Patent Application
20210230702
The invention relates to detecting chromosome interactions.
Disease processes are complex and outcomes cannot be predicted using available methods. In particular it is difficult to predict how patients will react to specific therapies.
Specific Chromosome Conformation Signatures (CCSs) at loci either exist or are absent due to the regulatory epigenetic control settings associated with pathology or treatment. CCSs have mild off-rates and when representing a particular phenotype or pathology, they will only change with a physiologically signalled transition to a new phenotype, or as a result of external intervention. In addition, the measurement of these events is binary, and so this read-out is in stark contrast to the continuum readout of varying levels of DNA methylation, histone modifications and most of the non-coding RNAs. The continuum read-out used for most molecular biomarkers to date offers a challenge to data analysis, in that the magnitude of change for particular biomarkers varies greatly from patient to patient, which causes problems for classification statistics when they are used to stratify cohorts of patients. These classification statistics are better-suited to using biomarkers that are absent of magnitude and offer just a “yes or no” binary score of phenotypic differences—signifying that chromosome conformation (EpiSwitch™) biomarkers are an excellent resource for potential diagnostic, prognostic and predictive biomarkers.
The inventors have identified regions of the genome with chromosomal interactions relevant to immunoresponsiveness using an approach which allows identification of subgroups in a population. The identified regions, genes and specific chromosome interactions from two separate studies using different therapies to treat two different conditions have been found to determine general immunoresponsiveness in a patient, including through the immune response regulating cell surface signalling pathway and regulation of T cell activation. The inventors' work allows changes in immunoresponsiveness to be followed, for example during the course of disease or therapy.
Accordingly, the invention provides a process for detecting a chromosome state which represents a subgroup in a population comprising determining whether a chromosome interaction relating to that chromosome state is present or absent within a defined region of the genome, wherein said subgroup relates to how immunoresponsive individuals are; and
The invention also provides a process for detecting a chromosome state which represents a subgroup in a population comprising determining whether a chromosome interaction relating to that chromosome state is present or absent within a defined region of the genome, wherein said subgroup relates to how immunoresponsive individuals are; and
The invention further provides a process for detecting a chromosome state which represents a subgroup in a population comprising determining whether a chromosome interaction relating to that chromosome state is present or absent within a defined region of the genome, wherein said subgroup relates to how immunoresponsive individuals are; and
The inventions concerns a panel of epigenetic markers which relate to the regulation of the immune system, in particular via cell surface signalling pathways and T cell activation.
The invention also includes monitoring the state of the immune system to determine its responsiveness to particular therapies. That means appropriate therapies can be given to a patient, and whether or not the patient is retaining or losing ‘responder’ status can be determined. The invention therefore provides in one embodiment a ‘live’ ongoing readout of ‘responder’ status allowing a personalised therapy to be given to the patient which accurately reflects the patient's needs.
The invention relates to determining immunoresponsiveness. This is preferably responsiveness to a therapy which comprises a molecule or cell that is relevant to the immune system, such as administration of a composition that comprises an antibody or immune cell (for example a T cell or dendritic cell) or administration of any therapeutic substance mentioned herein. It may be responsiveness to a substance that modulates or stimulates the immune system, such as a vaccine therapy. The immunoresponsiveness is thus preferably the responsiveness to an immunotherapy. The immunotherapy may modulate, block or stimulate an immune checkpoint, and thus may target or modulate PD-L1, PD-L2 or CTLA4 or any other immune checkpoint molecule disclosed herein, and thus may be an immunocheckpoint therapy. Preferably the immunoresponsiveness is responsiveness to an antibody therapy, or to any specific therapy disclosed herein (see specific drugs in later sections). The therapy may be a combination therapy.
In one embodiment immunoresponsiveness to responsiveness to a PD-1 inhibitor or PD-L1 inhibitor, including an antibody specific for PD-1 or PD-L1. PD-1 is ‘programmed cell death protein’ and PD-L1 is ‘programmed death-ligand 1’.
The immunoresponsiveness is preferably to a cancer therapy, and so typically is relevant to whether a specific individual is responsive to the therapy, where the individual may or may not have cancer, or may be at risk of cancer. The cancer is typically any cancer mentioned herein, and for example is melanoma, lung cancer, non-small cell lung carcinoma (NSCLC), diffuse large B-cell lymphoma, liver cancer, hepatocellular carcinoma, prostate cancer, breast cancer, leukaemia, acute myeloid leukaemia, pancreatic cancer, thyroid cancer, nasal cancer, brain cancer, bladder cancer, cervical cancer, non-Hodgkin lymphoma, ovarian cancer, colorectal cancer or kidney cancer.
The term ‘antibody’ includes all fragments and derivatives of an antibody that retain the ability to bind the antigen target, for example single chain scFV's or Fab's.
As will be discussed later immunoresponsiveness can be determined for any therapy, cell or drug which is mentioned herein. In some embodiments any therapy, cell or drug that is mentioned herein may be administered to individuals whose immunoresponsiveness has been determined.
The process of the invention comprises a typing system for detecting chromosome interactions relevant to immunoresponsiveness. This typing may be performed using the EpiSwitch™ system mentioned herein which is based on cross-linking regions of chromosome which have come together in the chromosome interaction, subjecting the chromosomal DNA to cleavage and then ligating the nucleic acids present in the cross-linked entity to derive a ligated nucleic acid with sequence from both the regions which formed the chromosomal interaction. Detection of this ligated nucleic acid allows determination of the presence or absence of a particular chromosome interaction.
The chromosomal interactions may be identified using the above described method in which populations of first and second nucleic acids are used. These nucleic acids can also be generated using EpiSwitch™ technology.
As used herein, the term ‘epigenetic’ and ‘chromosome’ interactions typically refers to interactions between distal regions of a chromosome, said interactions being dynamic and altering, forming or breaking depending upon the status of the region of the chromosome.
In particular processes of the invention chromosome interactions are typically detected by first generating a ligated nucleic acid that comprises sequence from both regions of the chromosomes that are part of the interactions. In such processes the regions can be cross-linked by any suitable means. In a preferred embodiment, the interactions are cross-linked using formaldehyde, but may also be cross-linked by any aldehyde, or D-Biotinoyl-e-aminocaproic acid-N-hydroxysuccinimide ester or Digoxigenin-3-O-methylcarbonyl-e-aminocaproic acid-N-hydroxysuccinimide ester. Para-formaldehyde can cross link DNA chains which are 4 Angstroms apart. Preferably the chromosome interactions are on the same chromosome and optionally 2 to 10 Angstroms apart.
The chromosome interaction may reflect the status of the region of the chromosome, for example, if it is being transcribed or repressed in response to change of the physiological conditions. Chromosome interactions which are specific to subgroups as defined herein have been found to be stable, thus providing a reliable means of measuring the differences between the two subgroups.
In addition, chromosome interactions specific to a characteristic (such as immunoresponsiveness) will normally occur early in a biological process, for example compared to other epigenetic markers such as methylation or changes to binding of histone proteins. Thus the process of the invention is able to detect early stages of a biological process. This allows early intervention (for example treatment) which may as a consequence be more effective. Chromosome interactions also reflect the current state of the individual and therefore can be used to assess changes to immunoresponsiveness. Furthermore there is little variation in the relevant chromosome interactions between individuals within the same subgroup. Detecting chromosome interactions is highly informative with up to 50 different possible interactions per gene, and so processes of the invention can interrogate 500,000 different interactions.
Herein the term ‘marker’ or ‘biomarker’ refers to a specific chromosome interaction which can be detected (typed) in the invention. Specific markers are disclosed herein, any of which may be used in the invention. Further sets of markers may be used, for example in the combinations or numbers disclosed herein. The specific markers disclosed in the tables herein are preferred as well as markers presents in genes and regions mentioned in the tables herein are preferred. These may be typed by any suitable method, for example the PCR or probe based methods disclosed herein, including a qPCR method. The markers are defined herein by location or by probe and/or primer sequences.
Epigenetic chromosomal interactions may overlap and include the regions of chromosomes shown to encode relevant or undescribed genes, but equally may be in intergenic regions. It should further be noted that the inventors have discovered that epigenetic interactions in all regions are equally important in determining the status of the chromosomal locus. These interactions are not necessarily in the coding region of a particular gene located at the locus and may be in intergenic regions.
The chromosome interactions which are detected in the invention could be caused by changes to the underlying DNA sequence, by environmental factors, DNA methylation, non-coding antisense RNA transcripts, non-mutagenic carcinogens, histone modifications, chromatin remodelling and specific local DNA interactions. The changes which lead to the chromosome interactions may be caused by changes to the underlying nucleic acid sequence, which themselves do not directly affect a gene product or the mode of gene expression. Such changes may be for example, SNPs within and/or outside of the genes, gene fusions and/or deletions of intergenic DNA, microRNA, and non-coding RNA. For example, it is known that roughly 20% of SNPs are in non-coding regions, and therefore the process as described is also informative in non-coding situation. In one embodiment the regions of the chromosome which come together to form the interaction are less than 5 kb, 3 kb, 1 kb, 500 base pairs or 200 base pairs apart on the same chromosome.
The chromosome interaction which is detected is preferably within any of the genes mentioned in Table 1. However it may also be upstream or downstream of the gene, for example up to 50,000, up to 30,000, up to 20,000, up to 10,000 or up to 5000 bases upstream or downstream from the gene or from the coding sequence.
The chromosome interaction which is detected is preferably within any of the genes mentioned in Table 13. However it may also be upstream or downstream of the gene, for example up to 50,000, up to 30,000, up to 20,000, up to 10,000 or up to 5000 bases upstream or downstream from the gene or from the coding sequence.
The chromosome interaction which is detected is preferably within any of the genes mentioned in Table 16. However it may also be upstream or downstream of the gene, for example up to 50,000, up to 30,000, up to 20,000, up to 10,000 or up to 5000 bases upstream or downstream from the gene or from the coding sequence.
The aim of the present invention is to determine the level of immunoresponsiveness. This may be at one or more defined time points, for example at at least 1, 2, 5, 8 or 10 different time points. The durations between at least 1, 2, 5 or 8 of the time points may be at least 5, 10, 20, 50, 80 or 100 days. Typically at least 1, 2 or 5 time points are before therapy begins and/or at least 1, 2 or 5 time points are after the beginning of therapy.
As used herein, a “subgroup” preferably refers to a population subgroup (a subgroup in a population), more preferably a subgroup in the population of a particular animal such as a particular eukaryote, or mammal (e.g. human, non-human, non-human primate, or rodent e.g. mouse or rat). Most preferably, a “subgroup” refers to a subgroup in the human population.
The invention includes detecting and treating particular subgroups in a population. The inventors have discovered that chromosome interactions differ between subsets (for example at least two subsets) in a given population. Identifying these differences will allow physicians to categorize their patients as a part of one subset of the population as described in the process. The invention therefore provides physicians with a process of personalizing medicine for the patient based on their epigenetic chromosome interactions.
In one embodiment the invention relates to testing whether an individual is a ‘responder’. Once a person is found to be a ‘responder’ they can be given the relevant therapy which is typically a therapy that targets an immune checkpoint molecule such as PD-1, PD-L1 or CTLA4. In one embodiment if an individual is found to be a non-responder then they will be given a combination therapy, such as any combination therapy listed herein. Typically a combination therapy comprises an antibody and a small molecule.
Certain embodiments of the invention utilise ligated nucleic acids, in particular ligated DNA. These comprise sequences from both of the regions that come together in a chromosome interaction and therefore provide information about the interaction. The EpiSwitch™ method described herein uses generation of such ligated nucleic acids to detect chromosome interactions.
Thus a process of the invention may comprise a step of generating ligated nucleic acids (e.g. DNA) by the following steps (including a method comprising these steps):
(i) cross-linking of epigenetic chromosomal interactions present at the chromosomal locus, preferably in vitro;
(ii) optionally isolating the cross-linked DNA from said chromosomal locus;
(iii) subjecting said cross-linked DNA to cutting, for example by restriction digestion with an enzyme that cuts it at least once (in particular an enzyme that cuts at least once within said chromosomal locus);
(iv) ligating said cross-linked cleaved DNA ends (in particular to form DNA loops); and
(v) optionally identifying the presence of said ligated DNA and/or said DNA loops, in particular using techniques such as PCR (polymerase chain reaction), to identify the presence of a specific chromosomal interaction.
These steps may be carried out to detect the chromosome interactions for any embodiment mentioned herein. The steps may also be carried out to generate the first and/or second set of nucleic acids mentioned herein.
PCR (polymerase chain reaction) may be used to detect or identify the ligated nucleic acid, for example the size of the PCR product produced may be indicative of the specific chromosome interaction which is present, and may therefore be used to identify the status of the locus. In preferred embodiments at least 1, 2 or 3 primers or primer pairs as shown in Table 4 are used in the PCR reaction. In other embodiments at least 1, 2 or 3 primers or primer pairs as shown in Table 13 are used in the PCR reaction. In other embodiments at least 1, 2 or 3 primers or primer pairs as shown in Table 17 are used in the PCR reaction. The skilled person will be aware of numerous restriction enzymes which can be used to cut the DNA within the chromosomal locus of interest. It will be apparent that the particular enzyme used will depend upon the locus studied and the sequence of the DNA located therein. A non-limiting example of a restriction enzyme which can be used to cut the DNA as described in the present invention is Taql.
The EpiSwitch™ Technology also relates to the use of microarray EpiSwitch™ marker data in the detection of epigenetic chromosome conformation signatures specific for phenotypes. Embodiments such as EpiSwitch™ which utilise ligated nucleic acids in the manner described herein have several advantages. They have a low level of stochastic noise, for example because the nucleic acid sequences from the first set of nucleic acids of the present invention either hybridise or fail to hybridise with the second set of nucleic acids. This provides a binary result permitting a relatively simple way to measure a complex mechanism at the epigenetic level. EpiSwitch™ technology also has fast processing time and low cost. In one embodiment the processing time is 3 hours to 6 hours.
The process of the invention will normally be carried out on a sample. The sample may be obtained at a defined time point, for example at any time point defined herein. The sample will normally contain DNA from the individual. It will normally contain cells. In one embodiment a sample is obtained by minimally invasive means, and may for example be a blood sample. DNA may be extracted and cut up with a standard restriction enzyme. This can pre-determine which chromosome conformations are retained and will be detected with the EpiSwitch™ platforms. Due to the synchronisation of chromosome interactions between tissues and blood, including horizontal transfer, a blood sample can be used to detect the chromosome interactions in tissues, such as tissues relevant to disease. For certain conditions, such as cancer, genetic noise due to mutations can affect the chromosome interaction ‘signal’ in the relevant tissues and therefore using blood is advantageous.
The invention relates to certain nucleic acids, such as the ligated nucleic acids which are described herein as being used or generated in the process of the invention. These may be the same as, or have any of the properties of, the first and second nucleic acids mentioned herein. The nucleic acids of the invention typically comprise two portions each comprising sequence from one of the two regions of the chromosome which come together in the chromosome interaction. Typically each portion is at least 8, 10, 15, 20, 30 or 40 nucleotides in length, for example 10 to 40 nucleotides in length. Preferred nucleic acids comprise sequence from any of the genes mentioned in any of the tables. Typically preferred nucleic acids comprise the specific probe sequences mentioned in Table 1; or fragments and/or homologues of such sequences. The preferred nucleic acids may comprise the specific probe sequences mentioned in Table 13; or fragments and/or homologues of such sequences. The preferred nucleic acids may comprise the specific probe sequences mentioned in Table 16; or fragments and/or homologues of such sequences.
Preferably the nucleic acids are DNA. It is understood that where a specific sequence is provided the invention may use the complementary sequence as required in the particular embodiment. Preferably the nucleic acids are DNA. It is understood that where a specific sequence is provided the invention may use the complementary sequence as required in the particular embodiment.
The primers shown in Table 4 may also be used in the invention as mentioned herein. In one embodiment primers are used which comprise any of: the sequences shown in Table 4; or fragments and/or homologues of any sequence shown in Table 4. The primers shown in Table 13 may also be used in the invention as mentioned herein. In one embodiment primers are used which comprise any of: the sequences shown in Table 13; or fragments and/or homologues of any sequence shown in Table 13. The primers shown in Table 17 may also be used in the invention as mentioned herein. In one embodiment primers are used which comprise any of: the sequences shown in Table 17; or fragments and/or homologues of any sequence shown in Table 17.
The second set of nucleic acid sequences has the function of being a set of index sequences, and is essentially a set of nucleic acid sequences which are suitable for identifying subgroup specific sequence. They can represents the ‘background’ chromosomal interactions and might be selected in some way or be unselected. They are in general a subset of all possible chromosomal interactions.
The second set of nucleic acids may be derived by any suitable process. They can be derived computationally or they may be based on chromosome interaction in individuals. They typically represent a larger population group than the first set of nucleic acids. In one particular embodiment, the second set of nucleic acids represents all possible epigenetic chromosomal interactions in a specific set of genes. In another particular embodiment, the second set of nucleic acids represents a large proportion of all possible epigenetic chromosomal interactions present in a population described herein. In one particular embodiment, the second set of nucleic acids represents at least 50% or at least 80% of epigenetic chromosomal interactions in at least 20, 50, 100 or 500 genes, for example in 20 to 100 or 50 to 500 genes.
The second set of nucleic acids typically represents at least 100 possible epigenetic chromosome interactions which modify, regulate or in any way mediate a phenotype in population. The second set of nucleic acids may represent chromosome interactions that affect a disease state (typically relevant to diagnosis or prognosis) in a species. The second set of nucleic acids typically comprises sequences representing epigenetic interactions both relevant and not relevant to an immunoresponsiveness subgroup.
In one particular embodiment the second set of nucleic acids derive at least partially from naturally occurring sequences in a population, and are typically obtained by in silico processes. Said nucleic acids may further comprise single or multiple mutations in comparison to a corresponding portion of nucleic acids present in the naturally occurring nucleic acids. Mutations include deletions, substitutions and/or additions of one or more nucleotide base pairs. In one particular embodiment, the second set of nucleic acids may comprise sequence representing a homologue and/or orthologue with at least 70% sequence identity to the corresponding portion of nucleic acids present in the naturally occurring species. In another particular embodiment, at least 80% sequence identity or at least 90% sequence identity to the corresponding portion of nucleic acids present in the naturally occurring species is provided.
In one particular embodiment, there are at least 100 different nucleic acid sequences in the second set of nucleic acids, preferably at least 1000, 2000 or 5000 different nucleic acids sequences, with up to 100,000, 1,000,000 or 10,000,000 different nucleic acid sequences. A typical number would be 100 to 1,000,000, such as 1,000 to 100,000 different nucleic acids sequences. All or at least 90% or at least 50% or these would correspond to different chromosomal interactions.
In one particular embodiment, the second set of nucleic acids represent chromosome interactions in at least 20 different loci or genes, preferably at least 40 different loci or genes, and more preferably at least 100, at least 500, at least 1000 or at least 5000 different loci or genes, such as 100 to 10,000 different loci or genes. The lengths of the second set of nucleic acids are suitable for them to specifically hybridise according to Watson Crick base pairing to the first set of nucleic acids to allow identification of chromosome interactions specific to subgroups. Typically the second set of nucleic acids will comprise two portions corresponding in sequence to the two chromosome regions which come together in the chromosome interaction. The second set of nucleic acids typically comprise nucleic acid sequences which are at least 10, preferably 20, and preferably still 30 bases (nucleotides) in length. In another embodiment, the nucleic acid sequences may be at the most 500, preferably at most 100, and preferably still at most 50 base pairs in length. In a preferred embodiment, the second set of nucleic acids comprises nucleic acid sequences of between 17 and 25 base pairs. In one embodiment at least 100, 80% or 50% of the second set of nucleic acid sequences have lengths as described above. Preferably the different nucleic acids do not have any overlapping sequences, for example at least 100%, 90%, 80% or 50% of the nucleic acids do not have the same sequence over at least 5 contiguous nucleotides.
Given that the second set of nucleic acids acts as an ‘index’ then the same set of second nucleic acids may be used with different sets of first nucleic acids which represent subgroups for different characteristics, i.e. the second set of nucleic acids may represent a ‘universal’ collection of nucleic acids which can be used to identify chromosome interactions relevant to different characteristics.
The first set of nucleic acids are typically from subgroups relevant to immunoresponsiveness. The first nucleic acids may have any of the characteristics and properties of the second set of nucleic acids mentioned herein. The first set of nucleic acids is normally derived from samples from the individuals which have undergone treatment and processing as described herein, particularly the EpiSwitch™ cross-linking and cleaving steps. Typically the first set of nucleic acids represents all or at least 80% or 50% of the chromosome interactions present in the samples taken from the individuals.
Typically, the first set of nucleic acids represents a smaller population of chromosome interactions across the loci or genes represented by the second set of nucleic acids in comparison to the chromosome interactions represented by second set of nucleic acids, i.e. the second set of nucleic acids is representing a background or index set of interactions in a defined set of loci or genes.
Any of the types of nucleic acid populations mentioned herein may be present in the form of a library comprising at least 200, at least 500, at least 1000, at least 5000 or at least 10000 different nucleic acids of that type, such as ‘first’ or ‘second’ nucleic acids. Such a library may be in the form of being bound to an array.
The invention requires a means for allowing wholly or partially complementary nucleic acid sequences from the first set of nucleic acids and the second set of nucleic acids to hybridise. In one embodiment all of the first set of nucleic acids is contacted with all of the second set of nucleic acids in a single assay, i.e. in a single hybridisation step. However any suitable assay can be used.
The nucleic acids mentioned herein may be labelled, preferably using an independent label such as a fluorophore (fluorescent molecule) or radioactive label which assists detection of successful hybridisation. Certain labels can be detected under UV light. The pattern of hybridisation, for example on an array described herein, represents differences in epigenetic chromosome interactions between the two subgroups, and thus provides a process of comparing epigenetic chromosome interactions and determination of which epigenetic chromosome interactions are specific to a subgroup in the population of the present invention.
The term ‘pattern of hybridisation’ broadly covers the presence and absence of hybridisation between the first and second set of nucleic acids, i.e. which specific nucleic acids from the first set hybridise to which specific nucleic acids from the second set, and so it not limited to any particular assay or technique, or the need to have a surface or array on which a ‘pattern’ can be detected.
Selecting a Subgroup with Particular Characteristics
The invention provides a process which comprises detecting the presence or absence of chromosome interactions, typically 5 to 20 or 5 to 500 such interactions, preferably 20 to 300 or 50 to 100 interactions, in order to determine the presence or absence of a characteristic relating to immunoresponsiveness in an individual. Preferably the chromosome interactions are those in any of the genes mentioned herein. In one embodiment the chromosome interactions which are typed are those represented by the nucleic acids in Table 1. In another embodiment the chromosome interactions are those represented in Table 13. In another embodiment the chromosome interactions are those represented in Table 16. The column titled ‘Loop Detected’ in the tables shows which subgroup is detected (i.e. responder or non-responder) by each probe.
The Individual that is Tested
Examples of the species that the individual who is tested is from are mentioned herein. In addition the individual that is tested in the process of the invention may have been selected in some way. The individual may be susceptible to any condition mentioned herein and/or may be in need of any therapy mentioned in. The individual may be receiving any therapy mentioned herein.
In one embodiment the individual that is tested has shown a lack of response to therapy, and the purpose of testing them is to discover whether they are a ‘pseudo-progressor’ that will respond to therapy in the second stage of disease, though they have not responded at an earlier stage.
For all aspects of the invention preferred gene regions, loci, genes and chromosome interactions are mentioned in the tables, for example in Table 1. Typically in the processes of the invention chromosome interactions are detected from at least 1, 2, 10, 50, 100, 150, 200 or 300 of the relevant genes listed in Table 1. Preferably the presence or absence of at least 1, 2, 10, 50, 100, 150, 200 or 300 of the relevant specific chromosome interactions represented by the probe sequences in Table 1 are detected. The chromosome interaction may be upstream or downstream of any of the genes mentioned herein, for example 50 kb upstream or 20 kb downstream, for example from the coding sequence.
In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 1.a are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 1.b are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 1.c are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 1.d are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 1.e are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 1.f are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 1.g are typed.
Typically at least 5, 10, 15, 20, 30, 40 or 70 chromosome interactions are typed from any of genes or regions disclosed the tables herein, or parts of tables disclosed herein. Typically the chromosome interactions which are typed are present in at least 20, 50, 100, 200, 300 or all of the genes mentioned in Table 2. Typically the chromosome interactions which are typed are present in at least 10, 20, 50, 70 or all of the genes mentioned in Table 3.
For all aspects of the invention preferred gene regions, loci, genes and chromosome interactions are mentioned in Table 13. Typically in the processes of the invention chromosome interactions are detected from at least 1, 2, 10, 50, 100, 150, 200 or 300 of the relevant genes listed in Table 13. Preferably the presence or absence of at least 1, 2, 10, 50, 100, 150, 200 or 300 of the relevant specific chromosome interactions represented by the probe sequences in Table 13 are detected. The chromosome interaction may be upstream or downstream of any of the genes mentioned herein, for example 50 kb upstream or 20 kb downstream, for example from the coding sequence.
In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 13.a are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 13.b are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 13.c are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 13.d are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 13.e are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 13.f are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 13.g are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 13.h are typed. In one embodiment at least 5, 10, 15, 20 or all of the chromosome interactions in Table 13.i are typed.
Typically at least 5, 10, 15, 20, 30, 40 or 70 chromosome interactions are typed from any of genes or regions disclosed the tables herein, or parts of tables disclosed herein. Typically the chromosome interactions which are typed are present in at least 20, 50, 100, 200, 300 or all of the genes mentioned in Table 13. Typically the chromosome interactions which are typed are present in at least 10, 20, 50, 70 or all of the genes mentioned in Table 13.
For all aspects of the invention preferred gene regions, loci, genes and chromosome interactions are mentioned in Table 16. Typically in the processes of the invention chromosome interactions are detected from at least 1, 2, 10, 20. 30 or 40 of the relevant genes listed in Table 16. Preferably the presence or absence of at least 1, 2, 10, 20, 30 or 40 of the relevant specific chromosome interactions represented by the probe sequences in Table 16 are detected. The chromosome interaction may be upstream or downstream of any of the genes mentioned herein, for example 50 kb upstream or 20 kb downstream, for example from the coding sequence.
In one embodiment at least 5, 10 or 15 or all of the chromosome interactions in Table 18 are typed.
Typically at least 5, 10, 15, 20, 30, 40 or 70 chromosome interactions are typed from any of genes or regions disclosed the tables herein, or parts of tables disclosed herein. Typically the chromosome interactions which are typed are present in at least 20, 50, 100, 200, 300 or all of the genes mentioned in Table 13. Typically the chromosome interactions which are typed are present in at least 10, 20, 50, 70 or all of the genes mentioned in Table 13.
In one embodiment at least 5, 10, 15, 20, 30, 40 or 70 different chromosome interactions are typed from those defined in any of Tables 1, 13 and 16. In another embodiment at least 50%, 80% or all of the chromosome interactions which are typed are from Tables 1, 13 and 16.
In one embodiment the locus (including the gene and/or place where the chromosome interaction is detected) may comprise a CTCF binding site. This is any sequence capable of binding transcription repressor CTCF. That sequence may consist of or comprise the sequence CCCTC which may be present in 1, 2 or 3 copies at the locus. The CTCF binding site sequence may comprise the sequence CCGCGNGGNGGCAG (in IUPAC notation). The CTCF binding site may be within at least 100, 500, 1000 or 4000 bases of the chromosome interaction or within any of the chromosome regions shown Table 1. The CTCF binding site may be within at least 100, 500, 1000 or 4000 bases of the chromosome interaction or within any of the chromosome regions shown Table 13.
In one embodiment the chromosome interactions which are detected are present at any of the gene regions shown Table 13. In the case where a ligated nucleic acid is detected in the process then sequence shown in any of the probe sequences in Table 13 may be detected.
Thus typically sequence from both regions of the probe (i.e. from both sites of the chromosome interaction) could be detected. In preferred embodiments probes are used in the process which comprise or consist of the same or complementary sequence to a probe shown in any table. In some embodiments probes are used which comprise sequence which is homologous to any of the probe sequences shown in the tables.
Table 1 shows probe (Episwitch™ marker) data and gene data representing chromosome interactions relevant to immunoresponsiveness. The probe sequences show sequence which can be used to detect a ligated product generated from both sites of gene regions that have come together in chromosome interactions, i.e. the probe will comprise sequence which is complementary to sequence in the ligated product. The first two sets of Start-End positions show probe positions, and the second two sets of Start-End positions show the relevant 4 kb region. The following information is provided in the probe data table:
Table 1 shows genes where a relevant chromosome interaction has been found to occur. Other tables show similar data. The p-value in the loci table is the same as the HyperG_Stats (p-value for the probability of finding that number of significant EpiSwitch™ markers in the locus based on the parameters of hypergeometric enrichment). The LS column shows presence or absence of the relevant interaction with that particular responder status.
The probes are designed to be 30 bp away from the Taq1 site. In case of PCR, PCR primers are typically designed to detect ligated product but their locations from the Taq1 site vary.
Start 1-30 bases upstream of Taql site on fragment 1
End 1—Taql restriction site on fragment 1
Start 2—Taql restriction site on fragment 2
End 2-30 bases downstream of Taql site on fragment 2
Start 1-4000 bases upstream of Taql site on fragment 1
End 1—Taql restriction site on fragment 1
Start 2—Taql restriction site on fragment 2
End 2-4000 bases downstream of Taql site on fragment 2
GLMNET values related to procedures for fitting the entire lasso or elastic-net regularization (Lambda set to 0.5 (elastic-net)).
Tables 1 and 4 relates to detection of immunoresponsiveness. Table 2 shows the overlap between the two studies that were done, and Table 3 shows the overlap with markers relating to interferon gamma. The invention can be carried out using markers as disclosed/represented in any of the tables. Other tables, including Tables 13 and 1, can be interpreted in a similar manner as set out for Table 1 above.
Methods of preparing samples and detecting chromosome conformations are described herein. Optimised (non-conventional) versions of these methods can be used, for example as described in this section.
Typically the sample will contain at least 2×105 cells. The sample may contain up to 5×105 cells. In one embodiment, the sample will contain 2×105 to 5.5×105 cells
Crosslinking of epigenetic chromosomal interactions present at the chromosomal locus is described herein. This may be performed before cell lysis takes place. Cell lysis may be performed for 3 to 7 minutes, such as 4 to 6 or about 5 minutes. In some embodiments, cell lysis is performed for at least 5 minutes and for less than 10 minutes.
Digesting DNA with a restriction enzyme is described herein. Typically, DNA restriction is performed at about 55° C. to about 70° C., such as for about 65° C., for a period of about 10 to 30 minutes, such as about 20 minutes.
Preferably a frequent cutter restriction enzyme is used which results in fragments of ligated DNA with an average fragment size up to 4000 base pair. Optionally the restriction enzyme results in fragments of ligated DNA have an average fragment size of about 200 to 300 base pairs, such as about 256 base pairs. In one embodiment, the typical fragment size is from 200 base pairs to 4,000 base pairs, such as 400 to 2,000 or 500 to 1,000 base pairs.
In one embodiment of the EpiSwitch method a DNA precipitation step is not performed between the DNA restriction digest step and the DNA ligation step.
DNA ligation is described herein. Typically the DNA ligation is performed for 5 to 30 minutes, such as about 10 minutes.
The protein in the sample may be digested enzymatically, for example using a proteinase, optionally Proteinase K. The protein may be enzymatically digested for a period of about 30 minutes to 1 hour, for example for about 45 minutes. In one embodiment after digestion of the protein, for example Proteinase K digestion, there is no cross-link reversal or phenol DNA extraction step.
In one embodiment PCR detection is capable of detecting a single copy of the ligated nucleic acid, preferably with a binary read-out for presence/absence of the ligated nucleic acid.
The process of the invention can be described in different ways. It can be described as a method of making a ligated nucleic acid comprising (i) in vitro cross-linking of chromosome regions which have come together in a chromosome interaction; (ii) subjecting said cross-linked DNA to cutting or restriction digestion cleavage; and (iii) ligating said cross-linked cleaved DNA ends to form a ligated nucleic acid, wherein detection of the ligated nucleic acid may be used to determine the chromosome state at a locus, and wherein preferably:
The process of the invention can be described as a process for detecting chromosome states which represent different subgroups in a population comprising determining whether a chromosome interaction is present or absent within a defined epigenetically active region of the genome, wherein preferably:
The process of the invention can be described as a method of making a ligated nucleic acid comprising (i) in vitro cross-linking of chromosome regions which have come together in a chromosome interaction; (ii) subjecting said cross-linked DNA to cutting or restriction digestion cleavage; and (iii) ligating said cross-linked cleaved DNA ends to form a ligated nucleic acid, wherein detection of the ligated nucleic acid may be used to determine the chromosome state at a locus, and wherein preferably:
The process of the invention can be described as a process for detecting chromosome states which represent different subgroups in a population comprising determining whether a chromosome interaction is present or absent within a defined epigenetically active region of the genome, wherein preferably:
The process of the invention can be described as a method of making a ligated nucleic acid comprising (i) in vitro cross-linking of chromosome regions which have come together in a chromosome interaction; (ii) subjecting said cross-linked DNA to cutting or restriction digestion cleavage; and (iii) ligating said cross-linked cleaved DNA ends to form a ligated nucleic acid, wherein detection of the ligated nucleic acid may be used to determine the chromosome state at a locus, and wherein preferably:
The process of the invention can be described as a process for detecting chromosome states which represent different subgroups in a population comprising determining whether a chromosome interaction is present or absent within a defined epigenetically active region of the genome, wherein preferably:
The invention includes detecting chromosome interactions at any locus, gene or regions mentioned Table 1. The invention includes use of the nucleic acids and probes mentioned herein to detect chromosome interactions, for example use of at least 1, 5, 10, 50, 100, 200, 250, 300 such nucleic acids or probes to detect chromosome interactions, preferably in at least 1, 5, 10, 50, 100, 200, 250, 300 different loci or genes. The invention includes detection of chromosome interactions using any of the primers or primer pairs listed in Table 4 or using variants of these primers as described herein (sequences comprising the primer sequences or comprising fragments and/or homologues of the primer sequences).
The invention includes detecting chromosome interactions at any locus, gene or regions mentioned Table 13. The invention includes use of the nucleic acids and probes mentioned herein to detect chromosome interactions, for example use of at least 1, 5, 10, 50, 100, 200, 250, 300 such nucleic acids or probes to detect chromosome interactions, preferably in at least 1, 5, 10, 50, 100, 200, 250, 300 different loci or genes. The invention includes detection of chromosome interactions using any of the primers or primer pairs listed in Table 13 or using variants of these primers as described herein (sequences comprising the primer sequences or comprising fragments and/or homologues of the primer sequences).
The invention includes detecting chromosome interactions at any locus, gene or regions mentioned Table 16. The invention includes use of the nucleic acids and probes mentioned herein to detect chromosome interactions, for example use of at least 1, 5, 10, 50, 100, 200, 250, 300 such nucleic acids or probes to detect chromosome interactions, preferably in at least 1, 5, 10, 50, 100, 200, 250, 300 different loci or genes. The invention includes detection of chromosome interactions using any of the primers or primer pairs listed in Table 17 or using variants of these primers as described herein (sequences comprising the primer sequences or comprising fragments and/or homologues of the primer sequences).
When analysing whether a chromosome interaction occurs ‘within’ a defined gene, region or location, either both the parts of the chromosome which have together in the interaction are within the defined gene, region or location or in some embodiments only one part of the chromosome is within the defined, gene, region or location.
Knowledge of chromosome interactions can be used to identify new treatments for conditions. The invention provides methods and uses of chromosomes interactions defined herein to identify or design new therapeutic agents, for example relating to immunotherapy.
Homologues of polynucleotide/nucleic acid (e.g. DNA) sequences are referred to herein. Such homologues typically have at least 70% homology, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% homology, for example over a region of at least 10, 15, 20, 30, 100 or more contiguous nucleotides, or across the portion of the nucleic acid which is from the region of the chromosome involved in the chromosome interaction. The homology may be calculated on the basis of nucleotide identity (sometimes referred to as “hard homology”).
Therefore, in a particular embodiment, homologues of polynucleotide/nucleic acid (e.g. DNA) sequences are referred to herein by reference to percentage sequence identity. Typically such homologues have at least 70% sequence identity, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity, for example over a region of at least 10, 15, 20, 30, 100 or more contiguous nucleotides, or across the portion of the nucleic acid which is from the region of the chromosome involved in the chromosome interaction.
For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology and/or % sequence identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUP and BLAST algorithms can be used to calculate homology and/or % sequence identity and/or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W5 T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
The homologous sequence typically differs by 1, 2, 3, 4 or more bases, such as less than 10, 15 or 20 bases (which may be substitutions, deletions or insertions of nucleotides). These changes may be measured across any of the regions mentioned above in relation to calculating homology and/or % sequence identity.
Homology of a ‘pair of primers’ can be calculated, for example, by considering the two sequences as a single sequence (as if the two sequences are joined together) for the purpose of then comparing against the another primer pair which again is considered as a single sequence.
The second set of nucleic acids may be bound to an array, and in one embodiment there are at least 15,000, 45,000, 100,000 or 250,000 different second nucleic acids bound to the array, which preferably represent at least 300, 900, 2000 or 5000 loci. In one embodiment one, or more, or all of the different populations of second nucleic acids are bound to more than one distinct region of the array, in effect repeated on the array allowing for error detection. The array may be based on an Agilent SurePrint G3 Custom CGH microarray platform. Detection of binding of first nucleic acids to the array may be performed by a dual colour system.
Therapeutic Agents (for which Responsiveness is Determined or which are Selected Based on Testing According to the Invention)
Therapeutic agents are mentioned herein. The invention provides such agents for use in preventing or treating a disease condition in certain individuals, for example those identified by a process of the invention. This may comprise administering to an individual in need a therapeutically effective amount of the agent. The invention provides use of the agent in the manufacture of a medicament to prevent or treat a condition in certain individuals.
The formulation of the agent will depend upon the nature of the agent. The agent will be provided in the form of a pharmaceutical composition containing the agent and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. Typical oral dosage compositions include tablets, capsules, liquid solutions and liquid suspensions. The agent may be formulated for parenteral, intravenous, intramuscular, subcutaneous, transdermal or oral administration.
The dose of an agent may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the individual to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular agent. A suitable dose may however be from 0.1 to 100 mg/kg body weight such as 1 to 40 mg/kg body weight, for example, to be taken from 1 to 3 times daily.
In one embodiment the invention comprises detecting responsiveness to a therapeutic agent, for example any therapeutic agent mentioned herein. This may be before treatment has started and/or during the course of therapy.
The therapy may be mono or combination therapy, for example with immunocheckpoint modulators (inhibitors) for PD-1 and or its ligand, PD-L1. The therapy could be a combination of an anti-PD-1 or anti-PD-L1 combined with another drug that targets a checkpoint like CTLA4 (Ipilimumab/Yervoy) or small molecules. The PD-1 inhibitors could be pembrolizumab (Keytruda) or nivolumab (Opdivo). The modulator of PD-L1 or therapeutic agent could be Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), CA-170, Ipilimumab, Tremelimumab, Nivolumab, Pembrolizumab, Pidilizumab, BMS935559, GVAXMPDL3280A, MEDI4736, MSB0010718C, MDX-1105/BMS-936559, AMP-224, MEDI0680.
The therapy may comprise administering agents that target and/or modulate interferon gamma or the JAK-START pathway.
The therapeutic agent may be any such agent disclosed in any table herein (for example in Table 9, 10 or 11), or may target any ‘target’ disclosed herein, including any protein disclosed herein in any table (including Table 1, 13 or 16). It is understood that any agent that is disclosed in a combination should be seen as also disclosed for administration individually.
Any of the substances, such as nucleic acids or therapeutic agents, mentioned herein may be in purified or isolated form. They may be in a form which is different from that found in nature, for example they may be present in combination with other substance with which they do not occur in nature. The nucleic acids (including portions of sequences defined herein) may have sequences which are different to those found in nature, for example having at least 1, 2, 3, 4 or more nucleotide changes in the sequence as described in the section on homology. The nucleic acids may have heterologous sequence at the 5′ or 3′ end. The nucleic acids may be chemically different from those found in nature, for example they may be modified in some way, but preferably are still capable of Watson-Crick base pairing. Where appropriate the nucleic acids will be provided in double stranded or single stranded form. The invention provides all of the specific nucleic acid sequences mentioned herein in single or double stranded form, and thus includes the complementary strand to any sequence which is disclosed.
The invention provides a kit for carrying out any process of the invention, including detection of a chromosomal interaction relating to immunoresponsiveness. Such a kit can include a specific binding agent capable of detecting the relevant chromosomal interaction, such as agents capable of detecting a ligated nucleic acid generated by processes of the invention. Preferred agents present in the kit include probes capable of hybridising to the ligated nucleic acid or primer pairs, for example as described herein, capable of amplifying the ligated nucleic acid in a PCR reaction.
The invention provides a device that is capable of detecting the relevant chromosome interactions. The device preferably comprises any specific binding agents, probe or primer pair capable of detecting the chromosome interaction, such as any such agent, probe or primer pair described herein.
In one embodiment quantitative detection of the ligated sequence which is relevant to a chromosome interaction is carried out using a probe which is detectable upon activation during a PCR reaction, wherein said ligated sequence comprises sequences from two chromosome regions that come together in an epigenetic chromosome interaction, wherein said method comprises contacting the ligated sequence with the probe during a PCR reaction, and detecting the extent of activation of the probe, and wherein said probe binds the ligation site. The method typically allows particular interactions to be detected in a MIQE compliant manner using a dual labelled fluorescent hydrolysis probe.
The probe is generally labelled with a detectable label which has an inactive and active state, so that it is only detected when activated. The extent of activation will be related to the extent of template (ligation product) present in the PCR reaction. Detection may be carried out during all or some of the PCR, for example for at least 50% or 80% of the cycles of the PCR.
The probe can comprise a fluorophore covalently attached to one end of the oligonucleotide, and a quencher attached to the other end of the nucleotide, so that the fluorescence of the fluorophore is quenched by the quencher. In one embodiment the fluorophore is attached to the 5′end of the oligonucleotide, and the quencher is covalently attached to the 3′ end of the oligonucleotide. Fluorophores that can be used in the methods of the invention include FAM, TET, JOE, Yakima Yellow, HEX, Cyanine3, ATTO 550, TAMRA, ROX, Texas Red, Cyanine 3.5, LC610, LC 640, ATTO 647N, Cyanine 5, Cyanine 5.5 and ATTO 680. Quenchers that can be used with the appropriate fluorophore include TAM, BHQ1, DAB, Eclip, BHQ2 and BBQ650, optionally wherein said fluorophore is selected from HEX, Texas Red and FAM. Preferred combinations of fluorophore and quencher include FAM with BHQ1 and Texas Red with BHQ2.
Use of the Probe in a qPCR Assay
Hydrolysis probes of the invention are typically temperature gradient optimised with concentration matched negative controls. Preferably single-step PCR reactions are optimized. More preferably a standard curve is calculated. An advantage of using a specific probe that binds across the junction of the ligated sequence is that specificity for the ligated sequence can be achieved without using a nested PCR approach. The methods described herein allow accurate and precise quantification of low copy number targets. The target ligated sequence can be purified, for example gel-purified, prior to temperature gradient optimization. The target ligated sequence can be sequenced. Preferably PCR reactions are performed using about 10 ng, or 5 to 15 ng, or 10 to 20 ng, or 10 to 50 ng, or 10 to 200 ng template DNA.
Forward and reverse primers are designed such that one primer binds to the sequence of one of the chromosome regions represented in the ligated DNA sequence, and the other primer binds to other chromosome region represented in the ligated DNA sequence, for example, by being complementary to the sequence.
The invention includes selecting primers and a probe for use in a PCR method as defined herein comprising selecting primers based on their ability to bind and amplify the ligated sequence and selecting the probe sequence based properties of the target sequence to which it will bind, in particular the curvature of the target sequence.
Probes are typically designed/chosen to bind to ligated sequences which are juxtaposed restriction fragments spanning the restriction site. In one embodiment of the invention, the predicted curvature of possible ligated sequences relevant to a particular chromosome interaction is calculated, for example using a specific algorithm referenced herein. The curvature can be expressed as degrees per helical turn, e.g. 10.5° per helical turn. Ligated sequences are selected for targeting where the ligated sequence has a curvature propensity peak score of at least 5° per helical turn, typically at least 10°, 15° or 20° per helical turn, for example 5° to 20° per helical turn. Preferably the curvature propensity score per helical turn is calculated for at least 20, 50, 100, 200 or 400 bases, such as for 20 to 400 bases upstream and/or downstream of the ligation site. Thus in one embodiment the target sequence in the ligated product has any of these levels of curvature. Target sequences can also be chosen based on lowest thermodynamic structure free energy.
In one embodiment only intrachromosomal interactions are typed/detected, and no extrachromosomal interactions (between different chromosomes) are typed/detected.
In particular embodiments certain chromosome interactions are not typed, for example any specific interaction mentioned herein (for example as defined by any probe or primer pair mentioned herein). In some embodiments chromosome interactions are not typed in any of the genes mentioned here, for example in any gene mentioned in the Figures, including any or all genes mentioned in
In one embodiment none of the chromosome interactions represented by the probes or primers of any or all of Tables 5 to 7 are typed. In another embodiment no chromosome interactions present in any of the genes listed in any or all of Tables 5 to 7 are typed. In a further embodiment no chromosome interactions present in any of the regions listed in any or all of Tables 5 to 7 are typed.
In one embodiment the immunoresponsiveness does not relate to antibody therapy. In another embodiment the immunoresponsiveness does not relate to anti-PD-1 therapy, for example anti-PD-1 therapy of melanoma. In another embodiment the immunoresponsiveness does not relate to therapy one or more of the following: a blood cancer, leukaemia, prostate cancer, breast cancer, diffuse large B cell lymphoma.
The invention provides a method of determining which chromosomal interactions are relevant to a chromosome state corresponding to an immunoresponsive subgroup of the population, comprising contacting a first set of nucleic acids from subgroups with different states of the chromosome with a second set of index nucleic acids, and allowing complementary sequences to hybridise, wherein the nucleic acids in the first and second sets of nucleic acids represent a ligated product comprising sequences from both the chromosome regions that have come together in chromosomal interactions, and wherein the pattern of hybridisation between the first and second set of nucleic acids allows a determination of which chromosomal interactions are specific to an immunoresponsive subgroup. The subgroup may be any of the specific subgroups defined herein, for example with reference to particular conditions or therapies.
The contents of all publications mentioned herein are incorporated by reference into the present specification and may be used to further define the features relevant to the invention.
Table 1 shows the final set of markers for testing immunoresponsiveness.
Table 2 shows the shared markers between the anti-PD-1 and anti-PD-L1 studies.
Table 3 shows markers which overlap with interferon gamma activated ORF's.
Table 4 shows primer pairs that can be used to detect markers relating to immunoresponsiveness.
Tables 5 to 7 show markers, genes and regions which are not included in certain embodiments.
Table 8 shows immune checkpoint molecules.
Table 9 provides example of cancer therapies.
Tables 10 and 11 show combination and mono therapies for certain embodiments of the invention. In some embodiments these are the therapies for which responsiveness is tested. In other embodiments these therapies are given to the patient depending on the outcome of testing according to the invention.
Table 12 provides a description of genes relevant to the invention.
Table 13 shows a further set of markers for testing immunoresponsiveness.
Tables 14 and 15 describe genes that relate to embodiments of the invention.
Tables 16 to 18 shows markers for testing immunoresponsiveness.
Paragraph A. A process for detecting a chromosome state which represents a subgroup in a population comprising determining whether a chromosome interaction relating to that chromosome state is present or absent within a defined region of the genome, wherein said subgroup relates to how immunoresponsive individuals are; and
Paragraph B. A process according to paragraph A wherein a specific combination of chromosome interactions are typed:
(i) comprising all of the chromosome interactions represented by the probes in Table 1; and/or
(ii) comprising at least 10, 50, 100, 150, 200 or 300 of the chromosome interactions represented by the probes in Table 1; and/or
(iii) which together are present in at least 10, 50, 100, 150 or 200 of the regions or genes listed in Table 1; and/or
(iv) wherein at least 10, 50, 100, 150, 200 or 300 chromosome interactions are typed which are present in a 4,000 base region which comprises or which flanks the chromosome interactions represented by the probes in Table 1.
Paragraph C. A process according to paragraph A wherein a specific combination of chromosome interactions are typed:
(i) comprising all of the chromosome interactions represented by the probes in Table 13; and/or
(ii) comprising at least 10, 50, 100, 150, 200 or 300 of the chromosome interactions represented by the probes in Table 13; and/or
(iii) which together are present in at least 10, 50, 100, 150 or 200 of the regions or genes listed in Table 13; and/or
(iv) wherein at least 10, 50, 100, 150, 200 or 300 chromosome interactions are typed which are present in a 4,000 base region which comprises or which flanks the chromosome interactions represented by the probes in Table 13.
D. A process according to any one of the preceding paragraphs in which the chromosome interactions are typed:
E. A process according to any one of the preceding paragraphs, wherein:
F. A process according to any one of the preceding paragraphs wherein:
G. A process according to any one of the preceding paragraphs, wherein the first set of nucleic acids is obtainable in a process comprising the steps of:—
(i) cross-linking of chromosome regions which have come together in a chromosome interaction;
(ii) subjecting said cross-linked regions to cleavage, optionally by restriction digestion cleavage with an enzyme; and
(iii) ligating said cross-linked cleaved DNA ends to form the first set of nucleic acids (in particular comprising ligated DNA).
H. A process according to any one of the preceding paragraphs:
I. A process according to any one of the preceding paragraphs wherein said defined region of the genome:
(i) comprises a single nucleotide polymorphism (SNP); and/or
(ii) expresses a microRNA (miRNA); and/or
(iii) expresses a non-coding RNA (ncRNA); and/or
(iv) expresses a nucleic acid sequence encoding at least 10 contiguous amino acid residues; and/or
(v) expresses a regulating element; and/or
(vii) comprises a CTCF binding site.
J. A process according to any one of the preceding paragraphs which is carried out to identify or design a therapeutic agent for immunotherapy;
K. A process according to any one of the preceding paragraphs which is carried out to identify or design a therapeutic agent for immunotherapy;
L. A process according to paragraph J or K which comprises selecting a target based on detection of chromosome interactions, and preferably screening for a modulator of the target to identify a therapeutic agent for immunotherapy, wherein said target is optionally a protein.
M. A therapeutic agent for use in a method of immunotherapy in an individual that has been identified as being in need of the therapeutic agent by a process according to any one of paragraphs A to I.
N. A process according to any one of paragraphs A to K or a therapeutic agent for use according to paragraph M, wherein the typing or detecting comprises specific detection of the ligated product by quantitative PCR (qPCR) which uses primers capable of amplifying the ligated product and a probe which binds the ligation site during the PCR reaction, wherein said probe comprises sequence which is complementary to sequence from each of the chromosome regions that have come together in the chromosome interaction, wherein preferably said probe comprises:
an oligonucleotide which specifically binds to said ligated product, and/or
a fluorophore covalently attached to the 5′ end of the oligonucleotide, and/or
a quencher covalently attached to the 3′ end of the oligonucleotide, and
optionally
said fluorophore is selected from HEX, Texas Red and FAM; and/or
said probe comprises a nucleic acid sequence of length 10 to 40 nucleotide bases, preferably a length of 20 to 30 nucleotide bases.
The EpiSwitch™ platform technology detects epigenetic regulatory signatures of regulatory changes between normal and abnormal conditions at loci. The EpiSwitch™ platform identifies and monitors the fundamental epigenetic level of gene regulation associated with regulatory high order structures of human chromosomes also known as chromosome conformation signatures. Chromosome signatures are a distinct primary step in a cascade of gene deregulation. They are high order biomarkers with a unique set of advantages against biomarker platforms that utilize late epigenetic and gene expression biomarkers, such as DNA methylation and RNA profiling.
The custom EpiSwitch™ array-screening platforms come in 4 densities of, 15K, 45K, 100K, and 250K unique chromosome conformations, each chimeric fragment is repeated on the arrays 4 times, making the effective densities 60K, 180K, 400K and 1 Million respectively.
The 15K EpiSwitch™ array can screen the whole genome including around 300 loci interrogated with the EpiSwitch™ Biomarker discovery technology. The EpiSwitch™ array is built on the Agilent SurePrint G3 Custom CGH microarray platform; this technology offers 4 densities, 60K, 180K, 400K and 1 Million probes. The density per array is reduced to 15K, 45K, 100K and 250K as each EpiSwitch™ probe is presented as a quadruplicate, thus allowing for statistical evaluation of the reproducibility. The average number of potential EpiSwitch™ markers interrogated per genetic loci is 50; as such the numbers of loci that can be investigated are 300, 900, 2000, and 5000.
The EpiSwitch™ array is a dual colour system with one set of samples, after EpiSwitch™ library generation, labelled in Cy5 and the other of sample (controls) to be compared/analyzed labelled in Cy3. The arrays are scanned using the Agilent SureScan Scanner and the resultant features extracted using the Agilent Feature Extraction software. The data is then processed using the EpiSwitch™ array processing scripts in R. The arrays are processed using standard dual colour packages in Bioconductor in R: Limma*. The normalisation of the arrays is done using the normalisedWithinArrays function in Limma* and this is done to the on chip Agilent positive controls and EpiSwitch™ positive controls. The data is filtered based on the Agilent Flag calls, the Agilent control probes are removed and the technical replicate probes are averaged, in order for them to be analysed using Limma*. The probes are modelled based on their difference between the 2 scenarios being compared and then corrected by using False Discovery Rate. Probes with Coefficient of Variation (CV)<=30% that are <=−1.1 or =>1.1 and pass the p<=0.1 FDR p-value are used for further screening. To reduce the probe set further Multiple Factor Analysis is performed using the FactorMineR package in R. * Note: LIMMA is Linear Models and Empirical Bayes Processes for Assessing Differential Expression in Microarray Experiments. Limma is an R package for the analysis of gene expression data arising from microarray or RNA-Seq.
The pool of probes is initially selected based on adjusted p-value, FC and CV<30% (arbitrary cut off point) parameters for final picking. Further analyses and the final list are drawn based only on the first two parameters (adj. p-value; FC).
EpiSwitch™ screening arrays are processed using the EpiSwitch™ Analytical Package in R in order to select high value EpiSwitch™ markers for translation on to the EpiSwitch™ PCR platform.
Probes are selected based on their corrected p-value (False Discovery Rate, FDR), which is the product of a modified linear regression model. Probes below p-value<=0.1 are selected and then further reduced by their Epigenetic ratio (ER), probes ER have to be <=−1.1 or =>1.1 in order to be selected for further analysis. The last filter is a coefficient of variation (CV), probes have to be below <=0.3.
The top 40 markers from the statistical lists are selected based on their ER for selection as markers for PCR translation. The top 20 markers with the highest negative ER load and the top 20 markers with the highest positive ER load form the list.
The resultant markers from step 1, the statistically significant probes form the bases of enrichment analysis using hypergeometric enrichment (HE). This analysis enables marker reduction from the significant probe list, and along with the markers from step 2 forms the list of probes translated on to the EpiSwitch™ PCR platform.
The statistical probes are processed by HE to determine which genetic locations have an enrichment of statistically significant probes, indicating which genetic locations are hubs of epigenetic difference.
The most significant enriched loci based on a corrected p-value are selected for probe list generation. Genetic locations below p-value of 0.3 or 0.2 are selected. The statistical probes mapping to these genetic locations, with the markers from step 2, form the high value markers for EpiSwitch™ PCR translation.
EpiSwitch™ biomarker signatures demonstrate high robustness, sensitivity and specificity in the stratification of complex disease phenotypes. This technology takes advantage of the latest breakthroughs in the science of epigenetics, monitoring and evaluation of chromosome conformation signatures as a highly informative class of epigenetic biomarkers. Current research methodologies deployed in academic environment require from 3 to 7 days for biochemical processing of cellular material in order to detect CCSs. Those procedures have limited sensitivity, and reproducibility; and furthermore, do not have the benefit of the targeted insight provided by the EpiSwitch™ Analytical Package at the design stage.
CCS sites across the genome are directly evaluated by the EpiSwitch™ Array on clinical samples from testing cohorts for identification of all relevant stratifying lead biomarkers. The EpiSwitch™ Array platform is used for marker identification due to its high-throughput capacity, and its ability to screen large numbers of loci rapidly. The array used was the Agilent custom-CGH array, which allows markers identified through the in silico software to be interrogated.
Potential markers identified by EpiSwitch™ Array are then validated either by EpiSwitch™ PCR or DNA sequencers (i.e. Roche 454, Nanopore MinION, etc.). The top PCR markers which are statistically significant and display the best reproducibility are selected for further reduction into the final EpiSwitch™ Signature Set, and validated on an independent cohort of samples. EpiSwitch™ PCR can be performed by a trained technician following a standardised operating procedure protocol established. All protocols and manufacture of reagents are performed under ISO 13485 and 9001 accreditation to ensure the quality of the work and the ability to transfer the protocols. EpiSwitch™ PCR and EpiSwitch™ Array biomarker platforms are compatible with analysis of both whole blood and cell lines. The tests are sensitive enough to detect abnormalities in very low copy numbers using small volumes of blood.
In the first study patients with melanoma were treated with anti-PD-1 (Pembrolizumab) for 12 weeks. Their epigenetic status, measured by EpiSwitch was assessed first at baseline prior to treatment and then at 12 weeks, along with clinical readout of response or no-response. We then screened, evaluated and validated parts of 3D genome architecture profiles, chromosome conformation signatures, at base line to identify profiles conducive to response or no-response to treatment at over 332 genetic locations across the genome. These were evaluate on an EpiSwitch array looking at local multiple 3C interactions at those genetic locations in technical and biological repeats, comparing samples from responders and non-responders at base line. Over 14,000 EpiSwitch markers were directly evaluated on the array. The best markers were translated into PCR and evaluated on the independent patient cohort.
Stage 1 consist of the initial array screening and evaluation for the 14,000 marker leads (all predicted by pattern recognition). Stage 2 is the translation of statistically significant marker leads from array into PCR. Further evaluation by PCR of the markers by regression analysis for reduction of the final marker number in the final signature. Stage 3 is validation of the final signature of 6 best biomarkers (shown in
The third study relates to anti-PD-L1 treatment in lung cancer. At the array stage of screening 12 responders and 12 non-responders were compared at base line on the array for a total of 180,000 readouts (in technical and biological repeats). There was then PCR translation for the top 100 marker leads from the array followed by validation.
There was a comparison of marker leads from the arrays for the PD-1 (study I), PD-L1 (study II) with the PD-L1 study III.
Having established common epigenetic settings as markers for responders in PD-1 and PD-L1 treatments, we followed the genetic locations observed under control of epigenetic settings to their protein products and investigated the network relationship for those proteins in the context of known protein-protein networks and their functional roles. Over-imposing the genetic loci affected by EpiSwitch markers of response onto the String database network shows that epigenetically controlled proteins in responders are involved in the close network associated with the two functions: Immune response-regulating cell surface receptor singling pathway and regulation of T-cell activation.
Markers have been identified that have statistically significant disseminating powers to identify in baseline patients those who are set up for good response to immunotherapies (including immunocheckpoint therapies), i.e. responders, and those who are not, i.e. non-responders, or progressors. The markers of Table 1 represent a common universal profile of responders across three independent cohorts with either melanoma or lung cancer (NSCLC). Melanoma patients were treated with PD-1 (pembrolizumab), NSCLC with two different PD-L1. These are agnostic makers predictive of treatment and present across different types of cancers. There is also one important agnostic and universal predictive marker for non-response, located between PD-L1 and PD-L2 loci.
The list of markers in Table 1 reflects a common network regulation in responders. These can be used as agnostic markers to monitor response/non-response across various treatments and conditions, including in response to treatments targeting standard immunocheckpoint molecules controlling the immunological network response.
Continuing the goal of identifying epigenetic changes based on chromosome conformation signatures (CCSs) that will be able to select a priori patients who will respond to anti PD-1 therapy, either alone or in combination, for the treatment of non-small cell lung cancer (NSCLC). The discovered EpiSwitch™ markers from this array screen were then screened and validated using the EpiSwitch™ PCR platform using three immunotherapies cohorts, two anti-PD-L1 therapies and one anti-PD-1 therapy (Pembrolizumab) in baseline patients' samples. The two anti-PD-L1 response CCSs were developed on NSCLC patients and the Pembrolizumab response CCSs on melanoma patients and further validated on NSCLC patients. Tables 13, 16 and 18 provide the results of further work, and shows a further set of markers that can be used to determine immunoresponsiveness.
In the metastatic melanoma study, a total number of 32 peripheral blood mononuclear cells (PBMC) samples from 16 patients with metastatic melanoma were studied. These patients had received anti-PD1 therapy. The patients were assessed at 2 time-points, baseline and 12-week tumour assessment. A total number of 16 samples were used for the discovery stage using the EpiSwitch Array. The top 100 EpiSwitch Markers identified from the array screening were verified in the baseline and 12 week samples to identify response to anti-PD1 therapy and pharmacodynamics effects. The top EpiSwitch markers were classified into two group of responders and non-responders to anti-PD-1 therapy.
In the NSCLC study a total number of 16 baseline PBMCs from non-small-cell lung carcinoma (NSCLC) patients treated with an anti-PD-L1 therapy were studied. 30 EpiSwitch™ predictive markers were evaluated. The aim of this project was to confirm predictive capability of EpiSwitch markers common for an anti-PD-1 and anti-PD-L1 predictive profile in baseline NSCLC patients who have been treated with anti-PD-L1 therapy.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/GB2018/052818 | Oct 2018 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2018/053196 | 11/2/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62581287 | Nov 2017 | US |
