# US 2014/0194295 A1 · Method of Measuring Adaptive Immunity

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2014/0194295 A1
- Document ID: US20140194295A1
- Status: Published application
- Kind: A1
- Application number: 14/095,629
- Filed: 2013-12-03
- Published: 2014-07-10
- Earliest claimed: 2009-06-25
- Assignees: Fred Hutchinson Cancer Research Center
- Applicants: Fred Hutchinson Cancer Research Center
- Inventors: Harlan S. Robins; Edus H. Warren, III; Christopher Scott Carlson
- Web page: https://patentgrep.com/patent/US20140194295A1

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C12Q 1/6883: for diseases caused by alterations of genetic material
- G16B 40/00: ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- C12Q 2600/16: Primer sets for multiplex assays
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

A method of measuring immunocompetence is described. This method provides a means for assessing the effects of diseases or conditions that compromise the immune system and of therapies aimed to reconstitute it. This method is based on quantifying T-cell diversity by calculating the number of diverse T-cell receptor (TCR) beta chain variable regions from blood cells.

## Claims

### Claim 1

A method, comprising:
generating a plurality of amplicons comprising rearranged TCR or IG CDR3 nucleic acid sequences from a first sample comprising nucleic acid molecules obtained from lymphocytes from a mammalian subject, using a plurality of V-segment primers and a plurality of J-segment primers in a single multiplex PCR, wherein each V-segment primer is complementary to one or more functional TCR or IG V-region gene segments, wherein each J-segment primer is complementary to one or more functional TCR or IG J-region gene segments, wherein said plurality of V-segment primers and said plurality of J-segment primers are capable of amplifying in said single multiplex PCR at least 104 distinct amplicons representing a diversity of rearranged TCR or IG CDR3 nucleic acid sequences present in said first sample;
sequencing said plurality of amplicons; and
determining a total diversity of rearranged TCR or IG CDR3 nucleic acid sequences from said plurality of sequenced amplicons of said first sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein said determining comprises quantifying said plurality of sequenced amplicons generated in said single multiplex PCR.

### Claim 3 (depends on 2)

The method of claim 2, wherein said quantifying comprises quantifying the number of distinct rearranged TCR or IG CDR3 nucleic acid sequences from said first sample.

### Claim 4 (depends on 2)

The method of claim 2, wherein said determining comprises estimating a total diversity of rearranged TCR or IG CDR3 nucleic acid sequences.

### Claim 5 (depends on 1)

The method of claim 1, wherein said total diversity comprises at least 104 distinct rearranged TCR or IG CDR3 nucleic acid sequences.

### Claim 6 (depends on 1)

The method of claim 1, wherein said total diversity comprises at least 105 distinct rearranged TCR or IG CDR3 nucleic acid sequences.

### Claim 7 (depends on 1)

The method of claim 1, wherein said total diversity comprises at least 106 distinct rearranged TCR or IG CDR3 nucleic acid sequences.

### Claim 8 (depends on 1)

The method of claim 1, wherein said nucleic acid molecules comprise genomic DNA.

### Claim 9 (depends on 1)

The method of claim 1, wherein said nucleic acid molecules comprise cDNA.

### Claim 10 (depends on 1)

The method of claim 1, wherein said nucleic acid molecules comprise mRNA.

### Claim 11 (depends on 1)

The method of claim 1, wherein sequencing comprises high-throughput sequencing of said plurality of amplicons using a set of sequencing oligonucleotides that hybridize to a defined region in each of said plurality of amplicons.

### Claim 12 (depends on 1)

The method of claim 1, wherein said TCR V-region gene segment comprises a TCR Vδ segment and wherein said TCR J-region gene segment comprises a TCR Jδ segment.

### Claim 13 (depends on 1)

The method of claim 1, wherein said TCR V-region gene segment comprises a TCR Vγ segment and wherein said TCR J-region gene segment comprises a TCR Jγ segment.

### Claim 14 (depends on 1)

The method of claim 1, wherein said TCR V-region gene segment comprises a TCR Vα segment and wherein said TCR J-region gene segment comprises a TCR Jα segment.

### Claim 15 (depends on 1)

The method of claim 1, wherein said TCR V-region gene segment comprises a TCR Vβ segment and wherein said TCR J-region gene segment comprises a TCR Jβ segment.

### Claim 16 (depends on 1)

The method of claim 1, wherein said Ig V-region gene segment comprises an IGH V gene segment and wherein said Ig J-region gene segment comprises an IGH J gene segment.

### Claim 17 (depends on 1)

The method of claim 1, wherein said Ig V-region gene segment comprises an IGL V gene segment and wherein said Ig J-region gene segment comprises an IGL J gene segment.

### Claim 18 (depends on 1)

The method of claim 1, wherein said Ig V-region gene segment comprises an IGK V gene segment and wherein said Ig J-region gene segment comprises an IGK J gene segment.

### Claim 19 (depends on 1)

The method of claim 1, wherein each of said distinct rearranged TCR or IG CDR3 nucleic acid sequences spans a V-D-J or V-J junction.

### Claim 20 (depends on 1)

The method of claim 1, further comprising:
generating a plurality of amplicons comprising rearranged TCR or IG CDR3 nucleic acid molecules from a second sample comprising nucleic acid molecules obtained from lymphocytes from a mammalian subject, using said plurality of V-segment primers and said plurality of J-segment primers in a single multiplex PCR;
sequencing said plurality of amplicons; and
determining a total diversity of rearranged TCR or IG CDR3 nucleic acid sequences from said plurality of sequenced amplicons of said second sample.

### Claim 21 (depends on 20)

The method of claim 20, wherein said first sample and said second sample are from the same mammalian subject.

### Claim 22 (depends on 21)

The method of claim 21, wherein said first sample is obtained from said mammalian subject before a treatment and said second sample is obtained from said mammalian subject after said treatment.

### Claim 23 (depends on 21)

The method of claim 21, further comprising comparing the total diversity of rearranged TCR or IG CDR3 nucleic acid sequences of said first sample with the total diversity of rearranged TCR or IG CDR3 nucleic acid sequences of said second sample.

### Claim 24 (depends on 23)

The method of claim 23, further comprising assessing an effect of a treatment based on differences in measurements of diversity between said first and second samples.

### Claim 25 (depends on 23)

The method of claim 23, further comprising assessing an immunocompetence of said subject based on differences in measurements of diversity between said first and second samples.