WO2014100216A1 - Microarn associés au cancer et anticorps associés - Google Patents

Microarn associés au cancer et anticorps associés Download PDF

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WO2014100216A1
WO2014100216A1 PCT/US2013/076193 US2013076193W WO2014100216A1 WO 2014100216 A1 WO2014100216 A1 WO 2014100216A1 US 2013076193 W US2013076193 W US 2013076193W WO 2014100216 A1 WO2014100216 A1 WO 2014100216A1
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cancer
mir
mirna
binding
mirnas
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Gang Zeng
Shirley LOMELI
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University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • 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
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57557—Immunoassay; Biospecific binding assay; Materials therefor for cancer of other specific parts of the body, e.g. brain
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/50—Methods for regulating/modulating their activity
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • This invention generally relates to the field of microRNA (miRNA) and
  • miRNAs are single-stranded, non-coding 21-23 oligoribonucleotides
  • miR-21 has been reported to counteract the expression of putative tumor-suppressive targets, such as phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and
  • miRNAs are also known to play regulatory roles for the immune system, and can profoundly alter the phenotype and outcome of immune responses (13), including alleviation of immune suppression by miR-17 (14).
  • miR-155 plays the role of a proinflammatory regulator in clinical and experimental arthritis. In an animal model of arthritis, wild-type mice produced higher titre autoAb against collagen as compared to miRNA-155 knockout mice (15). miRNAs are generally around 6.9 to 7.6 kDa and often found in association with large RNA- binding proteins.
  • miRNAs seem to be stable because most of them are included in apoptotic bodies, microvesicles or exosomes and protected from known mRNA degradation factors (16).
  • miRNA folds into hairpin structures via complementary base pairing to form a secondary structure thereby further stabilizing its bioavailability and function in post-transcriptional regulation as well as promoting its potential interaction with the immune system to elicit an immunogenic response.
  • the above factors make miRNAs potentially immunogenic when they are encountered by the immune system alone, as foreign or neo-antigens, or as a complex in association with large miRNA binding proteins under some pathological conditions.
  • the present invention provides methods for detecting the presence of and/or diagnosing a cancer in a subject which comprise directly or indirectly detecting and/or quantifying one or more cancer-associated miRNAs in a sample from the subject.
  • BLItz interferometry is used to detect and/or quantify the one or more cancer-associated miRNAs.
  • the miRNAs are selected from the group consisting of miR-17, miR-21, and miR-191.
  • one or more antibodies that specifically bind the one or more cancer-associated miRNAs are used as the capture reagent.
  • one or more autoAbs are used to indirectly detect the one or more cancer-associated miRNAs.
  • the one or more autoAbs specifically bind one or more nucleic acid molecules which have the same or substantially the same sequence as the one or more cancer-associated miRNAs.
  • the capture reagents may be immobilized on a substrate.
  • the methods further comprise detecting and/or quantifying one or more additional biomarkers in the sample.
  • the cancer is selected from the group consisting of lung cancers (such as NSCLC), glioblastomas, breast cancer, hepatocellular cancers, gastric cancers, prostate cancer, and colon cancers.
  • the subject is mammalian, preferably human. In some
  • the subject is a test animal, such as a mouse. In some embodiments, the subject is suspected of having cancer or at risk for cancer.
  • the present invention provides methods for detecting the presence of and/or diagnosing a cancer in a subject which comprise directly or indirectly detecting and/or quantifying one or more autoantibodies against cancer- associated miRNAs in a sample from the subject.
  • BLItz interferometry is used to detect and/or quantify the one or more autoantibodies.
  • the miRNAs are selected from the group consisting of miR-17, miR-21, and miR-191.
  • one or more antibodies and/or one or more cancer-associated miRNAs that specifically bind the one or more autoantibodies are used as the capture reagent.
  • the one or more autoAbs specifically bind one or more nucleic acid molecules which have the same or substantially the same sequence as the one or more cancer-associated miRNAs.
  • the capture reagents may be immobilized on a substrate.
  • the methods further comprise detecting and/or quantifying one or more additional biomarkers in the sample.
  • the cancer is selected from the group consisting of lung cancers (such as NSCLC), glioblastomas, breast cancer, hepatocellular cancers, gastric cancers, prostate cancer, and colon cancers.
  • the subject is mammalian, preferably human.
  • the subject is a test animal, such as a mouse.
  • the subject is suspected of having cancer or at risk for cancer.
  • the present invention is directed to methods of
  • the miRNAs are cancer-associated miRNAs.
  • the miRNAs are selected from the group consisting of miR- 17, miR-21 , and miR- 191.
  • the methods comprise administering to the subject an immunogenic amount of miRNA in the form of being bound to streptavidin and/or a substrate particle such as a bead.
  • the miRNA is bound to a streptavidin coated particle.
  • the miRNA is bound to a magnetic particle.
  • the miRNA is bound to a streptavidin coated magnetic particle such as a streptavidin coated magnetic bead.
  • the miRNA is biotinylated.
  • the method comprises administering to the subject a second immunogenic amount of the miRNA a given period of time, e.g., two weeks, after the first administration.
  • the miRNA is administered to the subject by intraperitoneal injection.
  • an immunogenic amount is an amount that elicits an immune response in a subject.
  • the subject is mammalian, preferably human.
  • the subject is a test animal, such as a mouse.
  • the antibodies generated are used in assays to detect and/or measure the miRNA in test samples, e.g., samples obtained from a subject.
  • Figure 1A shows the primary structures of the cancer-associated miRNA involved in the study. Modification of the RNA backbone with 2'-0-methylation, represented by m, was introduced to minimize hydrolysis of the RNA backbone by RNases. A control miRNA was also synthesized as a randomized sequence and searched against the miRNA database to confirm that there is no identity or homology to any known miRNA. The control miRNA was designed and used as a negative control to ensure specificity of binding.
  • Figure IB schematically shows a 96-well plate for immunoassays.
  • enlarged well depicts 4 differently colored beads, 1 coated with human albumin or a randomized miRNA as negative control, 3 coated with targets of interest, miR-17, miR-21, and miR-191 for measuring reactive autoAb.
  • Figures 1C1-1E2 are graphs summarizing the results of various autoAb assays.
  • Figures 2A-2D graphically show the characterization of the humoral immune responses against cancer-associated miRNA. Binding kinetics to miR-191 or miR-17 ligand using BLltz interferometry (ForteBio) with (Figure 2A) seropositive lung cancer patient plasma, ( Figure 2B) BSA negative controls for binding specificity and affinity, (Figure 2C) healthy patient plasma, and ( Figure 2D) human serum albumin (HSA) non-specific binding controls. BSA and HSA lacked binding specificity and affinity to miR-191 or miR-17 even at or above the highest concentrations of autoantibody used (shown in nM).
  • LPS lipopolysaccharide
  • Figure 3F lipopolysaccharide
  • Figure 3G 100 ⁇ g/ml of polyLC
  • DC morphological changes such as elongation and attachment to the culture dish were correlated with the rearrangement of filamentous actin, reproduced in at least 3 donors, imaged by fluorescence microscopy, 200X magnification ( Figures 3H-3L).
  • Figure 3M are graphs showing that extracellular miRNA induced upregulation of maturation and co-stimulatory molecules on the surface of DC.
  • Human monocyte- derived DC were incubated with synthesized miR-control, -17, -21, and -191 (6 ⁇ g/mL) on day 6 for 48 hours, compared to media with PBS, and LPS (100 ng/niL) and stained for CD80, CD83, and CD86 markers (black line), compared to isotype- matched control Ab (grey filled-in). These results have been reproduced using more than 3 donors.
  • FIGS 5A and 5B show antibodies are present in mice immunized with miR-
  • Figures 6A-6C graphically show the characterization of the humoral immune response against miR-17 immunogen in miR-17-immunized and non-immunized mice. Binding kinetics to miR-17 ligand using BLItz interferometry (ForteBio) with ( Figure 6 A) mouse serum seropositive for miR-17antibody, ( Figure 6B) na ' ive, non- immunized mouse serum, and ( Figure 6C) comparison of seropositive (Ml) and na ' ive mouse (N) serum-derived antibody affinity to miR-17 ligand at 41 nM autoAb concentration.
  • Formula 6 A mouse serum seropositive for miR-17antibody
  • Figure 6B na ' ive, non- immunized mouse serum
  • Figure 6C comparison of seropositive (Ml) and na ' ive mouse (N) serum-derived antibody affinity to miR-17 ligand at 41 nM autoAb concentration.
  • the present invention generally relates to cancer-associated miRNAs and immune responses, e.g., autoantibodies (autoAbs), against the cancer-associated miRNAs.
  • autoantibodies e.g., autoantibodies
  • cancer-associated miRNAs such as miR-17, miR-21 and miR-191, were found to have a significant impact on the maturation of human DC.
  • autoAb responses against these cancer-associated miRNAs were found to be present in prostate and lung cancer patients, but not at statistically significant levels in healthy controls as evidenced by the diminished binding of healthy plasma to the miRNAs compared to lung cancer plasma demonstrated by xMAP-based assays and confirmed by BLItz interferometry.
  • cancer-associated miRNAs endogenous to subjects having cancer are recognized as an immunogens by the subjects' own immune systems and result in autoAbs specific against the cancer associated miRNAs. Therefore, in some embodiments, the present invention relates to cancer-associated miRNAs as immunogenic agents and in immunogenic compositions. In some embodiments, the present invention relates to cancer-associated miRNAs and autoAbs specific against the cancer-associated miRNAs as biomarkers for cancer.
  • the present invention relates to methods of detecting a cancer in a subject, diagnosing a subject as having a cancer, and/or characterizing the type of cancer in a subject which comprise using one or more cancer-associated miRNAs and/or one or more autoAbs specific against a cancer-associated miRNA as a biomarker to be detected and/or quantified in a sample from the subject.
  • miRNA array and quantitative real-time PCR have been the two approaches used for quantifying miRNA (11). While these approaches are straightforward, they have given rise to conflicting data in different studies. These discrepancies might be due to the lack of an established endogenous miRNA control for normalization. In addition, because of the small size of the miRNA and their attachment to lipids and proteins, efficient and reproducible extraction methods remain elusive (Schwarzenbach, et al. (2011) Nat Rev Cancer 11 :426-437). There has been no previously known art to quantify miRNA using antibodies or approaches to generating antibodies against target miRNA. However, as disclosed herein, cancer-associated miRNA are immunogenic ( Figures 1A-1E2). Accordingly, in some embodiments, miRNA can be used as immunogens to induce antibodies ( Figures 5A-5B) and/or for the
  • an “epitope” is the part of a molecule that is recognized by a given antibody.
  • autoantibody refers to an antibody produced by a subject that is directed against one or more of the subject's own antigens (e.g., a tumor antigen).
  • antibody refers to an immunoglobulin molecule and immunologically active portions thereof (i.e., molecules that contain an antigen binding site that specifically bind the molecule to which antibody is directed against).
  • the term antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments.
  • antibody examples include, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab' fragments, F(ab') 2 , disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain.
  • scFvs single chain Fvs
  • Fab fragments fragments
  • Fab' fragments F(ab') 2
  • sdFvs disulfide linked Fvs
  • Fvs fragments comprising or alternatively consisting of, either a VL or a VH domain.
  • Antibodies of the invention may also include multimeric forms of antibodies.
  • antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules.
  • the antibodies of the present invention can be natural or synthetic, polyclonal or monoclonal, or chimeric, and can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi , IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2 ) or subclass of
  • a molecule e.g., an antibody, that "specifically binds" another molecule, means that the interaction is dependent upon the presence of a specific structure, e.g., an epitope, on the molecule being bound.
  • a specific structure e.g., an epitope
  • an antibody which specifically binds a protein is recognizing and binding a specific structure on the protein rather than indiscriminate binding that gives rise to non-specific binding and/or background binding.
  • non-specific binding and background binding refer to an interaction that is not dependent on the presence of a specific structure (e.g., a particular epitope).
  • biomarker refers to a substance used as an indicator of a process, event, or condition.
  • a biomarker can be a biomolecule such as a nucleic acid molecule (e.g., miRNA, genomic DNA, etc.), a protein, a polysaccharide, and the like. Biomarkers include tumor antigens and tumor markers.
  • tumor antigens refer to tumor-specific antigens (TSAs), which generally classified as antigens present only on tumor cells and tumor-associated antigens (TAAs), which are generally classified as antigens present on some tumor cells and also some normal cells.
  • TSAs tumor-specific antigens
  • TAAs tumor-associated antigens
  • tumor marker is a substance that may be found in body tissues or bodily fluids that is produced by tumor cells or non-tumor cells in response to the presence of cancerous cells.
  • tumor markers include AFP (in liver cancer), CA 125 (in ovarian cancer), CA 15-3 (in breast cancer), CEA (in ovarian, lung, breast, pancreas, and gastrointestinal tract cancers), and PSA (in prostate cancer).
  • Tumor markers can be classified in two groups: cancer- specific markers and tissue-specific markers. Tumor markers include tumor antigens. However, tumor markers might not induce an immune response.
  • a "subject suspected of having cancer” refers to a subject that presents one or more symptoms indicative of a cancer (e.g., a detectable lump or mass).
  • a subject suspected of having cancer has generally not been tested for cancer.
  • a subject suspected of having cancer may encompass one who has received an initial diagnosis (e.g., a CT scan or X-ray showing a mass) but for whom the type or stage of cancer is not known.
  • a subject suspected of having cancer may also include one who once had cancer (e.g., individuals in remission).
  • a subject suspected of having cancer may also be a subject at risk for cancer.
  • a "subject at risk for cancer” refers to a subject with one or more risk factors for developing a specific cancer. Risk factors include genetic predisposition, environmental exposure, preexisting non-cancer diseases, previous cancers, and lifestyle.
  • a "subject” is used interchangeably with “patient” and refers to a mammal such as a human.
  • stage of cancer refers to the level of advancement of a given cancer as is recognized by those skilled in the art. Criteria used to determine the stage of a cancer include, but are not limited to, the size of the tumor, whether the tumor has spread to other parts of the body and where the cancer has spread (e.g., within the same organ or region of the body or to another organ).
  • detecting the presence of cancer in a subject refers to
  • a "subject diagnosed with a cancer” refers to a subject having cancerous cells.
  • the cancer may be diagnosed using any suitable method, including but not limited to, the diagnostic methods of the present invention.
  • isolated when used in relation to a nucleic acid molecule or a peptide or polypeptide refers to the given biomolecule that is separated from at least one component or contaminant with which it is ordinarily associated in nature.
  • a “purified” composition refers to the removal of components
  • sample is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases.
  • Biological samples include blood products, such as plasma, serum and the like.
  • capture reagent refers to a molecule which is used to
  • the capture reagent may be immobilized on a substrate.
  • the capture reagent may be an antibody which specifically binds the antigen and if the analyte of interest is an antibody, then the capture reagent may be an epitope which the antibody specifically binds.
  • Biotinylated miRNA were products of Bio-Synthesis Inc. and GenScript USA Inc. Conjugation of miRNA to xMAP Microspheres was conducted according to the manufacturer's recommendations (Luminex Corporation). As a result, xMAP Microsphere region 050 was conjugated with human albumin (Central Laboratory Blood Transfusion Service Swiss Red Cross) or randomized miRNA, region 060 with miR-21, region 070 with miR-191, and region 080 with miR-17. As a positive control, xMAP region 012 was conjugated with a long NY-ESO-1 peptide epitope containing amino acid residues 1-75.
  • human albumin Central Laboratory Blood Transfusion Service Swiss Red Cross
  • xMAP region 012 was conjugated with a long NY-ESO-1 peptide epitope containing amino acid residues 1-75.
  • MagPlex region 015 was conjugated with mixed NY-ESO-1 peptide epitopes containing 7 overlapping regions from amino acid residues 1-180 for binding to a commercially- available mouse anti-human NY-ESO-1 monoclonal Ab clone E978 (Upstate/EMD Millipore, Billerica, MA, USA).
  • MagPlex-based Ab assay was conducted using 96-well clear, flat bottom plates (Bio-Rad Laboratories, Hercules, CA, USA). The SeroMAP plate was washed using a manual 96-well vacuum- filtration system and the MagPlex plate was washed using the automated Bio-Rad magnetic plate washer. The plate was read using a Bioplex-100 instrument (Bio-Rad Laboratories) to measure the median fluorescence intensity (MFI) for each xMAP region included in the multiplex assay. Human albumin or control miRNA conjugated to an xMAP bead was used in each multiplex assay to measure its nonspecific MFI.
  • MFI median fluorescence intensity
  • Relative fiuorescence intensity (RFI) for each xMAP region was calculated using the MFI of a miRNA xMAP region over non-specific fluorescence and graphically displayed as a dot plot or bar chart.
  • RFI Relative fiuorescence intensity
  • the assays according to the present invention employ MagPlex materials, e.g., MagPlex beads.
  • Binding Kinetics of Seropositive Plasma AutoAb to miR-191 [59] Binding Kinetics of Seropositive Plasma AutoAb to miR-191 [60] BLItz interferometer instrument and software (ForteBio, Menlo Park, CA,
  • Streptavidin-coated sensor tips (ForteBio) were used to bind biotinylated miR-191 (20 ⁇ g/mL) for 200 seconds. A wash step for 30 or 250 seconds was used to remove unbound ligand from the sensor and establish the baseline prior to binding.
  • Seropositive plasma from a lung cancer patient was serially diluted from 18.6 ng/ ⁇ , to 1.55 pg/ ⁇ (proposed autoAb concentration based on the assumption that approximately 0.1% of human plasma is comprised of the specific IgG).
  • the autoAb (analyte) was allowed to bind to the biotinylated miR-191 or biotinylated miR-17 immobilized by the streptavidin-coated sensor tip for 250 seconds to load the tip.
  • the PBS buffer used as a diluent was added to the sensor tip for the dissociation of the analyte from the biotinylated miR-191 or biotinylated miR-17 immobilized to the streptavidin sensor tip for 250 seconds.
  • Disposable sensor tips were hydrated in PBS for a minimum of 10 minutes immediately prior to use per manufacturer recommendations. Each step was performed with a shaker speed of 2200 rpm.
  • Human PBMC were obtained from donors of the UCLA virology core under an IRB-approved protocol.
  • CD14 + monocytes were positively selected from fresh PBMC using a magnetic beads-based approach (Miltenyi Biotech Inc., Germany), followed by tissue culture in the presence of human GM-CSF and IL-4 as previously described (28).
  • DC was subjected to treatment with the addition of miRNA (6 ⁇ g/ml or otherwise indicated), polyLC (50-100 ⁇ g/ml unless otherwise indicated), or LPS (100 ng/ml).
  • Routine flow cytometry procedures (28) were performed for staining with cell surface markers such as CD40, CD54, CD80, CD83, CD86, HLA-DR, B7-H1 (PD-L1), B7-H2, and B7-H4 using DC (5xl0 5 /well) that were treated with miRNA, polyLC, or LPS for 48 hours.
  • cell surface markers such as CD40, CD54, CD80, CD83, CD86, HLA-DR, B7-H1 (PD-L1), B7-H2, and B7-H4 using DC (5xl0 5 /well) that were treated with miRNA, polyLC, or LPS for 48 hours.
  • the culture supernatant were collected and analyzed for cytokine/chemokine secretion (EVE Technologies, Calgary, Canada; Biolegend, San Diego, CA, USA). Analysis of protein phosphorylation was performed using lysate from 7xl0 5 DC that were treated with miRNA and polyLC for about 90 minutes.
  • mice of about 8 weeks old were used for immunization with a mixture of 4 ⁇ g of biotinylated miR-17 bound with streptavidin magnetic beads (Thermo Scientific Pierce, Rockford, IL, USA) in filter sterile PBS with 0.01% Tween-20 by intraperitoneal injection. Two weeks later, mice were boosted with the same amount of miRNA mixture.
  • mice Two weeks following, mice were bled and serum- derived Ab against each miRNA and non-specific albumin protein binding for normalization was measured using MagPlex-based approach (Luminex Corp.) with PE-conjugated goat-anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) as the detection Ab.
  • MagPlex-based approach Luminex Corp.
  • PE-conjugated goat-anti-mouse IgG Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA
  • mice were immunized with an miRNA, i.e., miR-17, and their serum was measured for antibody reactive against the miRNA.
  • miRNA small antigens
  • increasing the proximity and number of accessible antigen or miRNA for binding to immune cells is vital for successful immunization. Therefore, biotinylated miR-17 (Bio-Synthesis or GenScript) bound to streptavidin-coated magnetic beads (Thermo Scientific Pierce) in PBS with 0.01% Tween-20 was administered to 3 Balb/c female mice in efforts to generate an immunoglobulin-specific response to miR-17.
  • Tayapiwatana et al. have conducted immunization of mice using streptavidin magnetic beads for monoclonal antibody production previously (2006) (51).
  • mice serum were collected after boosting with miR-17 and stored at -20°C.
  • Serum was diluted 1 :10, 1 :50, and 1 :250 in PBS with 0.01% Tween-20 for measuring antibody reactive to the miRNA.
  • Mouse #1 serum diluted 1 : 10, 1 :50, and 1 :250 were found reactive to miR-17 (black bars) above the 99% confidence level. Na ' ive mice did not have such Ab responses (black bars); nor did the immunized mouse recognize miR-Control ( Figure 5A, white bars).
  • a positive result must include a mean fluorescence intensity (MFI) cut-off of 50 or above in the raw data to eliminate background binding and false positives.
  • MFI mean fluorescence intensity
  • the data shown in Figures 5A-5B are RFIs or MFIs of antibody binding to the respective miRNA shown in the legend, normalized by albumin for non-specific MFI background.
  • two of three dilutions of serum must be positive for antibody to the miRNA.
  • antibody detected for binding to miR-17 was found in at least two of three dilutions (1 : 10, 1 :50, Figure 5A).
  • mouse #1 was detected with relatively increased antibody binding to miR-17 ligand using BLItz interferometry (Figure 6A) compared to non-immunized, naive Balb/c mouse serum ( Figures 5 A and 6B) at estimated antibody concentrations between 0.1 nM-10.3 nM. Approximately three times greater binding of antibody to miR-17 was detected in seropositive mouse serum ( Figure 6A, about 1.5 nm shift) versus the na ' ive mouse serum ( Figure 6B, about 0.5 nm binding shift). As mentioned previously, binding above 1 nm indicates a significant binding interaction.
  • Luminex xMAP -based Ab detection approach (Figure IB) was used to measure Ab response in serum samples from 30 prostate cancer patients and the same number of healthy male controls, as well as plasma samples from 27 NSCLC patients and 15 sex- and age- matched healthy controls.
  • NSCLC patient plasma and 0 of 15 (0%) healthy donor plasma samples ( Figure 1C2), calculated with 99% confidence level (bars) or higher.
  • detection of autoantibody against miR-21 was found in 2 out of 27 (about 7%) NSCLC patient plasma, and r 0 of 15 (0%) healthy donor plasma samples (Figure 1D2), calculated with 99% confidence level (bars).
  • autoAb against miR-191 was detected in 3 out of 27 (11%) NSCLC patient plasma, and 0 of 15 (0%) healthy donor plasma samples (Figure 1E2), calculated with 99% confidence level (bars) or higher.
  • Binding kinetics is commonly measured using purified samples in large amounts using the widely accepted surface plasmon resonance (SPR)-based technique with instruments such as Biacore.
  • SPR surface plasmon resonance
  • Biacore surface plasmon resonance
  • unpurified, complex mixtures such as serum and plasma pose a challenge and further complicated by the low abundance of autoAb specific to a TAA. Therefore, the BLItz interferometer was an attractive alternative to Biacore to accomplish binding kinetics of an impure and low abundant molecule given its much greater sensitivity allowing for an extraordinarily reduced amount of sample required.
  • the BLItz instrument has capabilities to measure complex samples, including cell lysate.
  • the BLItz interferometer is a recently released singleplex instrument that measures one sample at a time, a smaller derivative of the more robust performing Octet instrument (ForteBio) for octoplexing samples.
  • the principle of the Octet instrumentation and BioLayer Interferometry (BLI) is described recently (49). Therefore, the data presented ( Figures 2A-2D and 6A-6C) using the newly released BLItz instrument (ForteBio) described here is the first report that we know of to date.
  • the y-axis represents the relative intensity in the wavelength shifted (nm) as a result of the increased number of analyte molecules (here, autoAb reactive to miR-191) bound to the biosensor tip's surface coated with ligand (here, streptavidin bound to biotinylated miR-191) as a function of time in seconds ( Figures 2A-2D).
  • the biosensor tip has an optic fiber that directs white light back to the opposite end of the tip where the detector is located.
  • Two beams of light are detected, one from the tip as a reference (a constant), and the second from the ligand or analyte molecular layer bound to the tip surface (varies as a function of the number of molecules bound).
  • the difference in the optical properties detected is a function of the thickness in the molecular layer formed and corresponds to the number of molecules binding on the sensor tip surface. This real-time measurement of binding was used to calculate the association and dissociation rates, which can be used to determine the dissociation constant K D in Equation 1 (49):
  • concentrations used to bind NSCLC autoAb 1.493 nM and 14.93 nM.
  • concentrations used to measure non-specific interactions is important when confirming this possible phenomenon of non-specific molecular interactions, normally seen at high concentrations.
  • HSA human serum albumin
  • the most abundant protein found in human blood plasma is human serum albumin (HSA), a soluble, monomeric, 67 kDa protein.
  • the range of albumin concentration in blood is 3.4-5.4 g/dL, equivalent to 34-54 g/L and 507-806 ⁇ (undiluted).
  • the NSCLC patient plasma was diluted 1 :3 (41.3 nM autoAb), then 1 :4 (10.33 nM autoAb), 1 : 10 (1.03 nM), and so forth. Therefore, the highest concentration range of albumin expected in the NSCLC patient plasma when diluted to 1.03 nM autoAb (1 : 120 total serial dilution) used for binding miR-191 was 6.72 ⁇ HSA.
  • the concentration of albumin present in the NSCLC patient plasma when diluted to 1.03 pM (0.00103 nM, a total serial dilution of 1 : 111,120) would have been equivalent to the approximate range of 4.56-7.25 nM human serum albumin.
  • the binding (nm) measured for 1.03 pM autoAb ( Figure 2A) is equivalent to contain between 1-10 nM human serum albumin. Therefore, the binding intensity (nm) of BSA between 1-10 nM ( Figure 2B) should be lower than the binding intensity for 1.03 pM autoAb ( Figure 2A). Otherwise, it is possible that the human serum albumin present in NSCLC plasma binds to miR-191 instead or in addition to autoAb present. However, nonspecific protein binding exhibits several fold orders in magnitude lower binding affinities than antibodies. It is doubtful that BSA would attribute a binding affinity greater than or similar to antibodies or autoAb in the ⁇ to pM range.
  • HSA human serum albumin
  • HSA protein at various concentrations that reflect the expected levels of HSA estimated in the dilutions used for human blood measurements by BLItz
  • HSA protein concentrations at 150 nM, 1.5 ⁇ , and 7.5 ⁇ were used to represent the equivalent HSA protein concentration in human plasma when diluted to roughly 1 :3,000, 1 :300, and 1 : 100 final dilutions, and roughly equivalent to 0.01 nM autoAb, 0.1 nM autoAb, 1.0 nM autoAb, respectively.
  • the estimated binding of 1.0 nM autoAb from NSCLC patient plasma to miRNA was nearly 1.5 nm in Figure 2A compared to nearly 0.4 nm by HSA in Figure 2D, suggesting that HSA non-specific binding to miR A does not attribute to the level of that observed with increased binding to miRNA in NSCLC patient plasma.
  • the binding affinity was estimated by BLItz software for HSA binding (Figure 2D) as 5 mM, significantly lower binding affinity than the autoAb estimated pM affinities ( Figure 2A) measured here by an estimated 9 orders in magnitude difference. Therefore, it is unlikely that non-specific proteins present in human plasma resulted in the observed binding to miRNA at the affinities measured, other than by an Ab.
  • the present invention is directed to methods for
  • the miRNAs are selected from the group consisting of miR-17, miR-21 and miR-191.
  • the subject is mammalian, preferably human.
  • the subject is suspected of having cancer.
  • the subject is at risk for cancer.
  • the cancer is selected from the group consisting of lung cancers (such as NSCLC), glioblastomas, breast cancer, hepatocellular cancers, gastric cancers, prostate cancer, and colon cancers.
  • one or more antibodies that specifically bind the miRNAs are used as the capture reagent.
  • the miRNAs are indirectly detected by detecting one or more autoAbs against the miRNAs.
  • the one or more autoAbs are detected using, as the capture reagent, one or more antibodies that specifically bind the autoAbs.
  • the one or more autoAbs are detected using, as the capture reagent, one or more nucleic acid molecules (e.g. synthetically made oligonucleotides having the same or substantially the same sequence(s) as the one or more miRNAs) to which the autoAbs specifically bind.
  • the capture reagents may be immobilized on a substrate, e.g. assay plate or well.
  • the capture reagents are provided in one or more assay panels which comprise a plurality of additional capture reagents for other biomarkers such as tumor antigens and tumor markers. See e.g., US 20110311998, which is herein incorporated by reference in its entirety.
  • the presence of a particular miRNA or a particular combination of miRNAs, alone or in combination with one or more other tumor antigens and/or tumor markers is used to characterize the type of cancer and/or the stage of the cancer in the subject.
  • compositions of the present invention are combined with other assays to form multiplex assays.
  • the methods and/or compositions of the present invention may be combined with that described in US 20110311998, which is herein incorporated by reference in its entirety.
  • NY-ESO-1 recognized by sera from a wide spectrum of cancer patients: implications as a potential biomarker. Int J Cancer 2005; 114: 268-73.
  • microspheres provide superior performance to non-magnetic beads in prostate cancer assays.
  • White paper Luminex Corporation.

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Abstract

L'invention concerne des anticorps et des auto-anticorps qui lient spécifiquement des miARN associés à un cancer, des procédés de test et des procédés d'immunisation de sujets contre des miARN.
PCT/US2013/076193 2012-12-19 2013-12-18 Microarn associés au cancer et anticorps associés Ceased WO2014100216A1 (fr)

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WO2018191645A1 (fr) * 2017-04-14 2018-10-18 The Regents Of The University Of California Procédés et dispositifs pour réaliser des dosages d'auto-anticorps
CN112251411A (zh) * 2020-09-24 2021-01-22 吉林大学 一种miR-17-92修饰的间充质干细胞、外泌体及其制备方法和应用
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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2018191645A1 (fr) * 2017-04-14 2018-10-18 The Regents Of The University Of California Procédés et dispositifs pour réaliser des dosages d'auto-anticorps
US20230314417A1 (en) * 2020-03-23 2023-10-05 Amgen Inc. Monoclonal antibodies to chemically-modified nucleic acids and uses thereof
CN112251411A (zh) * 2020-09-24 2021-01-22 吉林大学 一种miR-17-92修饰的间充质干细胞、外泌体及其制备方法和应用
CN112251411B (zh) * 2020-09-24 2024-02-13 吉林大学 一种miR-17-92修饰的间充质干细胞、外泌体及其制备方法和应用

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