Immunocompetence assessment by adaptive immune receptor diversity and clonality characterization

Abstract

Disclosed are methods for determining the immunological status of the adaptive immune system of a subject by identifying and quantifying rearranged DNA (and/or subsequently transcribed RNA) sequences encoding T cell receptor (TCR) and/or immunoglobulin (IG) polypeptides, in a lymphoid DNA-containing sample from the subject. TCR and/or IG sequence diversity and sequence distribution permit immunocompetence and immune repertoire assessment and reflect the degree of T cell or B cell clonality and clonal expansion in the sample. Methods for stratifying patient populations on the basis of immunocompetence including likelihood of responding to immunotherapy are also described.

1 . A method for predicting an immunological response of a test subject to an immunotherapy, comprising:
 amplifying a plurality of nucleic acid sequences from one or more solid tumor tissue samples obtained from a test subject at one or more time points prior to and after treatment with said immunotherapy comprising lymphoid cells from a test subject in a multiplex PCR assay using a plurality of adaptive immune receptor (AIR) V-segment oligonucleotide primers and either a plurality of AIR J-segment oligonucleotide primers or one or more AIR C-segment oligonucleotide primers to obtain a plurality of AIR amplicons;   sequencing said AIR amplicons to obtain nucleic acid sequence information;   determining a test subject immunological rating score for said one or more solid tumor tissue samples by quantifying said nucleic acid sequence information;   comparing said test subject immunological rating score with a set of immunological rating scores of subjects with known responses to said immunotherapy; and   predicting an immunological response for said test subject to said immunotherapy based on said comparison.   
     
     
         2 . The method of  claim 1 , wherein comparing said test subject immunological rating scores for said one or more samples to a set of immunological rating scores of subjects with known responses to said immunotherapy comprises determining whether said test subject immunological rating score is statistically significantly different from a threshold score determined from said set of immunological rating scores of subjects with known responses. 
     
     
         3 . The method of  claim 1 , wherein said nucleic acids comprise genomic DNA, cDNA or mRNA. 
     
     
         4 . The method of  claim 1 , wherein said nucleic acids comprise cDNA. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein determining a test subject immunological rating score comprises quantifying an AIR sequence distribution score for said test subject by:
 determining a number of unique rearranged AIR sequences that account for combined frequency of occurrence of up to 50%, 40%, 30%, 20% or 10% of the total number of observed rearranged sequences in said sample.   
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , further comprising quantifying an AIR sequence diversity of said one or more samples based on a total number of unique rearranged DNA sequences in said one or more samples and thereby determining a total number of unique clones in said one or more samples. 
     
     
         9 .- 10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein said test subject is predicted to have the same outcome of response to immunotherapy as compared to said subjects with known responses to said immunotherapy. 
     
     
         12 . The method of  claim 1 , wherein said test subject is predicted to have a different outcome of response to immunotherapy as compared to said subjects with known responses to said immunotherapy. 
     
     
         13 . The method of  claim 1 , wherein said immunotherapy comprises an immunotherapeutic antibody, a cytokine, an immunosuppressive agent, or a vaccine. 
     
     
         14 . The method of  claim 1 , wherein said immunotherapy comprises administration of an inhibitor of a negative regulator of the immune system or an agent that targets a potentiator of an immune response. 
     
     
         15 . The method of  claim 14 , wherein said negative regulator is selected from a group consisting of CTLA-4, LAG3 and PD-1. 
     
     
         16 .- 18 . (canceled) 
     
     
         19 . The method of  claim 14 , wherein said inhibitor is an anti-PD-1 antibody or a CTLA-4 antibody selected from the group consisting of ipilimumab and tremelimumab. 
     
     
         20 . The method of  claim 1 , further comprising determining a presence of a clonal expansion of at least one clone in a sample obtained from said test subject after said immunotherapy treatment, wherein said at least one clone has a frequency of occurrence that is statistically significantly different from a mean frequency of occurrence of a set of remaining clones in said sample and previously did not have a frequency of occurrence that was statistically significantly different from a mean frequency of occurrence of a set of remaining clones in a sample obtained from said test subject prior to said immunotherapy. 
     
     
         21 . The method of  claim 20 , wherein said set of remaining clones comprise clones each having a frequency of occurrence that is in the top 50%, 40%, 30%, 20% or 10% of the total clones in said sample. 
     
     
         22 .- 26 . (canceled) 
     
     
         27 . The method of  claim 20 , wherein said clonal expansion of said at least one clone is indicative of a poor response of said test subject to said immunotherapy treatment. 
     
     
         28 .- 36 . (canceled) 
     
     
         37 . The method of  claim 1 , wherein determining a test subject immunological rating score comprises determining an AIR sequence distribution score of said one or more samples by selecting a set of unique rearranged sequences in one of said samples having a frequency of occurrence that is statistically significantly higher compared with a threshold frequency of occurrence set by other unique rearranged sequences in said one or more samples. 
     
     
         38 .- 44 . (canceled) 
     
     
         45 . The method of  claim 1 , further comprising:
 categorizing said test subject based on said test subject immunological rating score in said one or more samples as a non-responder or responder to immunotherapy in comparison to a set of subjects having a known immunological responses to immunotherapy; and   stratifying a patient population of test subjects according to relative likelihood of responding to immunotherapy.   
     
     
         46 . The method of  claim 1 , wherein determining said test subject immunological rating score comprises extrapolating based on a mathematical model a total AIR repertoire diversity of said test subject by sequencing said nucleic acid sequences from one of said samples and determining a test subject immunological rating score from said total AIR repertoire diversity. 
     
     
         47 . The method of  claim 46 , wherein said mathematical model is an unseen species model. 
     
     
         48 . The method of  claim 1 , wherein determining said test subject immunological rating score comprises quantifying an AIR sequence distribution score, wherein said quantifying said AIR distribution ascore comprises calculating a Shannon entropy score using said nucleic acid sequence information from said one or more samples. 
     
     
         49 . The method of  claim 1 , wherein said adaptive immune receptor (AIR) polypeptide is a mammalian AIR polypeptide and is selected from a T cell receptor-gamma (TCRG) polypeptide, a T cell receptor-beta (TCRB) polypeptide, a T cell receptor-alpha (TCRA) polypeptide, a T cell receptor-delta (TCRD) polypeptide, an immunoglobulin heavy-chain (IGH) polypeptide, and an immunoglobulin light-chain (IGL) polypeptide. 
     
     
         50 . The method of  claim 49 , wherein said IGH polypeptide is selected from an IgM, an IgA polypeptide, an IgG polypeptide, an IgD polypeptide and an IgE polypeptide. 
     
     
         51 . The method of  claim 49 , wherein said IGL polypeptide is selected from an IGL-lambda polypeptide and an IGL-kappa polypeptide. 
     
     
         52 . (canceled) 
     
     
         53 . The method of  claim 49 , wherein said mammalian AIR polypeptide is selected from a human AIR polypeptide, non-human primate AIR polypeptide, a rodent AIR polypeptide, a canine AIR polypeptide, a feline AIR polypeptide and an ungulate AIR polypeptide. 
     
     
         54 . The method of  claim 1 , wherein said test subject is a subject having or suspected of having a malignant condition selected from a hematologic malignancy, a melanoma, a sarcoma and a carcinoma. 
     
     
         55 . (canceled) 
     
     
         56 . The method of  claim 54 , wherein said malignant condition is selected from malignant melanoma, small cell lung cancer, non-small cell lung cancer, renal cell carcinoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. 
     
     
         57 .- 64 . (canceled) 
     
     
         65 . The method of  claim 14 , wherein said potentiator of an immune response is selected from 41BB/CD137, OX40/CD 134 and CD40. 
     
     
         66 .- 117 . (canceled)