Rapid Isolation of Monoclonal Antibodies from Animals

• US 20110312505A1
• Filed: 05/17/2011
• Published: 12/22/2011
• Est. Priority Date: 05/17/2010
• Status: Active Grant

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Ellington, Andrew D., Marcotte, Edward M., Georgiou, George, Reddy, Sai, Ge, Xin, Lavinder, Jason, Boutz, Daniel
• Granted Patent: US 9,090,674 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C07K 16/065 Purification, fragmentation
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• 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: G01N 33/6857 Antibody fragments
• CPC Class Codes: 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
• CPC Class Codes: G16B 40/30 Unsupervised data analysis

Abstract

Methods and compositions for identification of candidate antigen-specific variable regions as well as generation of antibodies or antigen-binding fragments that could have desired antigen specificity are provided. For example, in certain aspects methods for determining amino acid sequences of serum antibody CDR and abundancy level are described. In some aspects, methods for determining nucleic acid sequences of antibody variable region sequences and frequency are provided. Furthermore, the invention provides methods for identification and generation of antibody or antigen-binding fragments that comprise highly-represented CDR.

Claims

1. A method for determining antibody sequences in circulation, comprising:

a) obtaining nucleic acid, and the corresponding amino acid, sequence information of VH and VL gene repertoires encoded by mature B cells of a subject;

b) obtaining mass spectra of peptides derived from serum antibodies of the subject; and

c) using the sequence information and the mass spectra to determine the amino acid sequence of VH and VL regions of one or more antibodies in circulation.

2. The method of claim 1, where in the mature B cells are from peripheral blood.

3. The method of claim 1, where in the mature B cells are from a lymphoid organ.

4. The method of claim 1, wherein step a) comprises determining the nucleic acid sequences and the corresponding amino acid sequences of rearranged antibody VH and VL.

5. The method of claim 4, wherein the nucleic acid sequences are determined by high throughput DNA sequencing.

7. The method of claim 1, wherein the mature B cells comprise memory B cells.

8. The method of claim 1, wherein the mature B cells comprise plasma cells.

9. The method of claim 1, wherein step b) comprises the use of high performance liquid chromatography (HPLC).

10. The method of claim 1, wherein step b) further comprises isolating or enriching a selected class of serum antibodies.

11. The method of claim 1, wherein step b) further comprises isolating or enriching serum antibodies that bind to a predetermined antigen.

12. The method of claim 1, wherein step b) further comprises isolating or enriching CDR3-containing fragments of serum antibodies.

13. The method of claim 1, wherein step b) further comprises preparing CDR3-containing peptide fragments from serum antibodies using a protease that is identified based on the sequence information.

14. The method of claim 1, wherein the subject has a tumor, an infectious disease, an autoimmune disease or has been immunized.

15. The method of claim 1, wherein the subject has or has been exposed to an antigen which is an infectious agent, a tumor antigen, a tumor cell or a self-antigen.

16. The method of claim 1, further comprising reporting the determination in step c).

17. The method of claim 1, wherein the method further comprises determining the abundancy level of the amino acid sequences of the serum antibodies.

18. The method of claim 1, wherein the method further comprises identifying the antibody sequences that exhibit at least a threshold level of abundancy.

19. The method of claim 18, further comprising generating one or more antibodies or antigen-binding fragments comprising one or more of the abundant amino acid sequences.

20. The method of claim 19, wherein each of the antibodies or antigen-binding fragments so generated comprises similarly abundant amino acid sequences of VH and VL or is part of a cluster of highly homologous amino acid sequence which are similarly abundant.

21. The method of claim 19, wherein the antibodies or antigen-binding fragments so generated in step c) bind an antigen the subject has or has been exposed to with a monovalent affinity of about 100 pM to 5 μ

M.

22. The method of claim 19, further comprising evaluating binding affinity of the antibody or antigen-binding fragments so generated toward a predetermined antigen.

6. (canceled)

23. A method for identifying abundant antibody sequences in circulation, comprising:

a) determining amino acid sequences corresponding to the VH and VL regions of antibodies in a serum-containing sample of a subject, to provide serum antibody sequences; and

b) identifying the antibody sequences that exhibit at least a threshold level of abundancy relative to other serum antibody sequences.

24-50. -50. (canceled)

51. A method for generating one or more antibodies or antigen-binding fragments, comprising:

a) obtaining sequence and abundancy information of antibody amino acid sequences corresponding to VH and VL regions of antibodies present in the serum of a subject;

b) identifying those sequences that exhibit at least a threshold level of abundancy as abundant amino acid sequences; and

c) generating one or more antibodies or antigen-binding fragments that comprise one or more of the abundant amino acid sequences so determined.

52-96. -96. (canceled)

97. A method for generating one or more antibodies or antigen-binding fragments, comprising:

a) obtaining sequence and abundancy information of nucleic acid sequences corresponding to VH and VL genes in mature B cells of a subject; and

b) identifying those sequences that exhibit a threshold level of abundancy; and

c) generating one or more antibodies or antigen-binding fragments comprising one or more of the amino acid sequences corresponding to identified nucleic acid sequences.

98-131. -131. (canceled)

132. A method for identifying candidate antigen-specific antibody variable region nucleic acid sequences, wherein the method comprises the following steps:

a) obtaining a pool of antibody variable region nucleic acids from a population of nucleic acids of a lymphoid tissue of a subject without separation of B cells from the lymphoid tissue, wherein the subject has been exposed to an antigen; and

b) determining sequences and occurrence frequency of antibody variable region nucleic acids in the pool;

c) identifying abundant variable region sequences that occur in total at a frequency of at least 0.5% in the sequences so determined at step b), wherein the abundant variable region sequences are candidate antigen-specific sequences.

133-159. -159. (canceled)