High-Throughput Immune Sequencing

• US 20130296535A1
• Filed: 10/11/2011
• Published: 11/07/2013
• 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
• Granted Patent: US 10,392,726 B2
• Time in Patent Office: Days
• US Class Current: 530/387.1
• 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

1-35. -35. (canceled)

36. A method for identifying a therapeutic antibody, comprising:

(a) linking polynucleotide sequences encoding a V_H and a V_L isolated from a single cell of a plurality of cells from a single subject to form paired sequences;

(b) sequencing polynucleotides amplified from the paired sequences;

(c) determining the frequency of two or more paired V_H and V_L sequences;

(d) determining a variance from a reference library of the paired sequences from (c); and

(e) identifying a therapeutic antibody based on (c) and (d).

37. The method of claim 36, further comprising screening the therapeutic antibody for functional specificity.

38. The method of claim 36, further comprising screening the therapeutic antibody for affinity.

39. The method of claim 36, further comprising screening the therapeutic antibody for neutralization ability.

40. The method of claim 36, further comprising cloning the therapeutic antibody directly into surface-display technology.

41. The method of claim 36, wherein the therapeutic antibody is a neutralizing antibody.

42. The method of claim 36, wherein the therapeutic antibody is identified without prior knowledge of an antigen to which it binds.

43. The method of claim 36, wherein the therapeutic antibody is a rapid response antibody.

44. The method of claim 36, further comprising determining the isotype of the therapeutic antibody.

45. An isolated and purified therapeutic antibody identified by the method of claim 36.

46. The method of claim 36, wherein the amplified polynucleotides are amplified by PCR with non-specific primers, degenerate primers, or specific primers.

47. The method of claim 36, wherein the amplified polynucleotides are 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.

48. The method of claim 36, wherein the amplified polynucleotides are amplified by PCR with two sets of primers, wherein the first set of primers hybridizes to V regions and the second set of primers hybridizes to C regions.

49. The method of claim 36, further comprising generating a database of paired V_H and V_L sequences.

50. The method of claim 49, wherein the identifying 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.

51. The method of claim 36, wherein the plurality of cells are a plurality of B-cells.

52. The method of claim 36, wherein the therapeutic antibody is fully human.

53. The method of claim 36, wherein the amplified polynucleotides are DNA.

54. The method of claim 36, wherein the amplified polynucleotides comprise sequence adaptors compatible with next-generation high-throughput sequencing.

55. The method of claim 36, wherein the sequencing is next-generation high-throughput sequencing.

56. The method of claim 36, wherein the linked polynucleotide sequences encoding a V_H and a V_L are bar-coded, wherein bar-coding can be used to achieve remapping of the amplified polynucleotides to their original starting molecules.

57. The method of claim 36, wherein in (c), a coefficient of variation of about 0.5 occurs at clone frequencies of <

10<sup>−

4</sup>-10<sup>−

3</sup>.

58. The method of claim 36, wherein determining a variance from a reference library of the paired sequences from (c) comprises aligning the paired sequences from (c) to the reference library sequences.

59. The method of claim 36, wherein in (c), a coefficient of variation approaches 1 at a sampling level of 10⁵.

60. The method of claim 36, further comprising clustering the paired sequences from (c) into unique clones.

61. The method of claim 39, further comprising identifying an antigen that interacts with the therapeutic antibody as a biomarker for a disease.