Method for the enrichment of cell free nucleosomes

Abstract

The present invention relates to the use of a histone H1 binding agent for detecting, isolating and/or purifying cell free nucleosomes of tumor origin from a biological sample. The invention also describes methods of negative or positive selection using histone H1 binding agents, in order to enrich a sample for cell free nucleosomes of tumor origin.

It is claimed: 
     
         1 . A method of treating cancer in an animal or a human subject, which comprises the following steps:
 (i) contacting a biological sample obtained from the subject with a histone H1 binding agent;   (ii) separating the nucleosome fractions which are not bound to the histone H1 binding agent and isolating the unbound fraction;   (iii) contacting the isolated nucleosomes obtained in step (ii) with a second binding agent which binds to an epitope of tumor derived nucleosomes;   (iv) detecting and/or measuring the level of binding of said second binding agent to said epitope;   (v) using the measured level of biomarker(s) in step (iv) as indicative of the presence of cancer in the subject; and   (vi) treating surgically or administering a therapeutic agent to a subject diagnosed in step (v) as a subject having cancer.   
     
     
         2 . The method according to  claim 1 , wherein the cancer is selected from: breast, bladder, colorectal, skin, ovarian, prostate, lung, pancreatic, bowel, liver, endometrial, lymphoma, oral, head and neck cancer, leukaemia and osteosarcoma. 
     
     
         3 . The method according to  claim 1 , wherein the histone H1 binding agent is selected from: a binding agent that binds to histone H1 protein, variant, isoform or modification thereof. 
     
     
         4 . The method of  claim 1 , wherein the histone H1 binding agent is selected from: a binding agent that binds to histone H1 protein or a histone H1 variant selected from H1.0, H1.10 or H1.X. 
     
     
         5 . The method according to  claim 1 , wherein the epitope comprises a histone modification. 
     
     
         6 . The method according to  claim 5 , wherein the histone modification comprises H3K27Ac and/or 5-methylcytosine. 
     
     
         7 . The method according to  claim 1 , wherein the epitope comprises a modified nucleotide. 
     
     
         8 . The method according to  claim 1 , wherein the epitope comprises a histone H2A, H2B, H3 or H4 isoform. 
     
     
         9 . The method according to  claim 1 , wherein the epitope comprises a nucleosome adduct or variant thereof. 
     
     
         10 . The method according to  claim 1 , wherein the biological sample comprises blood, serum, plasma, menstrual blood, cerebrospinal fluid (CSF), endometrial fluid, urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, condensed breath, or an extract or purification therefrom, or dilution thereof. 
     
     
         11 . The method of  claim 10 , wherein the biological sample comprises a blood, serum or plasma sample. 
     
     
         12 . A method for measuring the proportion of circulating cell free nucleosomes containing an epigenetic epitope in a biological sample taken from a subject that are of tumor origin, comprising the steps of;
 (i) obtaining a biological sample from a subject,   (ii) performing a first immunoassay comprising the steps of;
 (1A) contacting the sample with a first binding agent which binds to said epigenetic epitope; 
 (1B) contacting the nucleosomes or sample with a second binding agent which binds to histone H1; and 
 (1C) detecting or quantifying the binding of said second binding agent to the nucleosome in the sample; 
   (iii) performing a second immunoassay comprising the steps of;
 (2A) contacting the sample with the first binding agent which binds to said epigenetic epitope; 
 (2B) contacting the nucleosomes or sample with a third binding agent which binds to a non-histone H1 nucleosome or chromatin fragment epitope; and 
 (2C) detecting or quantifying the binding of said third binding agent to the nucleosome in the sample; 
   (iv) combining the measurements of steps (1C) and (2C) as an indicator of the amount or proportion of cell free nucleosomes in the sample that contain said epigenetic epitope and that are of tumor origin.   
     
     
         13 . The method according to  claim 12 , wherein the epigenetic epitope bound by the first binding agent is selected from: (i) an isoform of histone H2A, H2B, H3 or H4, (ii) a post translational modification present in histone H2A, H2B, H3 or H4, (iii) a nucleotide including a modified nucleotide, (iv) a non-histone protein incorporated in or adducted to a cell free chromatin fragment. 
     
     
         14 . The method according to  claim 12 , wherein the second binding agent is selected from: a binding agent that binds to histone H1 protein, variant, isoform or modification thereof. 
     
     
         15 . A method for measuring the proportion of circulating cell free nucleosomes containing an epigenetic epitope in a biological sample taken from a subject that are of tumor origin, comprising the steps of;
 (i) obtaining a biological sample from a subject,   (ii) performing a first immunoassay comprising the steps of;
 (1A) contacting the sample with a first binding agent which binds to histone H1; 
 (1B) contacting the nucleosomes or sample with a second binding agent which binds to said epigenetic epitope; and 
 (1C) detecting or quantifying the binding of said second binding agent to the nucleosome in the sample; 
   (iii) performing a second immunoassay comprising the steps of;
 (2A) contacting the sample with a third binding agent which binds to a non-histone H1 nucleosome or chromatin fragment epitope; 
 (2B) contacting the nucleosomes or sample with the second binding agent which binds to said epigenetic epitope; and 
 (2C) detecting or quantifying the binding of said second binding agent to the nucleosome in the sample; 
   (iv) combining the measurements of steps (1C) and (2C) as an indicator of the amount or proportion of cell free nucleosomes in the sample that contain said epigenetic epitope and that are of tumor origin.   
     
     
         16 . The method according to  claim 15 , wherein the epigenetic epitope bound by the first binding agent is selected from: (i) an isoform of histone H2A, H2B, H3 or H4, (ii) a post translational modification present in histone H2A, H2B, H3 or H4, (iii) a nucleotide including a modified nucleotide, (iv) a non-histone protein incorporated in or adducted to a cell free chromatin fragment. 
     
     
         17 . The method according to  claim 15 , wherein the second binding agent is selected from: a binding agent that binds to histone H1 protein, variant, isoform or modification thereof.