High-throughput immune sequencing

• US 10,392,726 B2
• Filed: 10/11/2011
• Issued: 08/27/2019
• Est. Priority Date: 10/08/2010
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Vigneault, Francois, Laserson, Uri, Bachelet, Ido
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US13/878,400
• Publication Number:
  US 20130296535A1
• Time in Patent Office: 2,877 Days
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/065 Purification, fragmentation
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2535/00 Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C40B 10/00 Directed molecular evolution of macromolecules, e.g. RNA, DNA or proteins
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Methods and compositions for determining and/or monitoring the immune state of an individual are provided.

Claims

1. A method comprising:

(a) linking a polynucleotide sequence encoding a heavy chain variable region (V_H) and a polynucleotide sequencing encoding a light changing variable region (V_L) from a single cell to form paired sequences;

(b) sequencing a polynucleotide amplified from the paired sequences, thereby forming paired sequence reads;

(c) selecting a therapeutic antibody candidate comprising a V_H and a V_L encoded by at least one sequence pair linked in (a); and

(d) screening the therapeutic antibody candidate for a property, wherein the property is selected from the group consisting of functional specificity, affinity and neutralization ability;

wherein (a) is performed for a plurality of polynucleotide sequences encoding heavy chain variable regions (V_H) and a plurality of polynucleotide sequences encoding light chain variable regions (V_L) each from a single cell to form a plurality of paired sequences, each representing a sequence pair from an individual cell, and wherein selecting is based on a frequency of each of two or more paired V_H and V_L sequences from among the plurality of paired sequences and a variance from a reference library for at least one of the two or more paired V_H and V_L sequences.

2. The method of claim 1, wherein the linking is a physical linking.

3. The method of claim 1, further comprising cloning the therapeutic antibody directly into surface-display technology.

4. The method of claim 1, wherein the therapeutic antibody is a neutralizing antibody.

5. The method of claim 1, wherein the therapeutic antibody is a rapid response antibody.

6. The method of claim 1, further comprising determining an isotype of the therapeutic antibody.

7. The method of claim 1, wherein the polynucleotide amplified from the paired sequences is amplified by PCR with non-specific primers, degenerate primers, or specific primers.

8. The method of claim 1, wherein the polynucleotide amplified from the paired sequences is amplified by PCR with specific primer sets that hybridize to the heavy and light chains of:

B-cells, T-cells, or B-cells and T-cells.

9. The method of claim 1, wherein the polynucleotide amplified from the paired sequences is amplified by PCR with two sets of primers after (a), wherein the first set of primers hybridizes to V regions and the second set of primers hybridizes to C regions.

10. The method of claim 1, further comprising generating a database of paired V_H and V_L sequences.

11. The method of claim 10, wherein selecting comprises aligning sequence data from the database against known or expected V, D and J segments in:

a personal database, a NCBI database, or an IMGT database.

12. The method of claim 1, wherein the cell is a B-cell.

13. The method of claim 1, wherein the therapeutic antibody is fully human.

14. The method of claim 1, wherein the polynucleotide amplified from the paired sequences is DNA.

15. The method of claim 1, wherein the polynucleotide amplified from the paired sequences comprises sequence adaptors compatible with next-generation high-throughput sequencing.

16. The method of claim 1, wherein the sequencing is next-generation high-throughput sequencing.

17. The method of claim 1, wherein the paired sequences encoding a V_H and a V_L are bar-coded.

18. The method of claim 1, wherein in (c), a coefficient of variation of about 0.5 occurs at clone frequencies of <

10<sup>−

4</sup>-10<sup>−

3</sup>.

19. The method of claim 1, wherein the variance is a variance of an alignment of a paired sequence to a sequence of the reference library.

20. The method of claim 1, wherein in (c), a coefficient of variation approaches 1 at a sampling level of 10⁵.

21. The method of claim 1, further comprising clustering the paired sequences into unique clones.

22. The method of claim 4, further comprising identifying an antigen that interacts with the therapeutic antibody as a biomarker for a disease.

23. The method of claim 1, wherein each paired sequence of the plurality of paired sequences represents a sequence pair from an individual cell of a plurality of individually isolated single cells.

24. The method of claim 23, wherein the single cell is isolated from a single subject.

25. The method of claim 1, further comprising isolating and purifying the therapeutic antibody candidate.