Quantification of adaptive immune cell genomes in a complex mixture of cells
First Claim
1. A method for quantifying a relative representation of adaptive immune cells in a solid tumor sample that comprises a mixture of cells comprising adaptive immune cells and cells that are not adaptive immune cells, the method comprising:
- (a) amplifying DNA molecules comprising rearranged CDR3 regions from said sample by performing a multiplex quantitative polymerase chain reaction (qPCR) using;
(1) a plurality of V-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs;
1-52, 66-68, 221-238, 255-260, 262-267, 269, 272, 283, 286, 291, 292, 294-297, 301-326, 330, 338, 382, 405, 447-484, 644-695, 709-839, and 843-879 that are each capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) V-region polypeptide or an immunoglobulin (Ig) V-region polypeptide, wherein the plurality of V-segment oligonucleotide primers specifically hybridize to at least 90% of-all functional TCR or Ig V-encoding gene segments that are present in the sample, and(2) a plurality of J-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs;
53-63, 65, 215-220, and 247 that are each capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) J-region polypeptide or an immunoglobulin (Ig) J-region polypeptide, wherein the plurality of J-segment oligonucleotide primers specifically hybridize to at least 90% of all functional TCR or Ig J-encoding gene segments that are present in the sample, to produce a multiplicity of amplified rearranged TCR or Ig CDR3 DNA molecules from the adaptive immune cells in the sample;
(b) measuring a plurality of first signal levels at a plurality of time points detected from said amplified rearranged TCR or Ig CDR3 DNA molecules;
(c) comparing the plurality of first signal levels with a plurality of second signal levels detected from amplification of rearranged TCR or Ig CDR3 DNA molecules of known concentrations, thereby quantifying a relative amount of adaptive immune cell DNA; and
(d) determining, from the relative amount of adaptive immune cell DNA quantified in (c), the relative representation of adaptive immune cells in the solid tumor sample.
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Abstract
Compositions and methods are described for highly sensitive quantification of the relative representation of DNA from adaptive immune cells (e.g., T and/or B lymphocytes) in DNA extracted from complex mixtures of cells that include cells which are not adaptive immune cells. Included are methods for determining the relative presence in a tumor of tumor infiltrating lymphocytes (TIL), the relative presence of lymphocytes infiltrating a somatic tissue that is the target of an autoimmune disease, and the relative presence of lymphocytes infiltrating a transplanted organ.
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Citations
51 Claims
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1. A method for quantifying a relative representation of adaptive immune cells in a solid tumor sample that comprises a mixture of cells comprising adaptive immune cells and cells that are not adaptive immune cells, the method comprising:
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(a) amplifying DNA molecules comprising rearranged CDR3 regions from said sample by performing a multiplex quantitative polymerase chain reaction (qPCR) using; (1) a plurality of V-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs;
1-52, 66-68, 221-238, 255-260, 262-267, 269, 272, 283, 286, 291, 292, 294-297, 301-326, 330, 338, 382, 405, 447-484, 644-695, 709-839, and 843-879 that are each capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) V-region polypeptide or an immunoglobulin (Ig) V-region polypeptide, wherein the plurality of V-segment oligonucleotide primers specifically hybridize to at least 90% of-all functional TCR or Ig V-encoding gene segments that are present in the sample, and(2) a plurality of J-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs;
53-63, 65, 215-220, and 247 that are each capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) J-region polypeptide or an immunoglobulin (Ig) J-region polypeptide, wherein the plurality of J-segment oligonucleotide primers specifically hybridize to at least 90% of all functional TCR or Ig J-encoding gene segments that are present in the sample, to produce a multiplicity of amplified rearranged TCR or Ig CDR3 DNA molecules from the adaptive immune cells in the sample;(b) measuring a plurality of first signal levels at a plurality of time points detected from said amplified rearranged TCR or Ig CDR3 DNA molecules; (c) comparing the plurality of first signal levels with a plurality of second signal levels detected from amplification of rearranged TCR or Ig CDR3 DNA molecules of known concentrations, thereby quantifying a relative amount of adaptive immune cell DNA; and (d) determining, from the relative amount of adaptive immune cell DNA quantified in (c), the relative representation of adaptive immune cells in the solid tumor sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 25, 26, 27, 28, 29, 32, 33, 34)
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18. A method for assessing an effect of a therapeutic treatment on at least one tissue of a subject, the method comprising:
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(I) obtaining one or a plurality of samples from a first tissue of the subject at one or a plurality of time points prior to administering the therapeutic treatment, wherein the sample contains DNA from a mixture of cells, the mixture comprising adaptive immune cells and cells that are not adaptive immune cells; (II) obtaining one or a plurality of samples from a second tissue of the subject at one or a plurality of time points after administering the therapeutic treatment, wherein the sample contains DNA from a mixture of cells, the mixture comprising adaptive immune cells and cells that are not adaptive immune cells; (III) for each of said samples from (I) and (II); (a) amplifying nucleic acid molecules from said sample by performing a quantitative multiplex polymerase chain reaction (qPCR) using (i) a plurality of V-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs;
1-52, 66-68, 221-238, 255-260, 262-267, 269, 272, 283, 286, 291, 292, 294-297, 301-326, 330, 338, 382, 405, 447-484, 644-695, 709-839, and 843-879 that are each capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) V-region polypeptide or an immunoglobulin (Ig) V-region polypeptide, wherein the plurality of V-segment oligonucleotide primers specifically hybridize to at least 90% of all functional TCR or Ig V-encoding gene segments that are present in the sample, and(ii) a plurality of J-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs;
53-63, 65, 215-220, and 247 that are each capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) J-region polypeptide or an immunoglobulin (Ig) J-region polypeptide, wherein the plurality of J-segment oligonucleotide primers specifically hybridize to at least 90% of all functional TCR or Ig J-encoding gene segments that are present in the sample, to produce a multiplicity of amplified rearranged TCR or Ig CDR3 DNA molecules from the adaptive immune cells in the sample;(b) measuring first DNA signal levels at a plurality of time points that are detectable in said multiplicity of amplified rearranged TCR or Ig DNA molecules of (a) to generate a qPCR signal profile; (c) comparing the qPCR signal profile in (b) to a standard signal profile detected from amplification of rearranged TCR or Ig CDR3 DNA molecules of known DNA concentrations, and therefrom quantifying a relative amount of adaptive immune cell DNA in the sample; and (d) determining, from the relative amount of adaptive immune cell DNA quantified in (c), the relative representation of adaptive immune cell DNA in the sample; and (IV) comparing the relative representation of adaptive immune cells in at least one sample obtained at a time point prior to administering the therapeutic treatment to the relative representation of adaptive immune cells in at least one sample obtained at a time point after administering the therapeutic treatment, and thereby assessing an effect of the therapeutic treatment on relative representation of adaptive immune cells in at least one tissue of a subject. - View Dependent Claims (19, 20, 21, 22, 23, 24, 30, 31)
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35. A method for quantifying the relative representation of adaptive immune cells in a sample that comprises a mixture of cells, the mixture comprising adaptive immune cells and cells that are not adaptive immune cells, the method comprising:
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(a) distributing template DNA extracted from the sample to form a set of assay samples, (b) amplifying said template DNA in the set of assay samples in a multiplex digital polymerase chain reaction (dPCR) that comprises; (1) (i) a plurality of V-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs;
1-52, 66-68, 221-238, 255-260, 262-267, 269, 272, 283, 286, 291, 292, 294-297, 301-326, 330, 338, 382, 405, 447-484, 644-695, 709-839, and 843-879 that are each independently capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) V-region polypeptide or an immunoglobulin (Ig) V-region polypeptide, and(ii) a plurality of J-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs;
53-63, 65, 215-220, and 247 that are each independently capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) J-region polypeptide or an immunoglobulin (Ig) J-region polypeptide,wherein the V-segment and J-segment primers are capable of amplifying in said multiplex dPCR at least 90% of all rearranged TCR or Ig CDR3-encoding regions in the sample to produce a multiplicity of amplified rearranged DNA molecules from the adaptive immune cells in the test sample; and (2) a set of control primers to produce an internal control gene amplification product, wherein the set of control primers amplifies an internal control gene segment that is not specific to adaptive immune cells; and (c) comparing a first number of assay samples that detectably contain said multiplicity of amplified rearranged DNA molecules of (b)(1) with a second number of assay samples that detectably contain said internal control gene amplification product of (b)(2), and therefrom quantifying the relative representation of adaptive immune cells in said test biological sample. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)
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48. A method for assessing an effect of a therapeutic treatment on relative representation of adaptive immune cells in at least one tissue of a subject, the tissue comprising adaptive immune cells and cells that are not adaptive immune cells, the method comprising:
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(I) obtaining one or a plurality of samples from a first tissue of the subject at one or a plurality of time points prior to administering the therapeutic treatment; (II) obtaining one or a plurality of samples from a second tissue of the subject at one or a plurality of time points after administering the therapeutic treatment; (III) for each of said samples from (I) and (II); (a) distributing template DNA extracted from the sample to form a set of assay samples, (b) amplifying said template DNA in the set of assay samples in a multiplex digital polymerase chain reaction (dPCR) that comprises; (1) (i) a plurality of V-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs;
1-52, 66-68, 221-238, 255-260, 262-267, 269, 272, 283, 286, 291, 292, 294-297, 301-326, 330, 338, 382, 405, 447-484, 644-695, 709-839, and 843-879 that are each independently capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) V-region polypeptide or an immunoglobulin (Ig) V-region polypeptide, and(ii) a plurality of J-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs;
53-63, 65, 215-220, and 247 that are each independently capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) J-region polypeptide or an immunoglobulin (Ig) J-region polypeptide,wherein the V-segment and J-segment oligonucleotide primers are capable of amplifying in said multiplex dPCR at least 90% of all rearranged TCR or Ig CDR3-encoding regions in the sample to produce a multiplicity of amplified rearranged DNA molecules from the adaptive immune cells in the test sample; and (2) a set of control primers capable of amplifying an internal control gene DNA segment present in every cell to produce internal control gene amplification products; and (c) comparing a first number of assay samples that detectably contain said multiplicity of amplified rearranged DNA molecules of (b)(1) with a second number of assay samples that detectably contain said internal control gene amplification product of (b)(2), and therefrom quantifying the relative representation of adaptive immune cells in said sample; and (IV) comparing the relative representation of adaptive immune cells in at least one sample obtained at a time point prior to administering the therapeutic treatment to the relative representation of adaptive immune cells in at least one sample obtained at a time point after administering the therapeutic treatment, and thereby assessing an effect of the therapeutic treatment on relative representation of adaptive immune cells in at least one tissue of a subject. - View Dependent Claims (49, 50, 51)
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Specification