, each time five days before BCG administration.
• Three months later similar, but reduced, treatment cycle will be repeated, including three weekly BCG and two AdCMVb2m administrations (two weeks apart).
• patients will be receiving more BCG instillations, but without gene therapy. We expect that this treatment will reduce the incidence of tumor recurrence as compared to patients treated only with BCG.
• the authors of the present invention studied the primary tumours and successive metastatic lesions of a melanoma patient who did not responded to treatment with a cancer vaccine.
• the patient was treated, with an individualized melanoma vaccine based on the transfection of autologous dendritic cells with autologous tumour-mRNA.
• NK cells might have eliminated melanoma cells but failed to migrate into the tumour, possibly attributable to suppressive factors in the tumour microenvironment. This highlights the fact that a better understanding of the aggressive phenotypes shall certainly contribute to a better selection of the most effective therapy.
• immunotherapy induces an additional cycle of immune selection of tumor cells with reduced or negative ⁇ 2 ⁇ expression, which are potentially able to escape from T-cell recognition.
• tumor cells have a reversible HLA alteration ("soft" lesion)
• immunotherapy induces the local release of cytokines leading to recovery of normal p2m expression and thus normal HLA class I expression, increased tumor cell immunogenicity, and immune rejection of tumor cells.
• hard lesions including LOH and/or gene mutations
• immunotherapy will fail to induce sufficient upregulation of tumor ⁇ 2 ⁇ expression and thus of HLA class I antigen expression, and the tumor cells will escape from immune recognition and will relapse. Therefore, activation of immune surveillance after immunotherapy leads to the imraunoselection and elimination of tumour cells with up- regulated HLA class I expression and to the outgrowth of cancer cells with structural ⁇ 2 ⁇ expression alterations.
• tumour cells with reduced expression of ⁇ 2 ⁇ after been treated with immunotherapy result, with time, in HLA-negative tumour cells, wherein HLA class I expression can only be restored with the introduction of wild-type ⁇ 2 ⁇ gene.
• adenovirus-mediated transfer of ⁇ 2 ⁇ gene restored HLA class I antigen expression and recognition, by peptide-specific HLA-A2 -restricted cytotoxic T lymphocytes in a melanoma cell line.
• reconstitution of a normal HLA class I phenotype is a pivotal strategy for tumour cells to circumvent an effective immune response and is associated with tumour progression in cancer patients. Therefore, the findings of the present invention support the importance of immunological tumour analysis in order to exclude those patients unlikely to respond to immunotherapy.
• the authors of the present invention have come to the conclusion that those subjects suffering from a cancer disease, wherein at least part, of their tumour ceils show hard lesions in the p2m gene, can only be successfully treated with immunotherapy by the introduction, of the wild-type p2m gene.
• the authors of the present invention have come to a particularly advantageous treatment protocol for those patients suffering from urothelial cancer. In this sense, patients with recurrent bladder carcinoma tumours despite BCG therapy, shall receive several cycles of local AdCMVb2m administration preceding BCG instillations. Each time, virus (10 n -10 12 viral particles) diluted in 50ml of PBS will be instilled into the bladder for a period of 60 min.
• the patient shall, continue the standard treatment with the local administration of BCG.
• the HLA-negative tumor escape variants will recover HLA class I expression leading to increased recognition and elimination of cancer cells by activated cytotoxic T-lymphocytes.
• a first aspect of the present invention refers to a method of predicting or prognosticating the response of a human subject to immunotherapy (from hereinafter method of the invention), wherein the subject is suffering from a cancer disease, and wherein the method comprises using, as an indicator, expression levels of [ ⁇ -microglobulin from the tumour cells of a biological sample of the subject; wherein the result is indicative of a negative response if the expression levels of p2-microglobulin are under-expressed in comparison to a reference sample and/or a positive control.
• the expression levels are determined after said subject has been treated with immunotherapy.
• said reduced expression levels are caused by gene alterations originated by the substitution of a guanine residue for a thymine residue in exon 2, codon 67, of the ⁇ 2- microglobulin gene sequence and/or the loss of heterozygosity (LOH) in chromosome 15.
• gene alterations originated by the substitution of a guanine residue for a thymine residue in exon 2, codon 67, of the ⁇ 2- microglobulin gene sequence and/or the loss of heterozygosity (LOH) in chromosome 15.
• the method of the present invention may be applied with samples from individuals of either sex, i.e. men or women, and at any age.
• the method of determining the result i.e. the expression level of ⁇ 2- microglobulin
• a gene profiling method such as a microarray, and/or a method comprising PGR, such as real time PGR; and/or Northern Blot or by using immunohistochemistry.
• PGR such as real time PGR
• Northern Blot or by using immunohistochemistry.
• the protein p2-micro globulin is used as an indicator.
• the result is obtained by using immunohistochemistry.
• the biological sample is fresh tissue or paraffin embedded tissue.
• the reference samples used in the above method can be selected from any type of autologous normal cells from, the subject.
• under-expressed is defined in comparison to a normal sample and/or a positive control as a level of expression lower than 2/3 of the maximum score achieved in the reference sample and/or positive control.
• under-expressed is defined as a level of expression lower or equal to 1 ⁇ 2 of the maximum score achieved in in the reference sample and/or positive control. More preferably, under-expressed is defined as a level of expression lower to 1/10 of the maximum score achieved in in the reference sample and/or positive control.
• a biological sample include different types of samples from tissues, as well as from biological fluids, such as blood, serum, plasma, cerebrospinal fluid, peritoneal fluid, faeces.
• said samples are samples from tissues and most preferably, said samples of tissues originate from tumour tissue of the individual the response of which is to be predicted, and may originate from biopsies.
• Response refers to the clinical outcome of the subject
• “Response” may be expressed as overall survival or progression-free survival. Survival of cancer patients is generally suitably expressed by Kaplan-Meier curves, named after Edward L. Kaplan and Paul Meier who first described it (Kaplan, Meier; Amer. Statist. Assn. 53:457- ⁇ 181).
• the Kaplan-Meier estimator is also known as the product limit estimator. It serves for estimating the survival function from life-time data.
• a plot of the Kaplan-Meier estimate of the survival function is a series of horizontal steps of declining magnitude which, when a large enough sample is taken, approaches the true survival function for that population.
• the Kaplan- Meier estimator may be used to measure the fraction of patients living for a certain amount of time after beginning of immunotherapy and chemotherapy and/or radiotherapy.
• the clinical outcome predicted may be the (overall/progression-free) survival in months/years, from the time point of taking the sample. It may be survival for a certain period from taking the sample, such as of six months or more, one year or more, two years or more, three years or more, four years or more, five years or more, six years or more.
• “survival” may refer to "overall survival” or "progression-free-survival".
• the response is clinical outcome, which is "overall survival” (OS).
• OS overall survival
• “Overall survival” denotes the chances of a patient of staying alive for a group of individuals suffering from a cancer. The decisive question is whether the individual is dead or alive at a given time point.
• the cancer disease as is a carcinoma, preferably a carcinoma selected from the list consisting of bladder, ovarian, neck. colon, stomach, cervix, thyroid gland, lung, uterus, rectum, breast or kidney carcinoma.
• the cancer disease is melanoma or bladder carcinoma.
• a second aspect of the invention refers to method for allocating a human subject suffering from cancer in one of two groups, wherein group 1 comprises subjects identifiable by the method of the invention; and wherein group 2 represents the remaining subjects.
• a third aspect of the invention refers to a composition comprising an expression system which in torn comprises a polynucleotide sequence coding for p2-microglobulin protein, for the treatment of a human subject of group 1 as identifiable by the method of the fourth aspect of the invention.
• an expression system is a system specifically designed for the production of the ( ⁇ -microglobulin.
• an expression system consists of the p2-microglobulin gene, normally encoded by DNA or cDNA, and the molecular machinery required to transcribe the DNA into mRNA and translate the mRNA into protein using the reagents provided.
• the expression system is a viral vector, more preferably the viral vector is an adenoviral vector.
• the composition is administered sequentially or simultaneously to a composition, suitable for cancer immunotherapy.
• said composition suitable for cancer immunotherapy comprises dendritic cells, obtained from the subject suffering the cancer disease, transfected with mRNA from the tumour cells of the biological sample obtained from the subject.
• said composition suitable for cancer immunotherapy comprises the use of Bacillus Calmette-Guerin (BCG), more particularly said treatment comprises the use of the BCG vaccine.
• BCG Bacillus Calmette-Guerin
• immunotherapeutic treatments are IL-2, 1FN, peptide based therapy or transfer of autologous activated T-cells.
• a fourth aspect of the invention refers to a composition suitable for cancer immunotherapy for the treatment of a human subject of group 2 as identifiable by the method of the fourth aspect of the invention.
• said composition suitable for cancer immunotherapy comprises dendritic cells, obtained from the subject suffering the cancer disease, transfected with mRNA from the tumour cells of the biological sample obtained from the subject.
• said composition suitable for cancer immunotherapy comprises the use of Bacillus Calraette-Guerin (BCG), more particularly said treatment comprises the use of the BCG vaccine.
• BCG Bacillus Calraette-Guerin
• the treatment of the subject further comprises chemo- or radiotherapy.
• the subject of any of the previous aspects of the invention suffers from a cancer disease, wherein said cancer disease is a melanoma.
• a cancer disease wherein said cancer disease is bladder carcinoma.
• said cancer disease is bladder carcinoma.
• Example 1 Material and methods 1.1. Tumour tissue samples and cells
• Tumour samples and the melanoma cell line DNR-DC-MOI O which was established from a lymph node fine needle biopsy, were obtained from a 72 years old patient with metastatic melanoma (received as part of European collaborative project ENACT).
• Autologous PBMCs were also received from the same patient. This patient was treated in Norwegian Radium Hospital using dendritic cells transfected with autologous tumour-mRNA.
• Table 1 below depicts a schematic summary of the patient's clinical time course from October 2001 to February 2003. In October 2001 , a primary tumour was surgically removed, and a second lesion, was removed two months later. In June 2002, several metastases from lymph nodes at both axillae and a cutaneous lesion were surgically removed.
• the cell line was grown in RPMI medium (Biochrom KG, Berlin, Germany) supplemented with 10% PCS (Gibco BRL, Life TECHSjKarlsruhe, Germany), 2% glutaraine (Biochrom KG), and 1% penicillin/streptomycin (Biochrom KG) at 37°C in a humidified atmosphere with 5% C02.
• Autologous PBMCs (A*0205, 3201 ; B*5001, 5101 ; Cw*0602, 1502) were used for LOH analysis and genome sequencing.
• HLA class I expression in the melanoma cell line was studied by flow cytometry using monoclonal antibody (mAb) which recognizes a cell surface complex of ⁇ 2- microglobulin with HLA heavy chain.
• mAb monoclonal antibody
• This antibody and an .anti-human ⁇ 2 ⁇ mAb L-368 were a kind gift from Dr. Bodmer (Tissue Antigen Laboratory, Imperial Cancer Research Fund Laboratories, London, UK), HC-10 mAb against free HLA heavy chain (Stam et al. 1986: J Immunol 137:2299-2306) was also used to detect free HLA heavy chain in melanoma cells.
• anti-p2m antibody L-368 and anti-pan-HLA class I mAb EMR8-5 which recognizes a common HLA- A,B,C heavy chain sequence (MBL, Naka-ku Nagoya, Japan).
• Antibodies against various components of antigen processing machinery (APM) (TAP-1, TAP-2, Tapasin., LMP-2, LMP- 7, calnexin, calreticulin and ERP-57) were purchased from Abeam (UK) and used for flow cytometry and immunocytochemistry.
• APM antigen processing machinery
• the mAbs against human NK ligands including anti- ULBP-1 -Fluorescein monoclonal antibody (for intracellular staining), anti-human ULBP-2- Phycoerythrin antibody, and the human ULBP-3 antibody were purchased from. R&D systems (Minneapolis, MN, USA), and anti-MlCA-MICB (clone 2C10) from Santa Cruz Biotechnology (Santa Cmz, CA).
• Antibodies to CD3, CD4, CDS, and CD56 markers for immunohistochemical detection of tumour infiltrating lymphocytes (TILs) were purchased from Master Diagnostica (Granada, Spain). Fluorescein isothiocyanate-conj ugatcd goat anti- mouse antibody (Sigma, St Louis, MO, USA) was used as a secondary antibody for flow cytometry.
• ImmuQocytochemical analysis of the DNR-DC-MOI O cell line was performed on cytospin glass slides as previously described. Immunohistochemical analysis of the tumour biopsies was performed by cutting consecutive 4-6 ⁇ thick sections from formalin-fixed paraffin- embedded tissue blocks using a SLEE MAINZ CUT 5062 microtome and mounting them on coated glass slides, After deparafmization and antigen retrieval using heat, EDTA, or citrate buffer treatment, depending on the antibody used, slides were rehydrated and used for antibody staining with the hiotin-streptavidin system (supersensitive Multilink-HRP/ DAB. kit, BioGenex).
• hiotin-streptavidin system supersensitive Multilink-HRP/ DAB. kit, BioGenex
• HLA class I expression was studied with the monoclonal antibodies L-368, EMR8-S, and HC-! O. TILs were analysed with antibodies against CD3, CD4, CDS and CDS 6. The primary antibody was replaced with PBS in negative controls, in which no labelling was detected. 1 .4. RNA isolation, reverse transcription, PGR, and quantitative real time-PCR
• Primers used for PGR amplification of C32- microglobulin cDNA were: forward 5 ' - GGGCATTCCTGAAGCTGACA-3 ', and backward: 5 ' - GGTTGCTCCACAGGTAGCTCTA-3 ' with 618 bp of predicted PGR product size.
• the primers used for HLA- A, HLA-B and HLA-C heavy chain amplification have been described by Ruano et al.
• G6PDH glucose-6-phosphate dehydrogenase
• beta-actin genes were also amplified. All PGR reactions were performed in a Light Cycler instrument using the LC-FastStart DNA Master SYBR Green I Kit (Roche Diagnostics, Manheim, Germany). The primer sequences, amplicon size, and annealing temperatures used are described in (Romero et al 2005),. with the exception, of G6PDH and beta-actin.
• DNA extraction from the cultured melanoma cells was performed with the blood and cell culture DNA Midi Kit (Qiagen Inc., Valencia, CA.) according to the manufacturer's instructions. Immunolabeled paraffin sections were micro-dissected using a laser micromanipulator (PALM Microlaser Systems, Olympus). Micro-dissected, fragments were collected in. PALM Adhesive Caps. These fragments were used for DNA extraction using the QIAamp®DNR FFPE Tissue Kit (QIAGEN, Westburg, Leusden, The Netherlands) following manufacturer's recommendations with few modifications.
• the p2m cDNA from the cell line was sequenced using the same primers as in the PGR analysis.
• the amplification of p2m gene from the micro-dissected tumour nests of paraffin- embedded samples was performed on genomic DNA with Illustra PuReTaq Ready-To-GoTM PGR Beads (GE Healthcare Life Sciences) using the following forward primers: 5'- CGATATTCCTC AGGTACTCC-3 ' and 5 ' - GGTGAATTCAGTGTAGTACAAG-3 ' » and with one backward primer: 5 '-ACACAACTTTCAGCAGCTTAC-3 '.
• the predicted PCR product sizes are 31 1 bp and 114 bp, respectively.
• Sequencing was performed by using the Big Dye Terminator vl. l Cycle Sequencing kit (Applied Biosystems, Warrington, UK) and the same primers as in the amplification reaction. The sequencing reaction was performed in Centri-Sep Columns (Applied Byosistems) following the manufacturer's recommendations. ABI 31 30x/ Genetic Analyzer and SequencingAnalysis v5.2 software (Applied Biosystems) were used for the analyses.
• STRs short tandem repeats
• D6S291, D6S273, C.1.2.C, C.1.2.5, D6S265, D6S 105, and D6S276 mapping the HLA region and an additional S I R, D6S3 1 1 , in the 6q region.
• the 2-microglobulin studies used five STR markers that flanked the gene in chromosome 15 (DS 15209, DS15126, DS15146, DS 151028 and DS 151 3).
• HLA-A2 PBMCs were primed with HLA-A2-restricted influenza A vims matrix 58-
• PBMCs 105/well were co-cultured in 96- well round- bottom plates with untreated DNR-DC-MOI O melanoma cells (5xl04cells/well) or with cells manipulated in one of the following ways; pulsed with flu peptide, transduced with AdCMVp2m 5 peptide-pulsed and transduced with AdCMVp2m, transduced with a control AdCMVLuc vector, or peptide-pulsed and transduced with AdCMVLuc. After 48 h of co- culture, T cells producing IFN-gamma were counted using an ELISPOT kit (BD-Pharmingen, San Diego, CA, USA) according to the manufacturer's instructions.
• ELISPOT kit BD-Pharmingen, San Diego, CA, USA
• peptide-pulsed PBMCs were co-cultured in 96-well round- bottom plates with HLA-A2 positive control melanoma cells (E-.7) and untreated DNR-DC- M010 melanoma cells (5xl04cells/well) or with cells manipulated in one of the following ways: pulsed with flu peptide, transduced with AdCMVb2m, peptide-pulsed and transduced with AdCMVb2m, transduced with a control AdCMVLuc vector, or peptide-pulsed and transduced with AdCMVLuc, Next day plates were washed with PBS and incubated with biotinlylated anti-IFN-g antibody for 4 h.
• Tumour cell lysis was measured by incubating the effector T-cells with 3000 Na251Cr04- labeled target cells at different tumor-effector cell ratios, Supematants were harvested 4 fa later and the percentage of specific lysis was calculated according to the formula: [(cpm experimental-cpm spontaneous) / (cpm maximum-cpm spontaneous) ⁇ 100, where spontaneous lysis corresponds to target cells incubated in the absence of effector cells, and maximum lysis is obtained by incubating target cells with 5% Triton X-1 0.
• HLA class I expression in primary tumours and five metastases (all paraffin-embedded) that have been surgically removed from the patient at different time points during disease progression before the administration of the DC vaccine (Table 1).
• the mRNA of lesion 17790 was used to prepare the DC vaccine. Positive labelling for HLA- A, B, C heavy chain (EMR 8-5 antibody) and p2m (L-368 antibody) was detected in the primary tumor (sample 13872) and metastatic lesions 8755 (lymph node metastasis), 9168-1 (lymph node lesions), and 9168-2 (cutaneous lesions) (Fig. 1). Sample 17790 (lesion used for immunotherapy) showed, a heterogeneous pattern of HLA- A, B, C labeling with P2m -negative areas.
• Monoclonal anti-pan HLA class I antibody EMR 8-5 detects a common domain, of HLA-A, -B, and -C heavy chain in formalin-fixed paraffin embedded tissues and does not recognize a heavy ehain ⁇ p2m complex, which is recognized by antibody W6/32 (Tsukaliara T et ah 2006).
• W6/32 Tsukaliara T et ah 2006.
• the second metastatic nodule (17130) was ' heterogeneous for both HLA class I complex and p2m (Fig. I ). In this sample, both positive and negative tumor nests were detected by antibodies directed against HLA heavy chain (HC-10 and BMR8-5) and by anti-p2m antibody (L-368) (Fig. 4).
• Example 3 Immunohistochemical detection of tumour infiltrating lymphocytes.
• TILs The presence of TILs was analysed in all studied paraffin-embedded samples and, as shown in Figure 2, a larger number of CD3+ and CD 8+ lymphocytes were found in ⁇ 2 ⁇ -positive samples (samples 8755 and 9168) than in P2m-negative tumour nests (biopsies 17130 and 17790), where they were only observed on the periphery of the tumour node.
• cytotoxic T cells have a better ability to penetrate HLA-positive tumours.
• NK cell aocumulation in the studied tumour specimens and found that tumour infiltration with CD56+ NK cells was practically negative in all the studied samples.
• expressions of activating NK ligands M1CA/B and ULBP-1 ,-2, and -3 were positive on D R-DC-MOl 0 cells as analysed by FACS.
• PBMC peripheral blood mononuclear cells
• LOH was defined by microsatellite analysis of the M010 cell line using five polymorphic markers spanning chromosome 15q, where the p2m gene has been mapped. Genomic DNA extracted from DNR-DC-M01Q patient's PBMC served as control. As shown in Figure 5A, LOH was detected at chromosome 15 for the five markers, showing a single allele at STR sites D15S1028 and D15S 146, which flank p2m gene, and also in STR D15S153, D15S126 and D15S209, whereas PBMC control showed retention of heterozygosity with two bands at the studied sites. We also checked the status of heterozygosity on chromosome 6.
• HLA-typieg was performed using a low-resolution genomic sequence-specific oligonucleotide analysis, which indicated LOH in chromosome 6 (HLA typing results for melanoma cell line DNR- DC-M010 were: A*0205, B*5001 , and Cw*0602) (Figure 5B); this finding was verified, by microsatellite analysis of this chromosome, which detected LOH in 6 out of 8 STR (the other two were non-informative), whereas the autologous PBMC retained heterozygosity (data not shown).
• Example 7 Reconstitution of normal HLA class I expression and its functional activity after transfer of wild-type p2m gene A non-replicating adenovirus (AdCMVp2m) expressing the wild-type human p2m gene under CMV promoter was constructed using the Cre-Lox recombination system as previously described (Efficient recovery of HLA class I expression in human tumor cells after beta2- microglobulin gene transfer using adenoviral vector: implications for cancer immunotherapy.del Campo AB, Aptsiauri N, Mendez R, Zinchenko S, Vales A, Paschen A, Ward S, Ruiz-Cabello F, Gonzalez-Aseguinolaza G, Garrido F.Scand J Immunol. 2009 Aug;70(2): 125-35)
• adenovims carrying human p2m (AdCMVb2m) under the control of cytomegalovirus (CMV) promoter were construed by homologous recombination between W5 (as a donor virus to supply the viral backbone) and pAdlox ⁇ 2 ⁇ (a shuttle plasmid with a single loxP site carrying the b2m gene) using the Cre-lox recombination system.
• RNA from peripheral blood mononuclear cells from a healthy donor was isolated with Ultraspec (Biotecx Laboratories,Houston 5 TX, USA) and amplified by reverse transcription- polymerase chain reaction (RT-PCR) using specific primers for human p2m (forward primer. 5 -AAGCTTGCCACCATGTCTCGCTCC- 30; reverse primer: 50- GGATCCTGCGGCATCTTCAAACCTCCATG-3 ) including the sites for restriction enzymes Hindlll and BamHX
• the 600-bp fragment corresponding to p2m cDNA was cloned into pCR4-TGPO plasmid generating TOPOb2m (TOPO TA cloning Kit; Invitrogen, Carlsbad, CA. USA),
• the Hindlll / BamHI P2m fragment excised from TOPO-b2m was ligated into HmdIII/BamHI -digested alkaline phosphatase-treated pAdlox.
• AdC Vb2m construction The new pAdlox p2m, linearized with Sfil, was co-transfected along with W5 DNA (W5 is an E1-E3 deleted version of Ad5-containing loxP sites flanking the packaging site) into 1 16 cells using jet PeiTM, (Redox Lab. S.L., Malaga, Spain) After development of complete cytopathic effect (7 or 8 days), the cell iysate was passaged three times in 116 cells to eliminate the W5 vims contamination.
• W5 is an E1-E3 deleted version of Ad5-containing loxP sites flanking the packaging site
• AdCMVb2m single clone was expanded to produce a large-scale concentrated stock in HEK293 cells, purified on CsCl gradient, desalted using PD-10 columns (Amersham Biosciences, Uppsala, Sweden) and stored in Tris-HCl 0.1 M and 10% glycerol at 80°C, Virus titres were determined by plaque assay in HEK293 cells based on the visual detection of infected cells stained with the mouse anti-adenovirus monoclonal antibody blend (Cliemicon Int., Temccula, CA, USA) and the titre was expressed as plaque-forming units.
• Concentrated stocks typically had titres in the range of l O 8 - 10 10 PFU / mL Adenoviral vectors containing green fluorescent protein (GFP) and Luciferase reporter genes under the control of CMV promoter (AdCM ' VGFP and AdCMVLuc) were produced similarly.
• GFP green fluorescent protein
• AdCM ' VGFP and AdCMVLuc Luciferase reporter genes under the control of CMV promoter
• IFN-gamma secretion by HLA-matched T-cells was measured by ELISPOT after 48 hours of co-culture with the original p2m-negative melanoma cells or AdCMVp2m-infected melanoma cells pulsed with HLA-A2-restricted virus matrix 58-66 peptide (G1LGFVFTL).
• ELISPOT As shown in Figure 6B, the number of IFNy-positive spots (per 100.000 PBMC) observed for T cell responses against non-transduced melanoma cells were as low as those in HLA-A2+ control melanoma cells (ESTDAB-064) not stimulated with peptide.

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